Maria Novatchkova

The increasing prevalence of diabetes has resulted in a global epidemic1. Diabetes is a major cause of blindness, kidney failure, heart attacks, stroke and amputation of lower limbs. These are often caused by changes in blood vessels, such as the expansion of the basement membrane and a loss of vascular cells2–4. Diabetes also impairs the functions of endothelial cells5 and disturbs the communication between endothelial cells and pericytes6. How dysfunction of endothelial cells and/or pericytes leads to diabetic vasculopathy remains largely unknown. Here we report the development of self-organizing three-dimensional human blood vessel organoids from pluripotent stem cells. These human blood vessel organoids contain endothelial cells and pericytes that self-assemble into capillary networks that are enveloped by a basement membrane. Human blood vessel organoids transplanted into mice form a stable, perfused vascular tree, including arteries, arterioles and venules. Exposure of blood vessel organoids to hyperglycaemia and inflammatory cytokines in vitro induces thickening of the vascular basement membrane. Human blood vessels, exposed in vivo to a diabetic milieu in mice, also mimic the microvascular changes found in patients with diabetes. DLL4 and NOTCH3 were identified as key drivers of diabetic vasculopathy in human blood vessels. Therefore, organoids derived from human stem cells faithfully recapitulate the structure and function of human blood vessels and are amenable systems for modelling and identifying the regulators of diabetic vasculopathy, a disease that affects hundreds of millions of patients worldwide. Organoids derived from human stem cells recapitulate the structure and functions of human blood vessels, and can be used to model and identify regulators of diabetic vasculopathy.

Autophagy is a mechanism by which starving cells can control their energy requirements and metabolic states, thus facilitating the survival of cells in stressful environments, in particular in the pathogenesis of cancer. Here we report that tissue-specific inactivation of Atg5, essential for the formation of autophagosomes, markedly impairs the progression of KRas(G12D)-driven lung cancer, resulting in a significant survival advantage of tumour-bearing mice. Autophagy-defective lung cancers exhibit impaired mitochondrial energy homoeostasis, oxidative stress and a constitutively active DNA damage response. Genetic deletion of the tumour suppressor p53 reinstates cancer progression of autophagy-deficient tumours. Although there is improved survival, the onset of Atg5-mutant KRas(G12D)-driven lung tumours is markedly accelerated. Mechanistically, increased oncogenesis maps to regulatory T cells. These results demonstrate that, in KRas(G12D)-driven lung cancer, Atg5-regulated autophagy accelerates tumour progression; however, autophagy also represses early oncogenesis, suggesting a link between deregulated autophagy and regulatory T cell controlled anticancer immunity.

Polycomb complexes establish chromatin modifications for maintaining gene repression and are essential for embryonic development in mice. Here we use pluripotent embryonic stem (ES) cells to demonstrate an unexpected redundancy between Polycomb-repressive complex 1 (PRC1) and PRC2 during the formation of differentiated cells. ES cells lacking the function of either PRC1 or PRC2 can differentiate into cells of the three germ layers, whereas simultaneous loss of PRC1 and PRC2 abrogates differentiation. On the molecular level, the differentiation defect is caused by the derepression of a set of genes that is redundantly repressed by PRC1 and PRC2 in ES cells. Furthermore, we find that genomic repeats are Polycomb targets and show that, in the absence of Polycomb complexes, endogenous murine leukemia virus elements can mobilize. This indicates a contribution of the Polycomb group system to the defense against parasitic DNA, and a potential role of genomic repeats in Polycomb-mediated gene regulation.

Apomixis, or asexual clonal reproduction through seeds, is of immense interest due to its potential application in agriculture. One key element of apomixis is apomeiosis, a deregulation of meiosis that results in a mitotic-like division. We isolated and characterised a novel gene that is directly involved in controlling entry into the second meiotic division. By combining a mutation in this gene with two others that affect key meiotic processes, we created a genotype called MiMe in which meiosis is totally replaced by mitosis. The obtained plants produce functional diploid gametes that are genetically identical to their mother. The creation of the MiMe genotype and apomeiosis phenotype is an important step towards understanding and engineering apomixis.

Worldwide, acute, and chronic pain affects 20% of the adult population and represents an enormous financial and emotional burden. Using genome-wide neuronal-specific RNAi knockdown in Drosophila, we report a global screen for an innate behavior and identify hundreds of genes implicated in heat nociception, including the α2δ family calcium channel subunit straightjacket (stj). Mice mutant for the stj ortholog CACNA2D3 (α2δ3) also exhibit impaired behavioral heat pain sensitivity. In addition, in humans, α2δ3 SNP variants associate with reduced sensitivity to acute noxious heat and chronic back pain. Functional imaging in α2δ3 mutant mice revealed impaired transmission of thermal pain-evoked signals from the thalamus to higher-order pain centers. Intriguingly, in α2δ3 mutant mice, thermal pain and tactile stimulation triggered strong cross-activation, or synesthesia, of brain regions involved in vision, olfaction, and hearing.

Genome-wide RNA interference (RNAi) screens have identified near-complete sets of genes involved in cellular processes. However, this methodology has not yet been used to study complex developmental processes in a tissue-specific manner. Here we report the use of a library of Drosophila strains expressing inducible hairpin RNAi constructs to study the Notch signalling pathway during external sensory organ development. We assigned putative loss-of-function phenotypes to 21.2% of the protein-coding Drosophila genes. Using secondary assays, we identified 6 new genes involved in asymmetric cell division and 23 novel genes regulating the Notch signalling pathway. By integrating our phenotypic results with protein interaction data, we constructed a genome-wide, functionally validated interaction network governing Notch signalling and asymmetric cell division. We used clustering algorithms to identify nuclear import pathways and the COP9 signallosome as Notch regulators. Our results show that complex developmental processes can be analysed on a genome-wide level and provide a unique resource for functional annotation of the Drosophila genome.

Organoids capable of forming tissue-like structures have transformed our ability to model human development and disease. With the notable exception of the human heart, lineage-specific self-organizing organoids have been reported for all major organs. Here, we established self-organizing cardioids from human pluripotent stem cells that intrinsically specify, pattern, and morph into chamber-like structures containing a cavity. Cardioid complexity can be controlled by signaling that instructs the separation of cardiomyocyte and endothelial layers and by directing epicardial spreading, inward migration, and differentiation. We find that cavity morphogenesis is governed by a mesodermal WNT-BMP signaling axis and requires its target HAND1, a transcription factor linked to developmental heart chamber defects. Upon cryoinjury, cardioids initiated a cell-type-dependent accumulation of extracellular matrix, an early hallmark of both regeneration and heart disease. Thus, human cardioids represent a powerful platform to mechanistically dissect self-organization, congenital heart defects and serve as a foundation for future translational research.

STAT5 and interleukin 7 (IL-7) signaling are thought to control B lymphopoiesis by regulating the expression of key transcription factors and by activating variable (V(H)) gene segments at the immunoglobulin heavy-chain (Igh) locus. Using conditional mutagenesis to delete the gene encoding the transcription factor STAT5, we demonstrate that the development of pro-B cells was restored by transgenic expression of the prosurvival protein Bcl-2, which compensated for loss of the antiapoptotic protein Mcl-1. Expression of the genes encoding the B cell-specification factor EBF1 and the B cell-commitment protein Pax5 as well as V(H) gene recombination were normal in STAT5- or IL-7 receptor alpha-chain (IL-7Ralpha)-deficient pro-B cells rescued by Bcl-2. STAT5-expressing pro-B cells contained little or no active chromatin at most V(H) genes. In contrast, rearrangements of the immunoglobulin-kappa light-chain locus (Igk) were more abundant in STAT5- or IL-7Ralpha-deficient pro-B cells. Hence, STAT5 and IL-7 signaling control cell survival and the developmental ordering of immunoglobulin gene rearrangements by suppressing premature Igk recombination in pro-B cells.

One week after fertilization, human embryos implant into the uterus. This event requires the embryo to form a blastocyst consisting of a sphere encircling a cavity lodging the embryo proper. Stem cells can form a blastocyst model that we called a blastoid1. Here we show that naive human pluripotent stem cells cultured in PXGL medium2 and triply inhibited for the Hippo, TGF-β and ERK pathways efficiently (with more than 70% efficiency) form blastoids generating blastocyst-stage analogues of the three founding lineages (more than 97% trophectoderm, epiblast and primitive endoderm) according to the sequence and timing of blastocyst development. Blastoids spontaneously form the first axis, and we observe that the epiblast induces the local maturation of the polar trophectoderm, thereby endowing blastoids with the capacity to directionally attach to hormonally stimulated endometrial cells, as during implantation. Thus, we propose that such a human blastoid is a faithful, scalable and ethical model for investigating human implantation and development3,4.

The balance between stem cell self-renewal and differentiation is precisely controlled to ensure tissue homeostasis and prevent tumorigenesis. Here we use genome-wide transgenic RNAi to identify 620 genes potentially involved in controlling this balance in Drosophila neuroblasts. We quantify all phenotypes and derive measurements for proliferation, lineage, cell size, and cell shape. We identify a set of transcriptional regulators essential for self-renewal and use hierarchical clustering and integration with interaction data to create functional networks for the control of neuroblast self-renewal and differentiation. Our data identify key roles for the chromatin remodeling Brm complex, the spliceosome, and the TRiC/CCT-complex and show that the alternatively spliced transcription factor Lola and the transcriptional elongation factors Ssrp and Barc control self-renewal in neuroblast lineages. As our data are strongly enriched for genes highly expressed in murine neural stem cells, they are likely to provide valuable insights into mammalian stem cell biology as well.

The reversible phosphorylation of proteins on serine, threonine, and tyrosine residues is an important biological regulatory mechanism. In the context of genome integrity, signaling cascades driven by phosphorylation are crucial for the coordination and regulation of DNA repair. The two serine/threonine protein kinases ataxia telangiectasia-mutated (ATM) and Ataxia telangiectasia-mutated and Rad3-related (ATR) are key factors in this process, each specific for different kinds of DNA lesions. They are conserved across eukaryotes, mediating the activation of cell-cycle checkpoints, chromatin modifications, and regulation of DNA repair proteins. We designed a novel mass spectrometry-based phosphoproteomics approach to study DNA damage repair in Arabidopsis thaliana. The protocol combines filter aided sample preparation, immobilized metal affinity chromatography, metal oxide affinity chromatography, and strong cation exchange chromatography for phosphopeptide generation, enrichment, and separation. Isobaric labeling employing iTRAQ (isobaric tags for relative and absolute quantitation) was used for profiling the phosphoproteome of atm atr double mutants and wild type plants under either regular growth conditions or challenged by irradiation. A total of 10,831 proteins were identified and 15,445 unique phosphopeptides were quantified, containing 134 up- and 38 down-regulated ATM/ATR dependent phosphopeptides. We identified known and novel ATM/ATR targets such as LIG4 and MRE11 (needed for resistance against ionizing radiation), PIE1 and SDG26 (implicated in chromatin remodeling), PCNA1, WAPL, and PDS5 (implicated in DNA replication), and ASK1 and HTA10 (involved in meiosis).

In numerous species, the formation of meiotic crossovers is largely under the control of a group of proteins known as ZMM. Here, we identified a new ZMM protein, HEI10, a RING finger-containing protein that is well conserved among species. We show that HEI10 is structurally and functionally related to the yeast Zip3 ZMM and that it is absolutely required for class I crossover (CO) formation in Arabidopsis thaliana. Furthermore, we show that it is present as numerous foci on the chromosome axes and the synaptonemal complex central element until pachytene. Then, from pachytene to diakinesis, HEI10 is retained at a limited number of sites that correspond to class I COs, where it co-localises with MLH1. Assuming that HEI10 early staining represents an early selection of recombination intermediates to be channelled into the ZMM pathway, HEI10 would therefore draw a continuity between early chosen recombination intermediates and final class I COs.

Genetic regulators and environmental stimuli modulate T cell activation in autoimmunity and cancer. The enzyme co-factor tetrahydrobiopterin (BH4) is involved in the production of monoamine neurotransmitters, the generation of nitric oxide, and pain1,2. Here we uncover a link between these processes, identifying a fundamental role for BH4 in T cell biology. We find that genetic inactivation of GTP cyclohydrolase 1 (GCH1, the rate-limiting enzyme in the synthesis of BH4) and inhibition of sepiapterin reductase (the terminal enzyme in the synthetic pathway for BH4) severely impair the proliferation of mature mouse and human T cells. BH4 production in activated T cells is linked to alterations in iron metabolism and mitochondrial bioenergetics. In vivo blockade of BH4 synthesis abrogates T-cell-mediated autoimmunity and allergic inflammation, and enhancing BH4 levels through GCH1 overexpression augments responses by CD4- and CD8-expressing T cells, increasing their antitumour activity in vivo. Administration of BH4 to mice markedly reduces tumour growth and expands the population of intratumoral effector T cells. Kynurenine-a tryptophan metabolite that blocks antitumour immunity-inhibits T cell proliferation in a manner that can be rescued by BH4. Finally, we report the development of a potent SPR antagonist for possible clinical use. Our data uncover GCH1, SPR and their downstream metabolite BH4 as critical regulators of T cell biology that can be readily manipulated to either block autoimmunity or enhance anticancer immunity.

Cells maintain the balance between homeostasis and inflammation by adapting and integrating the activity of intracellular signaling cascades, including the JAK-STAT pathway. Our understanding of how a tailored switch from homeostasis to a strong receptor-dependent response is coordinated remains limited. Here, we use an integrated transcriptomic and proteomic approach to analyze transcription-factor binding, gene expression and in vivo proximity-dependent labelling of proteins in living cells under homeostatic and interferon (IFN)-induced conditions. We show that interferons (IFN) switch murine macrophages from resting-state to induced gene expression by alternating subunits of transcription factor ISGF3. Whereas preformed STAT2-IRF9 complexes control basal expression of IFN-induced genes (ISG), both type I IFN and IFN-γ cause promoter binding of a complete ISGF3 complex containing STAT1, STAT2 and IRF9. In contrast to the dogmatic view of ISGF3 formation in the cytoplasm, our results suggest a model wherein the assembly of the ISGF3 complex occurs on DNA.

How targeted therapies and immunotherapies shape tumors, and thereby influence subsequent therapeutic responses, is poorly understood. In the present study, we show, in melanoma patients and mouse models, that when tumors relapse after targeted therapy with MAPK pathway inhibitors, they are cross-resistant to immunotherapies, despite the different modes of action of these therapies. We find that cross-resistance is mediated by a cancer cell-instructed, immunosuppressive tumor microenvironment that lacks functional CD103+ dendritic cells, precluding an effective T cell response. Restoring the numbers and functionality of CD103+ dendritic cells can re-sensitize cross-resistant tumors to immunotherapy. Cross-resistance does not arise from selective pressure of an immune response during evolution of resistance, but from the MAPK pathway, which not only is reactivated, but also exhibits an increased transcriptional output that drives immune evasion. Our work provides mechanistic evidence for cross-resistance between two unrelated therapies, and a scientific rationale for treating patients with immunotherapy before they acquire resistance to targeted therapy.

Evolutionary development of the human brain is characterized by the expansion of various brain regions. Here, we show that developmental processes specific to humans are responsible for malformations of cortical development (MCDs), which result in developmental delay and epilepsy in children. We generated a human cerebral organoid model for tuberous sclerosis complex (TSC) and identified a specific neural stem cell type, caudal late interneuron progenitor (CLIP) cells. In TSC, CLIP cells over-proliferate, generating excessive interneurons, brain tumors, and cortical malformations. Epidermal growth factor receptor inhibition reduces tumor burden, identifying potential treatment options for TSC and related disorders. The identification of CLIP cells reveals the extended interneuron generation in the human brain as a vulnerability for disease. In addition, this work demonstrates that analyzing MCDs can reveal fundamental insights into human-specific aspects of brain development.

During asymmetric cell division, the mitotic spindle must be properly oriented to ensure the asymmetric segregation of cell fate determinants into only one of the two daughter cells. In Drosophila neuroblasts, spindle orientation requires heterotrimeric G proteins and the Gα binding partner Pins, but how the Pins-Gαi complex interacts with the mitotic spindle is unclear. Here, we show that Pins binds directly to the microtubule binding protein Mud, the Drosophila homolog of NuMA. Like NuMA, Mud can bind to microtubules and enhance microtubule polymerization. In the absence of Mud, mitotic spindles in Drosophila neuroblasts fail to align with the polarity axis. This can lead to symmetric segregation of the cell fate determinants Brat and Prospero, resulting in the misspecification of daughter cell fates and tumor-like overproliferation in the Drosophila nervous system. Our data suggest a model in which asymmetrically localized Pins-Gαi complexes regulate spindle orientation by directly binding to Mud.

Allelic exclusion of immunoglobulin genes ensures the expression of a single antibody molecule in B cells through mostly unknown mechanisms. Large-scale contraction of the immunoglobulin heavy-chain (Igh) locus facilitates rearrangements between Igh variable (V(H)) and diversity gene segments in pro-B cells. Here we show that these long-range interactions are mediated by 'looping' of individual Igh subdomains. The Igk locus also underwent contraction by looping in small pre-B and immature B cells, demonstrating that immunoglobulin loci are in a contracted state in rearranging cells. Successful Igh recombination induced the rapid reversal of locus contraction in response to pre-B cell receptor signaling, which physically separated the distal V(H) genes from the proximal Igh domain, thus preventing further rearrangements. In the absence of locus contraction, only the four most proximal V(H) genes escaped allelic exclusion in immature mu-transgenic B lymphocytes. Pre-B cell receptor signaling also led to rapid repositioning of one Igh allele to repressive centromeric domains in response to downregulation of interleukin 7 signaling. These data link both locus 'decontraction' and centromeric recruitment to the establishment of allelic exclusion at the Igh locus.

We have identified a conserved sequence segment in transmembrane receptors (including SEFs, IL17Rs) and soluble factors (including CIKS/ACT1) in eukaryotes and bacteria – the SEFIR domain. This sequence domain is part of the new STIR domain superfamily comprising also the TIR domain known to mediate TIR–TIR homotypic interactions. In TOLL/IL1R-like pathways, the cytoplasmically localized TIR domain of a receptor and the TIR domain of a soluble adaptor interact physically and activate signalling. The similarity between the SEFIR and TIR domains involves the conserved boxes 1 and 2 of the TIR domain that are implicated in homotypic dimerization, but there is no sequence similarity between SEFIR domains and the TIR sequence box 3. By analogy, we suggest that SEFIR-domain proteins function as signalling components of Toll/IL-1R-similar pathways and that their SEFIR domain mediates physical protein–protein interactions between pathway components.

Heart diseases are the most common causes of morbidity and death in humans. Using cardiac-specific RNAi-silencing in Drosophila, we knocked down 7061 evolutionarily conserved genes under conditions of stress. We present a first global roadmap of pathways potentially playing conserved roles in the cardiovascular system. One critical pathway identified was the CCR4-Not complex implicated in transcriptional and posttranscriptional regulatory mechanisms. Silencing of CCR4-Not components in adult Drosophila resulted in myofibrillar disarray and dilated cardiomyopathy. Heterozygous not3 knockout mice showed spontaneous impairment of cardiac contractility and increased susceptibility to heart failure. These heart defects were reversed via inhibition of HDACs, suggesting a mechanistic link to epigenetic chromatin remodeling. In humans, we show that a common NOT3 SNP correlates with altered cardiac QT intervals, a known cause of potentially lethal ventricular tachyarrhythmias. Thus, our functional genome-wide screen in Drosophila can identify candidates that directly translate into conserved mammalian genes involved in heart function.

The noncoding Xist RNA triggers silencing of one of the two female X chromosomes during X inactivation in mammals. Gene silencing by Xist is restricted to a special developmental context in early embryos and specific hematopoietic precursors. Here, we show that Xist can initiate silencing in a lymphoma model. We identify the special AT-rich binding protein SATB1 as an essential silencing factor. Loss of SATB1 in tumor cells abrogates the silencing function of Xist. In lymphocytes Xist localizes along SATB1-organized chromatin and SATB1 and Xist influence each other's pattern of localization. SATB1 and its homolog SATB2 are expressed during the initiation window for X inactivation in ES cells. Importantly, viral expression of SATB1 or SATB2 enables gene silencing by Xist in embryonic fibroblasts, which normally do not provide an initiation context. Thus, our data establish SATB1 as a crucial silencing factor contributing to the initiation of X inactivation.

In this article, we summarize Arabidopsis genes encoding ubiquitin, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (E2s) and an additional selected set of proteins related to ubiquitylation. We emphasize comparisons to components from Saccharomyces cerevisiae, with occasional reference to animals. Among the E1 and E2s, Arabidopsis usually has two to four probable orthologs to one yeast gene. Also, Arabidopsis has genes with no likely ortholog in yeast, although they often have potential orthologs in animals. The large number of components with known function in ubiquitylation indicates that this process plays a complex role in cellular physiology.

Article23 August 2011free access A systematic analysis of Drosophila TUDOR domain-containing proteins identifies Vreteno and the Tdrd12 family as essential primary piRNA pathway factors Dominik Handler Dominik Handler Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria Search for more papers by this author Daniel Olivieri Daniel Olivieri Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria Search for more papers by this author Maria Novatchkova Maria Novatchkova Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria Institute of Molecular Pathology (IMP), Vienna, Austria Search for more papers by this author Franz Sebastian Gruber Franz Sebastian Gruber Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria Search for more papers by this author Katharina Meixner Katharina Meixner Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria Search for more papers by this author Karl Mechtler Karl Mechtler Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria Institute of Molecular Pathology (IMP), Vienna, Austria Search for more papers by this author Alexander Stark Alexander Stark Institute of Molecular Pathology (IMP), Vienna, Austria Search for more papers by this author Ravi Sachidanandam Ravi Sachidanandam Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Julius Brennecke Corresponding Author Julius Brennecke Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria Search for more papers by this author Dominik Handler Dominik Handler Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria Search for more papers by this author Daniel Olivieri Daniel Olivieri Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria Search for more papers by this author Maria Novatchkova Maria Novatchkova Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria Institute of Molecular Pathology (IMP), Vienna, Austria Search for more papers by this author Franz Sebastian Gruber Franz Sebastian Gruber Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria Search for more papers by this author Katharina Meixner Katharina Meixner Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria Search for more papers by this author Karl Mechtler Karl Mechtler Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria Institute of Molecular Pathology (IMP), Vienna, Austria Search for more papers by this author Alexander Stark Alexander Stark Institute of Molecular Pathology (IMP), Vienna, Austria Search for more papers by this author Ravi Sachidanandam Ravi Sachidanandam Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, NY, USA Search for more papers by this author Julius Brennecke Corresponding Author Julius Brennecke Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria Search for more papers by this author Author Information Dominik Handler1, Daniel Olivieri1, Maria Novatchkova1,2, Franz Sebastian Gruber1, Katharina Meixner1, Karl Mechtler1,2, Alexander Stark2, Ravi Sachidanandam3 and Julius Brennecke 1 1Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria 2Institute of Molecular Pathology (IMP), Vienna, Austria 3Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, NY, USA *Corresponding author. Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Dr Bohrgasse 3, Vienna 1030, Austria. Tel.: +43 179 044 4508; Fax: +43 179 044 110; E-mail: [email protected] The EMBO Journal (2011)30:3977-3993https://doi.org/10.1038/emboj.2011.308 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info PIWI proteins and their bound PIWI-interacting RNAs (piRNAs) form the core of a gonad-specific small RNA silencing pathway that protects the animal genome against the deleterious activity of transposable elements. Recent studies linked the piRNA pathway to TUDOR biology as TUDOR domains of various proteins bind symmetrically methylated Arginine residues in PIWI proteins. We systematically analysed the Drosophila TUDOR protein family and identified four previously not characterized TUDOR domain-containing proteins (CG4771, CG14303, CG11133 and CG31755) as essential piRNA pathway factors. We characterized CG4771 (Vreteno) in detail and demonstrate a critical role for this protein in primary piRNA biogenesis. Vreteno physically and/or genetically interacts with the primary pathway components Piwi, Armitage, Yb and Zucchini. Vreteno also interacts with the Tdrd12 orthologues CG11133 (Brother of Yb) and CG31755 (Sister of Yb), which are essential for the primary piRNA pathway in the germline and probably replace the function of the related but soma-specific factor Yb. Introduction The PIWI-interacting RNA (piRNA) pathway is an animal-specific small RNA pathway that silences selfish genetic elements such as transposons in gonads (Malone and Hannon, 2009; Khurana and Theurkauf, 2010; Senti and Brennecke, 2010). At the core of this pathway act Argonaute proteins from the PIWI clade and their bound small RNAs, generally referred to as piRNAs. Mutations in PIWI proteins or in factors involved in piRNA biogenesis or piRNA-mediated silencing lead to de-silencing of transposons, to widespread DNA damage and ultimately result in sterility. The analyses of piRNA populations from vertebrates and invertebrates have provided genuine insight into the genomic origin of piRNAs (Aravin et al, 2006, 2007, 2008; Girard et al, 2006; Lau et al, 2006; Vagin et al, 2006; Brennecke et al, 2007; Li et al, 2009; Malone et al, 2009; Robine et al, 2009; Saito et al, 2009). The three major piRNA sources are long RNAs originating from discrete genomic loci typically enriched in transposon sequences (piRNA clusters), transcripts from active transposons and finally mRNAs from numerous endogenous genes. The genetic and mechanistic principles of piRNA biogenesis are only poorly understood but sequence analyses of piRNA populations indicated that two modes of piRNA biogenesis exist (reviewed in Senti and Brennecke, 2010). On the one hand, during primary piRNA biogenesis presumably single-stranded precursor transcripts are processed in a seemingly random manner into 23–30 nt primary piRNAs (Lau et al, 2009; Li et al, 2009; Malone et al, 2009; Saito et al, 2009). On the other hand, transposon sense transcripts (typically from active elements) and antisense transcripts (typically from piRNA clusters) participate in the process of secondary piRNA biogenesis: Here, piRNA-mediated cleavage of the target transcript triggers the production of a novel piRNA with the reciprocal polarity (Brennecke et al, 2007; Gunawardane et al, 2007). Hallmarks of this so-called ping-pong amplification of piRNAs are conserved from sponges to mammals (Aravin et al, 2007; Grimson et al, 2008). The existence of two distinct piRNA biogenesis branches is particularly evident in the Drosophila ovary. Within ovarian germ cells, the three PIWI proteins Piwi, Aubergine and Argonaute 3 (Ago3) are co-expressed and piRNAs are generated via the primary and secondary pathways. The two major players of the secondary ping-pong pathway are Aubergine and Ago3 with Aubergine binding primarily cluster derived antisense piRNAs, while Ago3 is primarily complexed with transposon mRNA-derived sense piRNAs (Brennecke et al, 2007; Gunawardane et al, 2007; Li et al, 2009; Malone et al, 2009). In contrast, the surrounding follicle cells (somatic origin) express exclusively Piwi and piRNAs are produced only via the primary pathway (Lau et al, 2009; Li et al, 2009; Malone et al, 2009; Saito et al, 2009). As all three PIWI proteins are expressed in germline cells, accurate systems must be in place to guarantee controlled piRNA biogenesis and PIWI loading. Several recent studies indicate that modular interactions between PIWI proteins and TUDOR domain-containing proteins are part of this control system (Chen et al, 2009; Kirino et al, 2009, 2010; Nishida et al, 2009; Reuter et al, 2009; Vagin et al, 2009). The TUDOR domain is a member of the TUDOR ‘royal family’, which among others also contains Chromo, plant Agenet, MBT and PWWP domains (Maurer-Stroh et al, 2003). The core TUDOR domain spans ∼60 amino acids and folds into a strongly bent anti-parallel β-sheet with five strands forming a barrel-like fold (Sprangers et al, 2003; Chen et al, 2009; Friberg et al, 2009; Liu et al, 2010a, 2010b). A key function of this domain is to facilitate protein–protein interactions, which often depend on the post-translational methylation of Lysine or Arginine residues in target proteins. Indeed, several methylated Arginine residues have been identified in PIWI-family proteins and at least in some cases specific interactions between PIWI and TUDOR proteins require the symmetric di-methylation of Arginine residues (sDMAs) in PIWI proteins (Kirino et al, 2009, 2010; Nishida et al, 2009; Reuter et al, 2009; Vagin et al, 2009; Huang et al, 2011b). Based on the observed specificity of PIWI–TUDOR interactions, it is possible that an intricate sDMA code allows the controlled recruitment of selected TUDOR domain-containing proteins at specific points of the life cycle of PIWI–piRNA complexes. In Drosophila, six (Tudor, Spindle-E, Krimper, Tejas, Yb and Papi) out of the roughly 20 proteins implicated in the piRNA pathway contain TUDOR domains (Boswell and Mahowald, 1985; Gillespie and Berg, 1995; Lim and Kai, 2007; Malone et al, 2009; Nishida et al, 2009; Olivieri et al, 2010; Patil and Kai, 2010; Qi et al, 2010; Saito et al, 2010; Liu et al, 2011). We therefore decided to systematically analyse all fly TUDOR domain-containing proteins for an involvement in the piRNA pathway. This led to the identification of four novel TUDOR proteins as essential piRNA pathway factors. We characterized in detail the role of CG4771 (Vreteno), a tandem TUDOR domain-containing protein. Vreteno localizes to Yb bodies in follicle cells and to nuage in germline cells and is required for primary piRNA biogenesis in both cell types. Vreteno interacts with the three fly Tdrd12 proteins Yb, CG11133 (Brother of Yb) and CG31755 (Sister of Yb), which have partially overlapping functions in the somatic and germline piRNA pathways. Results Identification and classification of TUDOR domain-containing proteins in Drosophila We mined the Drosophila melanogaster proteome for TUDOR-clan domains (Pfam CL0049) using sensitive sequence–profile (HMMer) and profile–profile comparison methods (Soding et al, 2005). Supplementary Table SI lists all identified proteins and specifies the individual subclasses (see also Figure 1A). For further analysis we focused on the TUDOR-clan domains TUDOR and SMN, which both have been reported to bind sDMA residues (Selenko et al, 2001; Sprangers et al, 2003; Cote and Richard, 2005; Liu et al, 2010a, 2010b). This resulted in 22 proteins containing at least one TUDOR/SMN domain. Figure 1.Characterization of the Drosophila TUDOR proteins. (A) Cartoon showing all Drosophila melanogaster proteins containing TUDOR/SMN domains (blue boxes). All other significant protein domains identified via HHpred searches are indicated with coloured boxes and their identity is given to the right from N to C (ZnF: zinc finger; RRM: RNA recognition motif; BBC: B-Box C-terminal domain; DEAD: DEAD-Box RNA Helicase; Hel-C: Helicase C-terminal; HA2: Helicase associated domain; OB: oligo-nucleotide binding; CS: HSP20-like domain; DSRM: double-stranded RNA binding; TM: trans-membrane domain; KH: K homology; SNase: Staphylococcus nuclease; DUF: domain of unknown function; UBA: ubiquitin-associated domain). TUDOR proteins implicated in the piRNA pathway (including the ones from this study) marked with a black dot (left). The scale indicates amino-acid positions. The identified mouse orthologues (see Supplementary Figure S1), the number of identified TUDOR domains in fly (mouse) and the expression bias towards gonads in adult flies are shown to the right. Proteins with similar domain composition are grouped together. For CG14303, the ‘??’ indicate the non-annotated N-terminus. (B) The secondary structure cartoon (blue indicates β-strands, red α-helices) denotes the extended TUDOR domain and is based on Liu et al (2010a) (see also Supplementary Figure S1). The core TUDOR domain (SMART definition) is shown as an alignment for all identified TUDOR domains (‘e’ and ‘h’ above the alignment indicate β-strands and α-helices, respectively). The conserved Arginine and Aspartate residues present in all extended TUDOR domains are highlighted in green, aromatic cage residues in red, the Asparagine involved in sDMA binding in orange and a strongly conserved glycine in grey. To the left, the predicted likelihood of a domain to bind sDMA residues (based on the aromatic cage residues) is indicated with black (likely binder) and grey (potential binder) circles. Download figure Download PowerPoint An alignment of all TUDOR/SMN domains contained in this set indicates three subgroups (Supplementary Figure S1). Groups A (Smn, CG13472 and CG17454) and B (Otu, CG3251) show similarity only to the ∼60 amino-acid TUDOR core. All other sequences cluster together in group C and share significant similarity also N- and C-terminal to the TUDOR core. Characteristic for group C are two 100% conserved amino acids, an Arginine in β4 and an Aspartate in the loop linking β5 and β6 of the extended TUDOR structure (Supplementary Figure S1; marked in green in Figure 1B; Liu et al, 2010a). Based on structural studies, group C sequences represent extended TUDOR domains, which are characterized by a core TUDOR domain tightly interacting with an OB-fold that consists of the N-terminal and C-terminal extensions (Liu et al, 2010a, 2010b). So far, every TUDOR domain-containing protein that has been linked to the piRNA pathway belongs to the extended TUDOR group. To further characterize the set of proteins harbouring extended TUDOR domains, we annotated all additionally contained protein domains and searched for the corresponding mouse orthologues (Figure 1A; Supplementary Figure S2). Most of the fly proteins exhibit strong similarity to their mouse counterparts and the listed pairs in Supplementary Figure S2 are supported by multiple independent orthology assignment methods. CG14303 was linked to Rnf17 based on automated orthology identification (Inparanoid, Compara, OMA) and similarities in the TUDOR domains and an N-terminal B-Box C-terminal domain. We note that the N-terminus of CG14303 is not annotated in FlyBase (lack of EST data), indicating that the similarities between CG14303 and Rnf17 might also include the RING-type zinc finger found in Rnf17. Notably, an N-terminal RING finger could be identified in the Apis and Bombyx CG14303/Rnf17 orthologues. Murine Tdrd12 was assigned to CG11133 and CG31755 based on OrthoMCL (v2:OG2_82474 and v4:OG4_21213). Since both of these proteins share a similar domain composition with the piRNA pathway protein Yb, it appears that Tdrd12 has radiated in Drosophila into three proteins. Indeed, all three fly proteins are more related to each other than to the single mouse or human Tdrd12 proteins. Less obvious was the assignment of Tdrd1, a 4 × TUDOR domain protein with an N-terminal MYND-type zinc finger. Based on domain composition, Tdrd1 might be the single mammalian counterpart of fly CG9925, CG9864 and CG4771, all of which encode besides multiple TUDOR domains also a MYND zinc finger (CG4771 contains in addition an RRM domain). Fly proteins with no assignable mouse counterparts are Krimper as well as the two testes specific proteins CG15042 and CG15930 (the two TUDOR domains of Krimper and CG15042 are highly similar, potentially suggesting a common ancestor). Finally, mouse Tdrd8 seems to lack a detectable fly orthologue. TUDOR/SMN domains often bind peptides with sDMA residues in target proteins. The sDMA-binding pocket resides within the TUDOR core. It consists of four aromatic residues (Figure 1B, marked in red), whose aromatic rings form a cuboid cage and complex the di-methylated guanidine group (Selenko et al, 2001; Sprangers et al, 2003; Cote and Richard, 2005; Liu et al, 2010a, 2010b). An additional conserved Asparagine (Figure 1B, marked in orange) interacts with the sDMA residue via a hydrogen bond (Liu et al, 2010a, 2010b). In sDMA-binding TUDOR domains, the aromatic cage residues are highly conserved and are critical for sDMA binding. We inspected the Drosophila extended TUDOR domains for aromatic cage residues. The alignment in Figure 1B indicates that while a set of TUDOR domains harbours all of these important residues at the exact same position, numerous TUDOR domains seemingly lost the ability to bind sDMA residues due to multiple amino-acid exchanges at critical positions. Nevertheless, many of the identified proteins contain at least one TUDOR domain with an intact aromatic cage and therefore likely interact with sDMA residues. We finally analysed the RNA expression pattern of all TUDOR/SMN genes via the adult Drosophila Fly Atlas (Chintapalli et al, 2007). This showed a strong bias for genes with extended TUDOR domains to be expressed in ovaries and/or testes, further suggesting a link to piRNA biology (Figure 1A; Supplementary Figure S3). Defining the set of TUDOR proteins with critical roles in the ovarian piRNA pathway The implication of several TUDOR proteins in piRNA biology and their often gonad-specific expression prompted us to genetically test all proteins with extended TUDOR domains for their involvement in the piRNA pathway. Defects in the piRNA pathway lead to sterility and to a substantial accumulation of transposon transcripts in ovaries. We therefore assayed these phenotypes in females where individual TUDOR domain-containing proteins were knocked down via RNAi specifically in the ovarian soma (marked in green in Figure 2A and B) or in the germline (marked in beige in Figure 2A and B). Figure 2.The set of TUDOR proteins involved in the Drosophila piRNA pathway. (A) Cartoon of a Drosophila ovariole (somatic cells are in green, germline cells are in beige). The RNAi systems used for the two cell types are listed. (B) Immunostaining of Armitage (green) and DNA (blue) in egg chambers expressing RNAi constructs in a tissue-specific manner (left: wild type; middle: soma knockdown via tj-GAL4>hpRNA; right: germline knockdown via MTD-GAL4>shRNA or NGT-GAL4>Dcr-2+hpRNA). Monochrome panels show only the anti-Armitage channel. (C) Bright field images of ovarioles stained for β-GAL activity. The individual genotypes represent soma-specific knockdowns of the indicated genes in the background of the gypsy-lacZ sensor described in Sarot et al (2004). zucchini knockdown serves as a positive control and spindle-E as negative control. Of all TUDOR knockdowns, only those against CG4771 or Yb resulted in sensor de-repression. (D) Changes in steady-state levels of HeT-A and blood transposon transcripts upon knockdown of individual TUDOR proteins in the germline with the shRNA (black/gray) or the hpRNA (red/rose) knockdown systems (normalized to no-hairpin controls via rp49; log scale; n=3; error bars indicate s.d.). Identity of knocked down genes identical to the legend in (E). (E) Fertility rates of females with germline-specific knockdown of indicated TUDOR proteins using the shRNA (black) and the hpRNA (red) systems (∼200 eggs per experiment; n=3; error bars indicate s.d.). Download figure Download PowerPoint RNAi in the follicular epithelium (soma), where only the primary piRNA pathway is active, was based on tj-GAL4 driven dsRNA-hairpin constructs (hp-lines) from the VDRC (Vienna Drosophila RNAi Centre) library (Dietzl et al, 2007; Olivieri et al, 2010). For the germline we expressed short hairpin constructs (sh-lines) with the germline-specific MTD-GAL4 driver, which allows robust knockdowns (Haley et al, 2008; Ni et al, 2011). In addition, we took advantage of the observation that VDRC hp-lines induce potent RNAi in the germline if expressed in conjunction with Dicer-2 (Sidney Wang and Sarah Elgin, personal communication). Figure 2B illustrates specificity and efficacy of the soma and germline-specific knockdowns using the piRNA biogenesis factor Armitage as an example. Integrity of the somatic piRNA pathway was monitored via a gypsy-lacZ construct that accurately reports piRNA-mediated silencing in follicle cells (Figure 2C; Sarot et al, 2004; Olivieri et al, 2010). Integrity of the germline piRNA pathway was monitored via the steady-state RNA levels of the two transposons HeT-A and blood (Figure 2D). In addition, we determined female fertility rates (percentage of hatched eggs) for all knockdowns (Figure 2E). The two germline knockdown approaches yielded in nearly all cases identical results. We attribute the three exceptions (CG9925-hp, yu-sh, CG14303-sh; Figure 2D and E) to off-target effects or non-functional RNAi lines. Seven TUDOR proteins scored as putative piRNA pathway components (CG4771, CG11133, Tejas, CG14303, Spindle-E, Krimper, Yb). All four factors that had previously been shown to be essential pathway members (Spindle-E, Krimper, Tejas, Yb) were identified. In agreement with the literature, Spindle-E, Krimper and Tejas scored only in the germline knockdowns while Yb scored only in the soma assay (Lim and Kai, 2007; Malone et al, 2009; Szakmary et al, 2009; Olivieri et al, 2010; Patil and Kai, 2010). Papi and Tudor—though previously implicated in the pathway—did not result in transposon de-silencing or sterility. This is in agreement with the literature as both proteins are dispensable for fertility and corresponding mutant ovaries contain no or only slightly elevated transposon RNA levels (Nishida et al, 2009; Liu et al, 2011). We note that the grandchild-less phenotype for Tudor (Boswell and Mahowald, 1985) is recapitulated in the Tudor germline knockdowns. In addition to the known factors, germline knockdowns of three uncharacterized proteins (CG14303, CG4771, CG11133; Figure 2D and E) resulted in sterility and transposon silencing defects. Out of these, CG4771 was also identified as an essential component for the somatic piRNA pathway (Figure 2C) and we therefore decided to characterize this factor in more detail. Vreteno (CG4771) is an essential piRNA pathway factor CG4771 is localized on the third chromosome (Figure 3A) and encodes a protein with two extended TUDOR domains (Figure 3B). The C-terminal TUDOR domain might possess sDMA-binding activity (Figure 1B) and the relevant aromatic cage residues are conserved in distantly related Drosophila species (Figure 3B). In addition, CG4771 harbours an N-terminal RRM domain and a highly conserved zinc finger belonging to the MYND family (C2C4HC). Figure 3.Vreteno is a novel piRNA pathway member. (A) Overview of the CG4771 (vreteno) genomic locus indicating flanking genes (blue), the HP36220-insertion site (pink triangle) and the extent of the genomic rescue construct. (B) Cartoon of the CG4771 protein domain structure and sequence alignment of the C-terminal TUDOR domain in distantly related Drosophilids (virilis, mojavensis, grimshawi, willistoni, melanogaster, pseudoobscura). Aromatic cage residues and the conserved Arg/Asp residues colour coded as in Figure 1B. (C) Changes in steady-state transposon levels (n=3; s.d.) upon CG4771 knockdown (normalized to no-hairpin controls) in soma (green) or germline (beige) in comparison to those in CG4771[HP36220] mutants (black; normalized to heterozygotes). (D) Immunostaining of Piwi in wild-type and CG4771[HP36220] mutant egg chambers. (E) The occasionally observed egg chamber morphology of CG4771[Δ1] (vreteno) mutants, which originally led us to name the gene ‘avocado’ (DNA stained with DAPI). (F) RNA levels of CG4771, of the flanking genes HP1c and CG6985 and of actin-5C in vreteno[Δ1] mutant ovaries compared with vreteno[Δ1]; GFP–vreteno rescued ovaries (values normalized to w[1118] controls). (G) Immunostaining of Piwi in vreteno[Δ1] mutant egg chambers and in vreteno[Δ1] mutant egg chambers expressing a GFP–vreteno rescue construct. (H) Steady-state RNA levels of the HeT-A, blood and ZAM transposons in vreteno[Δ1] mutant ovaries compared with vreteno[Δ1]; EGFP–vreteno rescued ovaries (values normalized to heterozygous siblings; n=3; error bars indicate s.d.). (I) Immunostaining of Vreteno in wild-type and vreteno[Δ1] mutant egg chambers at identical microscope settings. Download figure Download PowerPoint To verify that CG4771 is a piRNA pathway factor, we obtained genetic alleles of this gene. Females homozygous for the P-insertion HP36220 (Bloomington), which is inserted into the 5′UTR of CG4771 (Figure 3A) were sterile and laid eggs that exhibited defects in dorso-ventral patterning as evidenced by a high percentage of fused dorsal appendages. This is a common phenotype of piRNA pathway mutants and stems from the activation of the Chk2 DNA damage pathway, presumably caused by widespread DNA damage originating from uncontrolled transposon activity (Chen et al, 2007; Klattenhoff et al, 2007). However, in HP36220 mutants only the blood element was de-repressed, although germline-specific and soma-specific knockdowns of CG4771 clearly de-repressed also HeT-A and ZAM, respectively (Figure 3C). This suggested that HP36220 is a hypomorphic allele. Indeed, nuclear Piwi localization in the mutant was impaired, yet to a lesser degree than in armitage or Yb mutants (Figure 3D; Olivieri et al, 2010). We therefore generated an additional allele by mobilizing the HP36220 element. Out of 280 analysed excision events, one line (CG4771[Δ1]) exhibited a more pronounced phenotype as homozygous females failed to lay eggs. The ovarian morphology of CG4771[Δ1] mutants strongly resembled those of armitage or zucchini null ovaries (Pane et al, 2007; Olivieri et al, 2010). In some egg chambers, we observed besides the oocyte nucleus a single giant nurse cell nucleus, indicating severe defects in cytokinesis. Based on the morphology of these egg chambers (Figure 3E), we initially named CG4771 ‘avocado’. While this work was under review, CG4771 was named ‘vreteno’ in FlyBase by the Lehmann group (‘vreteno’ means ‘spindle’ in Bulgarian, referring to the spindle class phenotype of eggs laid by CG4771 mutants) and we therefore adopted this name for consistency reasons. In vreteno[Δ1] homozygous ovaries, germline- and soma-specific transposons were severely de-repressed, indicating the stronger nature of this allele (Figure 3F). This was paralleled by a more pronounced defect in nuclear Piwi accumulation (compare Figure 3G and D). To verify that the vreteno[Δ1] phenotype is due to defects in the CG4771 locus, we restored fertility (not shown), transposon silencing (Figure 3F) and nuclear Piwi localization (Figure 3G) to wild-type levels by introducing a genomic rescue construct that expresses GFP-tagged Vreteno under its endogenous regulatory regions (Figure 3A). As this rescue construct also contained the complete loci for HP1c and CG6985, we measured steady-state RNA levels of all three genes in ovaries of vreteno[Δ1] mutants and of the GFP–vreteno rescued animals (Figure 3H). This confirmed the specificity of the Δ1 allele for the vreteno locus. Immunofluorescence analysis with an antibody recognizing the Vreteno N-terminus further indicated that also the protein is essentially not detectable in ovaries from vreteno[Δ1] mutants (Figure 3I). We also analysed the requirement of vreteno for the piRNA pathway in males. Towards this end, we measured steady-state levels of the transposons mdg1 and copia as well as of the repetitive Stellate locus that is under control of the piRNA pathway in testes. This indicated a requirement of vreteno for copia and Stellate silencing, supported by the observation that silencing was fully restored in males expressing a GFP–vreteno rescue construct (Supplementary Figure S4). Taken together, vreteno encodes a novel piRNA pathway factor that is essential for the ovarian and testes piRNA pathways. Vreteno is required for primary piRNA biogenesis in soma and germline Defects in primary piRNA biogenesis (e.g. in armitage or zucchini mutants) result in a collapse of piRNA populations in follicle cells, in a severe reduction of most germline piRNA species and in defects in Piwi's nuclear accumulation accompanied by a significant loss of Piwi protein (Pane et al, 2007; Malone et al, 2009; Haase et al, 2010; Olivieri et al, 2010; Saito et al, 2010). Delocalization and decreased levels of Piwi were also observed in the vreteno[Δ1] mutant (Figure 3D and G). We therefore analysed the effects of loss of Vreteno on PIWI-family proteins and on piRNA populations in the ovarian soma and germline. Similar to an RNAi-mediated Armitage knockdown, knockdown of Vreteno in follicle cells led to an almost complete loss of Piwi protein in these cells (Figure 4A), indicating defects in pr

We identify a highly specific mutation (jf18) in the Caenorhabditis elegans nuclear envelope protein matefin MTF-1/SUN-1 that provides direct evidence for active involvement of the nuclear envelope in homologous chromosome pairing in C. elegans meiosis. The reorganization of chromatin in early meiosis is disrupted in mtf-1/sun-1(jf18) gonads, concomitant with the absence of presynaptic homolog alignment. Synapsis is established precociously and nonhomologously. Wild-type leptotene/zygotene nuclei show patch-like aggregations of the ZYG-12 protein, which fail to develop in mtf-1/sun-1(jf18) mutants. These patches remarkably colocalize with a component of the cis-acting chromosomal pairing center (HIM-8) rather than the centrosome. Our data on this mtf-1/sun-1 allele challenge the previously postulated role of the centrosome/spindle organizing center in chromosome pairing, and clearly support a role for MTF-1/SUN-1 in meiotic chromosome reorganization and in homolog recognition, possibly by mediating local aggregation of the ZYG-12 protein in meiotic nuclei.

GPI lipid anchoring is an important post-translational modification of eukaryote proteins in the endoplasmic reticulum. In total, 19 genes have been directly implicated in the anchor synthesis and the substrate protein modification pathway. Here, the molecular functions of the respective proteins and their evolution are analyzed in the context of reported literature data and sequence analysis studies for the complete pathway (http://mendel.imp.univie.ac.at/SEQUENCES/gpi-biosynthesis/) and questions for future experimental investigation are discussed. Studies of two of these proteins have provided new mechanistic insights. The cytosolic part of PIG-A/GPI3 has a two-domain alpha/beta/alpha-layered structure; it is suggested that its C-terminal subsegment binds UDP-GlcNAc whereas the N-terminal domain interacts with the phosphatidylinositol moiety. The lumenal part of PIG-T/GPI16 apparently consists of a beta-propeller with a central hole that regulates the access of substrate protein C termini to the active site of the cysteine protease PIG-K/GPI8 (gating mechanism) as well as of a polypeptide hook that embraces PIG-K/GPI8. This structural proposal would explain the paradoxical properties of the GPI lipid anchor signal motif and of PIG-K/GPI8 orthologs without membrane insertion regions in some species.

<h2>Summary</h2> V_H-DJ_H recombination of the immunoglobulin heavy chain (<i>Igh</i>) locus is temporally and spatially controlled during early B cell development, and yet no regulatory elements other than the V_H gene promoters have been identified throughout the entire V_H gene cluster. Here, we discovered regulatory sequences that are interspersed in the distal V_H gene region. These conserved repeat elements were characterized by the presence of Pax5 transcription factor-dependent active chromatin by binding of the regulators Pax5, E2A, CTCF, and Rad21, as well as by Pax5-dependent antisense transcription in pro-B cells. The <i>P</i>ax5-<i>a</i>ctivated <i>i</i>ntergenic <i>r</i>epeat (PAIR) elements were no longer bound by Pax5 in pre-B and B cells consistent with the loss of antisense transcription, whereas E2A and CTCF interacted with PAIR elements throughout early B cell development. The pro-B cell-specific and Pax5-dependent activity of the PAIR elements suggests that they are involved in the regulation of distal V_H-DJ_H recombination at the <i>Igh</i> locus.

Polyploidy has had a considerable impact on the evolution of many eukaryotes, especially angiosperms. Indeed, most--if not all-angiosperms have experienced at least one round of polyploidy during the course of their evolution, and many important crop plants are current polyploids. The occurrence of 2n gametes (diplogametes) in diploid populations is widely recognised as the major source of polyploid formation. However, limited information is available on the genetic control of diplogamete production. Here, we describe the isolation and characterisation of the first gene, AtPS1 (Arabidopsis thaliana Parallel Spindle 1), implicated in the formation of a high frequency of diplogametes in plants. Atps1 mutants produce diploid male spores, diploid pollen grains, and spontaneous triploid plants in the next generation. Female meiosis is not affected in the mutant. We demonstrated that abnormal spindle orientation at male meiosis II leads to diplogamete formation. Most of the parent's heterozygosity is therefore conserved in the Atps1 diploid gametes, which is a key issue for plant breeding. The AtPS1 protein is conserved throughout the plant kingdom and carries domains suggestive of a regulatory function. The isolation of a gene involved in diplogamete production opens the way for new strategies in plant breeding programmes and progress in evolutionary studies.

Breast cancer is the most common female cancer, affecting approximately one in eight women during their life-time. Besides environmental triggers and hormones, inherited mutations in the breast cancer 1 (BRCA1) or BRCA2 genes markedly increase the risk for the development of breast cancer. Here, using two different mouse models, we show that genetic inactivation of the key osteoclast differentiation factor RANK in the mammary epithelium markedly delayed onset, reduced incidence, and attenuated progression of Brca1;p53 mutation-driven mammary cancer. Long-term pharmacological inhibition of the RANK ligand RANKL in mice abolished the occurrence of Brca1 mutation-driven pre-neoplastic lesions. Mechanistically, genetic inactivation of Rank or RANKL/RANK blockade impaired proliferation and expansion of both murine Brca1;p53 mutant mammary stem cells and mammary progenitors from human BRCA1 mutation carriers. In addition, genome variations within the RANK locus were significantly associated with risk of developing breast cancer in women with BRCA1 mutations. Thus, RANKL/RANK control progenitor cell expansion and tumorigenesis in inherited breast cancer. These results present a viable strategy for the possible prevention of breast cancer in BRCA1 mutant patients.

Drosophila neuroblasts (NBs) have emerged as a model for stem cell biology that is ideal for genetic analysis but is limited by the lack of cell-type-specific gene expression data. Here, we describe a method for isolating large numbers of pure NBs and differentiating neurons that retain both cell-cycle and lineage characteristics. We determine transcriptional profiles by mRNA sequencing and identify 28 predicted NB-specific transcription factors that can be arranged in a network containing hubs for Notch signaling, growth control, and chromatin regulation. Overexpression and RNA interference for these factors identify Klumpfuss as a regulator of self-renewal. We show that loss of Klumpfuss function causes premature differentiation and that overexpression results in the formation of transplantable brain tumors. Our data represent a valuable resource for investigating Drosophila developmental neurobiology, and the described method can be applied to other invertebrate stem cell lineages as well.

The immunoglobulin heavy-chain (Igh) locus undergoes large-scale contraction in pro-B cells, which facilitates VH-DJH recombination by juxtaposing distal VH genes next to the DJH-rearranged gene segment in the 3' proximal Igh domain. By using high-resolution mapping of long-range interactions, we demonstrate that local interaction domains established the three-dimensional structure of the extended Igh locus in lymphoid progenitors. In pro-B cells, these local domains engaged in long-range interactions across the Igh locus, which depend on the regulators Pax5, YY1, and CTCF. The large VH gene cluster underwent flexible long-range interactions with the more rigidly structured proximal domain, which probably ensures similar participation of all VH genes in VH-DJH recombination to generate a diverse antibody repertoire. These long-range interactions appear to be an intrinsic feature of the VH gene cluster, because they are still generated upon mutation of the Eμ enhancer, IGCR1 insulator, or 3' regulatory region in the proximal Igh domain.

The anaphase-promoting complex/cyclosome (APC/C) bound to CDC20 (APC/C(CDC20)) initiates anaphase by ubiquitylating B-type cyclins and securin. During chromosome bi-orientation, CDC20 assembles with MAD2, BUBR1 and BUB3 into a mitotic checkpoint complex (MCC) that inhibits substrate recruitment to the APC/C. APC/C activation depends on MCC disassembly, which was proposed to require CDC20 autoubiquitylation. Here we characterize APC15, a human APC/C subunit related to yeast Mnd2. APC15 is located near APC/C's MCC binding site; it is required for APC/C-bound MCC (APC/C(MCC))-dependent CDC20 autoubiquitylation and degradation and for timely anaphase initiation but is dispensable for substrate ubiquitylation by APC/C(CDC20) and APC/C(CDH1). Our results support the model wherein MCC is continuously assembled and disassembled to enable rapid activation of APC/C(CDC20) and CDC20 autoubiquitylation promotes MCC disassembly. We propose that APC15 and Mnd2 negatively regulate APC/C coactivators and report generation of recombinant human APC/C.

The PIWI-interacting RNA (piRNA) pathway protects genome integrity in part through establishing repressive heterochromatin at transposon loci. Silencing requires piRNA-guided targeting of nuclear PIWI proteins to nascent transposon transcripts, yet the subsequent molecular events are not understood. Here, we identify SFiNX (silencing factor interacting nuclear export variant), an interdependent protein complex required for Piwi-mediated cotranscriptional silencing in Drosophila. SFiNX consists of Nxf2–Nxt1, a gonad-specific variant of the heterodimeric messenger RNA export receptor Nxf1–Nxt1 and the Piwi-associated protein Panoramix. SFiNX mutant flies are sterile and exhibit transposon derepression because piRNA-loaded Piwi is unable to establish heterochromatin. Within SFiNX, Panoramix recruits heterochromatin effectors, while the RNA binding protein Nxf2 licenses cotranscriptional silencing. Our data reveal how Nxf2 might have evolved from an RNA transport receptor into a cotranscriptional silencing factor. Thus, NXF variants, which are abundant in metazoans, can have diverse molecular functions and might have been coopted for host genome defense more broadly. Identification of SFiNX, a complex of Nxf2–Nxt1, a variant of the mRNA export receptor Nxf1–Nxt1 and the Piwi-associated protein Panoramix, demonstrates an RNA export independent role for Nxf2 in piRNA-guided cotranscriptional transposon silencing.

There is considerable inter-individual variability in susceptibility to weight gain despite an equally obesogenic environment in large parts of the world. Whereas many studies have focused on identifying the genetic susceptibility to obesity, we performed a GWAS on metabolically healthy thin individuals (lowest 6th percentile of the population-wide BMI spectrum) in a uniquely phenotyped Estonian cohort. We discovered anaplastic lymphoma kinase (ALK) as a candidate thinness gene. In Drosophila, RNAi mediated knockdown of Alk led to decreased triglyceride levels. In mice, genetic deletion of Alk resulted in thin animals with marked resistance to diet- and leptin-mutation-induced obesity. Mechanistically, we found that ALK expression in hypothalamic neurons controls energy expenditure via sympathetic control of adipose tissue lipolysis. Our genetic and mechanistic experiments identify ALK as a thinness gene, which is involved in the resistance to weight gain.

Article24 June 2019Open Access Source DataTransparent process Apelin inhibition prevents resistance and metastasis associated with anti-angiogenic therapy Iris Uribesalgo Corresponding Author Iris Uribesalgo [email protected] orcid.org/0000-0001-9492-1000 Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter, Vienna, Austria Search for more papers by this author David Hoffmann David Hoffmann Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter, Vienna, Austria Search for more papers by this author Yin Zhang Yin Zhang Department of Microbiology, Tumor and Cell Biology, Biomedicum, Karolinska Institutet, Stockholm, Sweden Medicine and Pharmacy Research Center, Binzhou Medical University, Yantai, Shandong Province, China Search for more papers by this author Anoop Kavirayani Anoop Kavirayani VBCF Histopathology, Vienna BioCenter, Vienna, Austria Search for more papers by this author Jelena Lazovic Jelena Lazovic VBCF Preclinical Imaging, Vienna BioCenter, Vienna, Austria Search for more papers by this author Judit Berta Judit Berta Department of Tumor Biology, National Koranyi Institute of Pulmonology, Budapest, Hungary Search for more papers by this author Maria Novatchkova Maria Novatchkova Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter, Vienna, Austria Search for more papers by this author Tsung-Pin Pai Tsung-Pin Pai Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter, Vienna, Austria Search for more papers by this author Reiner A Wimmer Reiner A Wimmer Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter, Vienna, Austria Search for more papers by this author Viktória László Viktória László Division of Thoracic Surgery, Department of Surgery, Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria Division of Molecular and Gender Imaging, Department of Biomedical Imaging and Image-guided Therapy, Medical University of Vienna, Vienna, Austria Search for more papers by this author Daniel Schramek Daniel Schramek Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter, Vienna, Austria Department of Molecular Genetics, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, ON, Canada Search for more papers by this author Rezaul Karim Rezaul Karim Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter, Vienna, Austria Search for more papers by this author Luigi Tortola Luigi Tortola Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter, Vienna, Austria Search for more papers by this author Sumit Deswal Sumit Deswal Institute of Molecular Pathology (IMP), Vienna BioCenter, Vienna, Austria Search for more papers by this author Lisa Haas Lisa Haas Institute of Molecular Pathology (IMP), Vienna BioCenter, Vienna, Austria Search for more papers by this author Johannes Zuber Johannes Zuber Institute of Molecular Pathology (IMP), Vienna BioCenter, Vienna, Austria Search for more papers by this author Miklós Szűcs Miklós Szűcs Department of Urology, Semmelweis University, Budapest, Hungary Search for more papers by this author Keiji Kuba Keiji Kuba Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter, Vienna, Austria Department Biochemistry and Metabolic Science, Akita University Graduate School of Medicine, Akita, Japan Search for more papers by this author Balazs Dome Balazs Dome Department of Tumor Biology, National Koranyi Institute of Pulmonology, Budapest, Hungary Division of Thoracic Surgery, Department of Surgery, Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria Department of Thoracic Surgery, National Institute of Oncology-Semmelweis University, Budapest, Hungary Search for more papers by this author Yihai Cao Yihai Cao Department of Microbiology, Tumor and Cell Biology, Biomedicum, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Bernhard J Haubner Bernhard J Haubner Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter, Vienna, Austria Department of Internal Medicine III (Cardiology and Angiology), Medical University of Innsbruck, Innsbruck, Austria Search for more papers by this author Josef M Penninger Corresponding Author Josef M Penninger [email protected] orcid.org/0000-0002-8194-3777 Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter, Vienna, Austria Department of Medical Genetics, Life Science Institute, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Iris Uribesalgo Corresponding Author Iris Uribesalgo [email protected] orcid.org/0000-0001-9492-1000 Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter, Vienna, Austria Search for more papers by this author David Hoffmann David Hoffmann Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter, Vienna, Austria Search for more papers by this author Yin Zhang Yin Zhang Department of Microbiology, Tumor and Cell Biology, Biomedicum, Karolinska Institutet, Stockholm, Sweden Medicine and Pharmacy Research Center, Binzhou Medical University, Yantai, Shandong Province, China Search for more papers by this author Anoop Kavirayani Anoop Kavirayani VBCF Histopathology, Vienna BioCenter, Vienna, Austria Search for more papers by this author Jelena Lazovic Jelena Lazovic VBCF Preclinical Imaging, Vienna BioCenter, Vienna, Austria Search for more papers by this author Judit Berta Judit Berta Department of Tumor Biology, National Koranyi Institute of Pulmonology, Budapest, Hungary Search for more papers by this author Maria Novatchkova Maria Novatchkova Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter, Vienna, Austria Search for more papers by this author Tsung-Pin Pai Tsung-Pin Pai Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter, Vienna, Austria Search for more papers by this author Reiner A Wimmer Reiner A Wimmer Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter, Vienna, Austria Search for more papers by this author Viktória László Viktória László Division of Thoracic Surgery, Department of Surgery, Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria Division of Molecular and Gender Imaging, Department of Biomedical Imaging and Image-guided Therapy, Medical University of Vienna, Vienna, Austria Search for more papers by this author Daniel Schramek Daniel Schramek Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter, Vienna, Austria Department of Molecular Genetics, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, ON, Canada Search for more papers by this author Rezaul Karim Rezaul Karim Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter, Vienna, Austria Search for more papers by this author Luigi Tortola Luigi Tortola Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter, Vienna, Austria Search for more papers by this author Sumit Deswal Sumit Deswal Institute of Molecular Pathology (IMP), Vienna BioCenter, Vienna, Austria Search for more papers by this author Lisa Haas Lisa Haas Institute of Molecular Pathology (IMP), Vienna BioCenter, Vienna, Austria Search for more papers by this author Johannes Zuber Johannes Zuber Institute of Molecular Pathology (IMP), Vienna BioCenter, Vienna, Austria Search for more papers by this author Miklós Szűcs Miklós Szűcs Department of Urology, Semmelweis University, Budapest, Hungary Search for more papers by this author Keiji Kuba Keiji Kuba Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter, Vienna, Austria Department Biochemistry and Metabolic Science, Akita University Graduate School of Medicine, Akita, Japan Search for more papers by this author Balazs Dome Balazs Dome Department of Tumor Biology, National Koranyi Institute of Pulmonology, Budapest, Hungary Division of Thoracic Surgery, Department of Surgery, Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria Department of Thoracic Surgery, National Institute of Oncology-Semmelweis University, Budapest, Hungary Search for more papers by this author Yihai Cao Yihai Cao Department of Microbiology, Tumor and Cell Biology, Biomedicum, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Bernhard J Haubner Bernhard J Haubner Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter, Vienna, Austria Department of Internal Medicine III (Cardiology and Angiology), Medical University of Innsbruck, Innsbruck, Austria Search for more papers by this author Josef M Penninger Corresponding Author Josef M Penninger [email protected] orcid.org/0000-0002-8194-3777 Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter, Vienna, Austria Department of Medical Genetics, Life Science Institute, University of British Columbia, Vancouver, BC, Canada Search for more papers by this author Author Information Iris Uribesalgo *,1,‡, David Hoffmann1,‡, Yin Zhang2,3, Anoop Kavirayani4, Jelena Lazovic5, Judit Berta6, Maria Novatchkova1, Tsung-Pin Pai1, Reiner A Wimmer1, Viktória László7,8, Daniel Schramek1,9, Rezaul Karim1, Luigi Tortola1, Sumit Deswal10, Lisa Haas10, Johannes Zuber10, Miklós Szűcs11, Keiji Kuba1,12, Balazs Dome6,7,13, Yihai Cao2, Bernhard J Haubner1,14 and Josef M Penninger *,1,15 1Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter, Vienna, Austria 2Department of Microbiology, Tumor and Cell Biology, Biomedicum, Karolinska Institutet, Stockholm, Sweden 3Medicine and Pharmacy Research Center, Binzhou Medical University, Yantai, Shandong Province, China 4VBCF Histopathology, Vienna BioCenter, Vienna, Austria 5VBCF Preclinical Imaging, Vienna BioCenter, Vienna, Austria 6Department of Tumor Biology, National Koranyi Institute of Pulmonology, Budapest, Hungary 7Division of Thoracic Surgery, Department of Surgery, Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria 8Division of Molecular and Gender Imaging, Department of Biomedical Imaging and Image-guided Therapy, Medical University of Vienna, Vienna, Austria 9Department of Molecular Genetics, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, ON, Canada 10Institute of Molecular Pathology (IMP), Vienna BioCenter, Vienna, Austria 11Department of Urology, Semmelweis University, Budapest, Hungary 12Department Biochemistry and Metabolic Science, Akita University Graduate School of Medicine, Akita, Japan 13Department of Thoracic Surgery, National Institute of Oncology-Semmelweis University, Budapest, Hungary 14Department of Internal Medicine III (Cardiology and Angiology), Medical University of Innsbruck, Innsbruck, Austria 15Department of Medical Genetics, Life Science Institute, University of British Columbia, Vancouver, BC, Canada ‡These authors contributed equally to this work *Corresponding author. Tel: +43 (1)790 44; E-mail: [email protected] *Corresponding author. Tel: +43 (1)790 44; E-mail: [email protected] EMBO Mol Med (2019)11:e9266https://doi.org/10.15252/emmm.201809266 See also: L Claesson-Welsh (August 2019) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Angiogenesis is a hallmark of cancer, promoting growth and metastasis. Anti-angiogenic treatment has limited efficacy due to therapy-induced blood vessel alterations, often followed by local hypoxia, tumor adaptation, progression, and metastasis. It is therefore paramount to overcome therapy-induced resistance. We show that Apelin inhibition potently remodels the tumor microenvironment, reducing angiogenesis, and effectively blunting tumor growth. Functionally, targeting Apelin improves vessel function and reduces polymorphonuclear myeloid-derived suppressor cell infiltration. Importantly, in mammary and lung cancer, Apelin prevents resistance to anti-angiogenic receptor tyrosine kinase (RTK) inhibitor therapy, reducing growth and angiogenesis in lung and breast cancer models without increased hypoxia in the tumor microenvironment. Apelin blockage also prevents RTK inhibitor-induced metastases, and high Apelin levels correlate with poor prognosis of anti-angiogenic therapy patients. These data identify a druggable anti-angiogenic drug target that reduces tumor blood vessel densities and normalizes the tumor vasculature to decrease metastases. Synopsis Apelin is an angiogenic peptide implicated in embryonic and tumor angiogenesis. This study highlights Apelin targeting as a cancer therapy alone or in combination with current anti-angiogenic therapies to reduce tumour growth and improve vessel structure and functionality, and thus survival. Apelin deficiency reduced tumour growth and vessel number but improved vessel function. Apelin deficiency led to a remodelling of the tumour microenvironment by altering immune cell infiltration. Combining Apelin inhibition with the anti-angiogenic therapy Sunitinib markedly reduced tumour growth and improved survival in breast and lung cancer models. Combinatorial therapy reduced intratumoral vessel numbers compared with single treatments, but simultaneously improved blood vessel pericyte coverage, reduced hypoxia in the tumour microenvironment and prevented Sunitinib-induced metastasis. Introduction Angiogenesis, the sprouting of new blood vessels from the existing vasculature, is a hallmark of cancer that facilitates rapid tumor growth and metastasis (Hanahan & Weinberg, 2011). Activation of an “angiogenic switch” during cancer progression causes aberrant capillary sprouting, tortuous and excessive vessel branching, enlarged vessels, erratic blood flow, micro-hemorrhages, leakiness, and abnormal endothelial cell proliferation (Hanahan & Weinberg, 2011). Inhibition of this angiogenic switch has therefore been proposed as a key cancer treatment strategy. Given the importance of vascular endothelial growth factors (VEGFs) in angiogenesis, much attention has been focused on developing anti-angiogenic receptor tyrosine kinase (RTK) inhibitors targeting the VEGFR signaling pathway to treat a variety of cancers by blocking tumor angiogenesis. Although various clinical trials have demonstrated the efficacy of these therapies, the benefits are usually transitory and, in certain cases, VEGFR pathway inhibitors may even lead to a more aggressive disease (Carmeliet & Jain, 2011a). The large-scale eradication of tumor blood vessels results in necrosis and hypoxia, which can trigger several resistance mechanisms that drive tumor regrowth and malignancy (Bergers & Hanahan, 2008; Carmeliet & Jain, 2011a; Potente et al, 2011). In recent years, the concept of vessel normalization has been proposed to restore vascular abnormalities in tumors, with vessels becoming less permeable and better structured (Jain, 2001). Promoting vessel normalization has been linked to decreased metastasis and an increased efficacy of other therapies such as chemotherapies (Carmeliet & Jain, 2011b; Leite de Oliveira et al, 2012; Maes et al, 2014). However, treatment with VEGF/VEGFR inhibitors alone leads to a transient, usually rather short period of vessel normalization after which hypoxia recurs and acquired resistance emerges (Rivera & Bergers, 2015). In the last years, combination of VEGF and angiopoietin-2 (Ang2) blockage has shown greater effects than single targeting of both molecules in decreasing tumor growth, angiogenesis, vascular abnormality, and metastasis (Brown et al, 2010; Koh et al, 2010; Kienast et al, 2013; Rigamonti et al, 2014; Scholz et al, 2016; Allen et al, 2017; Schmittnaegel et al, 2017), thus increasing the promise of anti-angiogenic VEGF-targeting in combinatory therapies. However, this strategy increased hypoxia (Koh et al, 2010; Rigamonti et al, 2014; Scholz et al, 2016), which can worsen the tumor microenvironment and induce treatment resistance (Jain, 2014). Thus, there is a need to identify safe new agents that can retain the therapeutic advantages of current anti-angiogenic treatments, such as the reduction of angiogenesis and primary tumor growth, and at the same time prevent its resistance-associated features such as hypoxia and therapy-induced metastases. Apelin is an evolutionarily conserved peptide that acts as the endogenous ligand for the G protein-coupled Apelin receptor (Tatemoto et al, 1998). The Apelin/Apelin receptor signaling pathway has been implicated in developmental angiogenesis (Saint-Geniez et al, 2002; Kasai et al, 2004, 2010; Cox et al, 2006; Kälin et al, 2007; Kidoya et al, 2008; del Toro et al, 2010; Kidoya & Takakura, 2012). Although the Apelin/Apelin receptor pathway is downregulated in adulthood, it is frequently reactivated and upregulated in tumors (Sorli et al, 2007; Berta et al, 2010), including in endothelial cells within the tumor microenvironment (Seaman et al, 2007). Further, elevated levels of Apelin are associated with poor clinical outcome in certain human cancers (Berta et al, 2010). Although these observations make the Apelin/Apelin receptor pathway a potentially attractive target for anti-angiogenic cancer therapy, the detailed effects of its targeting for cancer treatment in vivo are poorly understood. In addition, some reports suggest that the Apelin/Apelin receptor pathway is not redundant with VEGFR signaling and that both have independent roles in angiogenesis (Kidoya et al, 2008; del Toro et al, 2010; Heo et al, 2012). Therefore, we wanted to explore whether combinations of Apelin blockade with current anti-angiogenic therapies may be of therapeutic benefit in cancer. Here, we show that genetic and pharmacological inhibition of Apelin is a feasible strategy to reduce tumor blood vessel formation, vessel leakiness, and hypoxia, as well as to reduce suppressive immune cell infiltration, thereby significantly diminishing growth of primary lung and mammary tumors. In 3D vascular sprouts, Apelin is essential for VEGF to trigger blood vessel outgrowth, indicating that Apelin might be a key pathway that interfaces with VEGF signaling. Combining targeting of Apelin with clinically relevant RTK inhibitors like sunitinib, in vivo not only reduced blood vessel density and leakage in tumors, but also decreased hypoxia and metastases induced by sunitinib treatment. Further, elevated Apelin levels in serum samples from renal cell cancer patients treated with sunitinib as a single agent were associated with a worse prognosis. Our findings unveil a new strategy that combines clinically relevant anti-angiogenic treatments with Apelin inhibition to diminish tumor growth, blood vessel density, and vessel abnormality within the tumor environment, and thus hypoxia, tumor resistance, and anti-angiogenic therapy-induced metastasis. Results Apelin blockage improves survival in mammary and lung cancer models To corroborate that Apelin expression is associated with outcome in human breast cancer, we performed an unbiased meta-analysis of multiple datasets using the Kmplot (Györffy et al, 2010) and PrognoScan (Mizuno et al, 2009) databases. We confirmed that high levels of Apelin expression in tumors are significantly associated with poor prognosis in breast cancer patients (Fig EV1A). Next, we determined whether Apelin blockage is a suitable strategy to ameliorate cancer progression by ablating its expression in mammary cancer. Apelin-deficient (Apln−/−) mice (Kuba et al, 2007) were crossed with MMTV-NeuT transgenic mice (Lucchini et al, 1992) to generate MMTV-NeuT; Apln−/− and MMTV-NeuT; Apln+/+ control littermates (termed NeuT;Apln−/− and NeuT;Apln+/+ hereafter). Apelin has been previously shown to be upregulated in tumor cells (Seaman et al, 2007; Wang et al, 2007; Liu et al, 2015). We confirmed that Apelin expression is enhanced in tumors of MMTV-NeuT mice compared to epithelial cells isolated from the mammary gland of healthy mice (Fig EV1B), recapitulating human breast cancer (Sorli et al, 2007) and validating our model. Importantly, NeuT;Apln−/− tumor-bearing mice displayed a delay in the onset of NeuT-driven mammary tumors and a significantly prolonged survival compared with NeuT;Apln+/+ littermates (Figs EV1C and 1A). In line with enhanced survival, Apelin-null mice displayed a decreased tumor burden in the mammary glands compared to age-matched controls (Fig EV1D). Click here to expand this figure. Figure EV1. Apelin inactivation improves survival and reduces tumor burden in mammary and lung cancer Kaplan–Meier survival plot from the KM-plotter database (Győrffy et al, 2013) for high and low Apelin (APLN)-expressing groups in human breast cancer. Relapse-free survival. Patients were split by the median. Affymetrix Apelin ID 222856_at. RT-qPCR of Apelin (Apln) expression in NeuT;Apln+/+ tumors (orange bar, n = 7) and purified normal (Apln+/+) mammary gland epithelial cells (gray bar, n = 3). Data points from individual tumors or mammary glands and means (black bars) are shown. Kaplan–Meier plot for mammary tumor onset in NeuT;Apln+/+ (n = 20) and NeuT;Apln−/− (n = 24) mice. P = 0.0019; log-rank test. Mean percentages ± SEM of tumor burden in NeuT;Apln+/+ (n = 6) and NeuT;Apln−/− (n = 9) mammary glands assessed 4 weeks after tumor onset. *P < 0.05; t-test. Kaplan–Meier plot from the KM-plotter database (Győrffy et al, 2013) for high and low APELIN-expressing groups in lung cancer patients. Overall survival. Patients were split by the median. Affymetrix Apelin ID 222856_at. Kaplan–Meier plot for survival in KRas;Apln+/y (n = 6) and KRas;Apln−/y (n = 4) mice with non-small cell lung cancer (NSCLC) after oncogenic KRas induction by AdenoCre inhalation. **P < 0.01; log-rank test. Kaplan–Meier plot for survival in p53f/f;KRas;Apln+/y (n = 7) and p53f/f;KRas;Apln−/y (n = 9) mice with NSCLC after AdenoCre inhalation. **P < 0.01; log-rank test. Percentages and representative histology of lung adenoma/adenocarcinoma and hyperplasia (±SEM) in age-matched KRas;Apln+/y (n = 4) and KRas;Apln−/y (n = 4) mice analyzed 18 weeks after AdenoCre inhalation. *P < 0.05, ns = not significant; t-test; three sections per lung were analyzed. Source data are available online for this figure. Download figure Download PowerPoint Figure 1. Genetic and pharmacological inhibition of Apelin impairs mammary tumor growth and tumor angiogenesis in a paracrine manner Kaplan–Meier plot for survival in NeuT;Apln+/+ (n = 11) and NeuT;Apln−/− (n = 10) mice with mammary cancer after tumor onset. *P = 0.0185; log-rank test. Mice were sacrificed when the tumor size reached 1 cm3, following ethical guidelines. Tumor volumes, followed over time, of control mammary tumor E0771 cells (shRenilla) and Apelin-depleted (shApln) E0771 cells orthotopically injected into both syngeneic C57BL/6J Apln+/+ and Apln−/− mice (5 × 105 cells/mouse), respectively. Tumor volumes were determined using calipers and are shown as mean tumor volumes ± SEM. Data shown are pooled from two independent experiments. Apln+/+;shRenilla (n = 18), Apln+/+;shApln (n = 17), Apln−/−;shRenilla (n = 14), Apln−/−;shApln (n = 15); **P < 0.01, ***P < 0.001; two-way ANOVA. Mean percentages (±SEM) of CD31+ area in E0771 shRenilla (n = 3) or shApln (n = 3) mammary tumors, assessed on day 23 post-orthotopic injection into C57BL/6J Apln+/+ or Apln−/− mice, respectively. **P < 0.01; t-test. Mean percentages (±SEM) of extravasated Dextran in E0771 shRenilla (n = 9) or shApln (n = 12) mammary tumors, assessed on day 19 post-orthotopic injection into C57BL/6J Apln+/+ or Apln−/− mice, respectively. **P < 0.01; t-test. Right panel shows representative immunofluorescence of Dextran (red), CD31+ vessels (green), and DAPI (blue). The white arrows indicate regions of Dextran extravasation. Scale bars = 100 μm. Mean counts (±SEM) of pimonidazole positive foci, assessed on day 26 post-orthotopic injection of E0771 shRenilla (n = 6) or shApln (n = 4) into C57BL/6J wild-type mice (5 × 105 cells/mouse). *P < 0.05; t-test. Right panels show representative immunohistochemical pimonidazole staining at two different magnifications; scale bars = 200 μm (upper panels) and 50 μm (lower panels). Mean percentage (±SEM) of tumor-infiltrating immune cells normalized to CD45+. E0771 shRenilla (n = 8) or shApln (n = 6) were orthotopically injected into C57BL/6J Apln+/+ or Apln−/− mice (5 × 105 cells/mouse), respectively, and tumors were harvested day 25 post-injection. *P < 0.05, **P < 0.01; t-test. All immune cell populations were gated for viable CD45+ cells and then further defined as: CD8 T cells (Thy1.2+, CD8+), CD4 T cells (Thy1.2+, CD4+), inflammatory monocytes (Ly6C+, Cd11b+, Ly6G−), PMN-MDSCs (Ly6G+, Cd11b+), natural killer cells (Thy1.2−, Ly6G−, NK1.1+), natural killer T cells (Thy1.2+, CD4−, CD8−, NK1.1+), and peripheral dendritic cells (Ly6G−,Ly6C+, Cd11b−, PDAC1+, B220+). Source data are available online for this figure. Source Data for Figure 1 [emmm201809266-sup-0004-SDataFig1.pdf] Download figure Download PowerPoint We further extended our studies to lung cancer as a second solid tumor model of epithelial origin. Similar to breast cancer, we confirmed that high levels of Apelin expression are significantly associated with poor prognosis in lung cancer patients (Fig EV1E) (Györffy et al, 2013). To be able to experimentally dissect the role of Apelin in lung cancer, Apln−/− mice were crossed to the Lox-Stop-Lox-KRasG12D lung cancer model (KRas;Apln+/y and KRas;Apln−/y hereafter; the Apelin gene is located on the X chromosome; Kuba et al, 2007) (Jackson, 2001). We also extended the investigation to a more aggressive form of non-small cell lung cancer (NSCLC) driven by the KRasG12D oncogene combined with loss of the tumor suppressor p53 (p53f/f;KRas;Apln+/y and p53f/f;KRas;Apln−/y; DuPage et al, 2009). Knockout of Apelin resulted in enhanced survival and reduced tumor burden in these lung cancer models (Fig EV1F–H). We also explored whether Apela, the recently described second ligand for Apelin receptor (Pauli et al, 2014), might also be overexpressed in NeuT-driven mammary tumors or KRas-driven lung tumors, but we failed to detect its expression, even using sensitive and multi-cycle qPCR analysis. Thus, we conclude that Apelin is the primary Apelin receptor ligand upregulated in our models of mammary and lung cancer. Of note, we did not detect abnormalities in mammary glands or lungs from non-tumor-bearing adult and background-matched Apln+/+ and Apln−/− mice without oncogenic drivers. These results not only extend the findings of previous overexpression studies (Sorli et al, 2007; Berta et al, 2010), but validate Apelin as a target in tumor models of epithelial origin. Thus, high Apelin levels correlate with a worse prognosis in breast and lung cancer patients, and Apelin inactivation increases the survival of mice with breast and lung cancer. Apelin modulates the tumor microenvironment through paracrine stimulation of tumor angiogenesis Apelin has previously been shown to stimulate tumor angiogenesis and is upregulated in tumor-associated endothelial cells (Seaman et al, 2007; Wang et al, 2007; Liu et al, 2015), which we could confirm in endothelial cells isolated from MMTV-NeuT tumors compared to normal mammary gland (Fig EV2A). Despite the known role of Apelin in tumor angiogenesis, the detailed effects of Apelin within the tumor cells and its microenvironment in vivo remain poorly understood. Click here to expand this figure. Figure EV2. Tumor cell-derived Apelin induces angiogenesis in a paracrine manner RT-qPCR of Apelin (Apln) expression in endothelial cells (ECs) isolated from NeuT;Apln+/+ tumors, normal Apln+/+ mammary glands. Data are shown as relative mRNA levels normalized to the normal mammary gland endothelium (set to 1) ± SEM. n = 2 per cohort; **P < 0.01, t-test. RT-qPCR of Apln and Aplnr in control E0771 mammary cancer cells (shRenilla) and Apelin or Apelin receptor-depleted E0771 cells (shApln and shAplnr, respectively). Data are shown as relative mRNA levels normalized to control E0771 cells (set to 1) ± SEM. n = 2 per cohort; **P < 0.01; t-test. Relative levels of AplnR mRNA (mean ± SEM) in isolated tumor endothelial cells compared to tumor epithelial cells from NeuT-driven Apln+/+ mouse mammary tumors; n = 4 per cohort. Data are shown as relative mRNA levels normalized to epithelial cells (set to 1) ± SEM. In vitro growth curves of shRenilla, shApln or shAplnr E0771 cells in the absence or presence of an active Apelin peptide (AplnPyr13, 1000 nM). No difference in growth was observed. A representative experiment is shown. Tumor volume, followed over time, of orthotopically injected shRenilla, shApln, and shAplnr E0771 cells. Data were determined using calipers and are shown as mean tumor volumes ± SEM. n = 4 syngeneic C57BL/6J mice per cohort; *P < 0.05, ***P < 0.001,

DExD/H box RNA helicases, such as the RIG-I-like receptors (RLR), are important components of the innate immune system. Here we demonstrate a pivotal and sex-specific role for the heterosomal isoforms of the DEAD box RNA helicase DDX3 in the immune system. Mice lacking DDX3X during hematopoiesis showed an altered leukocyte composition in bone marrow and spleen and a striking inability to combat infection with Listeria monocytogenes. Alterations in innate immune responses resulted from decreased effector cell availability and function as well as a sex-dependent impairment of cytokine synthesis. Thus, our data provide further in vivo evidence for an essential contribution of a non-RLR DExD/H RNA helicase to innate immunity and suggest it may contribute to sex-related differences in resistance to microbes and resilience to inflammatory disease.

A diverse array of vertebrate species employs the Earth’s magnetic field to assist navigation. Despite compelling behavioral evidence that a magnetic sense exists, the location of the primary sensory cells and the underlying molecular mechanisms remain unknown [1Johnsen S. Lohmann K.J. The physics and neurobiology of magnetoreception.Nat. Rev. Neurosci. 2005; 6: 703-712Crossref PubMed Scopus (309) Google Scholar]. To date, most research has focused on a light-dependent radical-pair-based concept and a system that is proposed to rely on biogenic magnetite (Fe3O4) [2Ritz T. Adem S. Schulten K. A model for photoreceptor-based magnetoreception in birds.Biophys. J. 2000; 78: 707-718Abstract Full Text Full Text PDF PubMed Scopus (827) Google Scholar, 3Kirschvink J.L. Walker M.M. Diebel C.E. Magnetite-based magnetoreception.Curr. Opin. Neurobiol. 2001; 11: 462-467Crossref PubMed Scopus (314) Google Scholar]. Here, we explore an overlooked hypothesis that predicts that animals detect magnetic fields by electromagnetic induction within the semicircular canals of the inner ear [4Viguier C. Le sens de l’orientation et ses organes chez les animaux et chez l’homme.Rev. Philos. France Let. 1882; : 1-36Google Scholar]. Employing an assay that relies on the neuronal activity marker C-FOS, we confirm that magnetic exposure results in activation of the caudal vestibular nuclei in pigeons that is independent of light [5Wu L.Q. Dickman J.D. Magnetoreception in an avian brain in part mediated by inner ear lagena.Curr. Biol. 2011; 21: 418-423Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar]. We show experimentally and by physical calculations that magnetic stimulation can induce electric fields in the pigeon semicircular canals that are within the physiological range of known electroreceptive systems. Drawing on this finding, we report the presence of a splice isoform of a voltage-gated calcium channel (CaV1.3) in the pigeon inner ear that has been shown to mediate electroreception in skates and sharks [6Bellono N.W. Leitch D.B. Julius D. Molecular tuning of electroreception in sharks and skates.Nature. 2018; 558: 122-126Crossref PubMed Scopus (18) Google Scholar]. We propose that pigeons detect magnetic fields by electromagnetic induction within the semicircular canals that is dependent on the presence of apically located voltage-gated cation channels in a population of electrosensory hair cells.

Successful pregnancies rely on adaptations within the mother1, including marked changes within the immune system2. It has long been known that the thymus, the central lymphoid organ, changes markedly during pregnancy3. However, the molecular basis and importance of this process remain largely obscure. Here we show that the osteoclast differentiation receptor RANK4,5 couples female sex hormones to the rewiring of the thymus during pregnancy. Genetic deletion of Rank (also known as Tnfrsf11a) in thymic epithelial cells results in impaired thymic involution and blunted expansion of natural regulatory T (Treg) cells in pregnant female mice. Sex hormones, in particular progesterone, drive the development of thymic Treg cells through RANK in a manner that depends on AIRE+ medullary thymic epithelial cells. The depletion of Rank in the mouse thymic epithelium results in reduced accumulation of natural Treg cells in the placenta, and an increase in the number of miscarriages. Thymic deletion of Rank also results in impaired accumulation of Treg cells in visceral adipose tissue, and is associated with enlarged adipocyte size, tissue inflammation, enhanced maternal glucose intolerance, fetal macrosomia, and a long-lasting transgenerational alteration in glucose homeostasis, which are all key hallmarks of gestational diabetes. Transplantation of Treg cells rescued fetal loss, maternal glucose intolerance and fetal macrosomia. In human pregnancies, we found that gestational diabetes also correlates with a reduced number of Treg cells in the placenta. Our findings show that RANK promotes the hormone-mediated development of thymic Treg cells during pregnancy, and expand the functional role of maternal Treg cells to the development of gestational diabetes and the transgenerational metabolic rewiring of glucose homeostasis. RANK promotes the hormone-mediated development of thymic regulatory T cells during pregnancy; loss of RANK is associated with impaired maturation of maternal regulatory T cells, leading to fetal loss and the development of gestational diabetes.

New SARS-CoV-2 variants are continuously emerging with critical implications for therapies or vaccinations. The 22 N-glycan sites of Spike remain highly conserved among SARS-CoV-2 variants, opening an avenue for robust therapeutic intervention. Here we used a comprehensive library of mammalian carbohydrate-binding proteins (lectins) to probe critical sugar residues on the full-length trimeric Spike and the receptor binding domain (RBD) of SARS-CoV-2. Two lectins, Clec4g and CD209c, were identified to strongly bind to Spike. Clec4g and CD209c binding to Spike was dissected and visualized in real time and at single-molecule resolution using atomic force microscopy. 3D modelling showed that both lectins can bind to a glycan within the RBD-ACE2 interface and thus interferes with Spike binding to cell surfaces. Importantly, Clec4g and CD209c significantly reduced SARS-CoV-2 infections. These data report the first extensive map and 3D structural modelling of lectin-Spike interactions and uncovers candidate receptors involved in Spike binding and SARS-CoV-2 infections. The capacity of CLEC4G and mCD209c lectins to block SARS-CoV-2 viral entry holds promise for pan-variant therapeutic interventions.

Organoids enable in vitro modeling of complex developmental processes and disease pathologies. Like most 3D cultures, organoids lack sufficient oxygen supply and therefore experience cellular stress. These negative effects are particularly prominent in complex models, such as brain organoids, and can affect lineage commitment. Here, we analyze brain organoid and fetal single-cell RNA sequencing (scRNAseq) data from published and new datasets, totaling about 190,000 cells. We identify a unique stress signature in the data from all organoid samples, but not in fetal samples. We demonstrate that cell stress is limited to a defined subpopulation of cells that is unique to organoids and does not affect neuronal specification or maturation. We have developed a computational algorithm, Gruffi, which uses granular functional filtering to identify and remove stressed cells from any organoid scRNAseq dataset in an unbiased manner. We validated our method using six additional datasets from different organoid protocols and early brains, and show its usefulness to other organoid systems including retinal organoids. Our data show that the adverse effects of cell stress can be corrected by bioinformatic analysis for improved delineation of developmental trajectories and resemblance to in vivo data.

The number one cause of human fetal death are defects in heart development. Because the human embryonic heart is inaccessible and the impacts of mutations, drugs, and environmental factors on the specialized functions of different heart compartments are not captured by in vitro models, determining the underlying causes is difficult. Here, we established a human cardioid platform that recapitulates the development of all major embryonic heart compartments, including right and left ventricles, atria, outflow tract, and atrioventricular canal. By leveraging 2D and 3D differentiation, we efficiently generated progenitor subsets with distinct first, anterior, and posterior second heart field identities. This advance enabled the reproducible generation of cardioids with compartment-specific in vivo-like gene expression profiles, morphologies, and functions. We used this platform to unravel the ontogeny of signal and contraction propagation between interacting heart chambers and dissect how mutations, teratogens, and drugs cause compartment-specific defects in the developing human heart.

Skeletal muscle plays a central role in the regulation of systemic metabolism during lifespan. With aging, this function is perturbed, initiating multiple chronic diseases. Our knowledge of mechanisms responsible for this decline is limited. Glycerophosphocholine phosphodiesterase 1 (Gpcpd1) is a highly abundant muscle enzyme that hydrolyzes glycerophosphocholine (GPC). The physiological functions of Gpcpd1 remain largely unknown. Here we show, in mice, that the Gpcpd1–GPC metabolic pathway is perturbed in aged muscles. Further, muscle-specific, but not liver- or fat-specific, inactivation of Gpcpd1 resulted in severely impaired glucose metabolism. Western-type diets markedly worsened this condition. Mechanistically, Gpcpd1 muscle deficiency resulted in accumulation of GPC, causing an 'aged-like' transcriptomic signature and impaired insulin signaling in young Gpcpd1-deficient muscles. Finally, we report that the muscle GPC levels are markedly altered in both aged humans and patients with type 2 diabetes, displaying a high positive correlation between GPC levels and chronological age. Our findings reveal that the muscle GPCPD1–GPC metabolic pathway has an important role in the regulation of glucose homeostasis and that it is impaired during aging, which may contribute to glucose intolerance in aging. Cikes et al. report dysregulation of glycerophosphocholine (GPC) metabolism in aged mouse muscle, which they functionally link to severe glucose intolerance. Correspondingly, muscle GPC levels are altered in both older adults and patients with type 2 diabetes.

E-cadherin-mediated cell-cell adhesion is frequently lost during the development of malignant epithelial cancers. Employing a transgenic mouse model of beta-cell carcinogenesis (Rip1Tag2) we have previously shown that the loss of E-cadherin is a rate-limiting step in the progression from adenoma to carcinoma. However, the mere loss of cell adhesion may not be sufficient and additional signals are required to cause tumor cells to permeate the basal membrane and to invade surrounding tissue. Besides being an important component of the E-cadherin cell-adhesion complex, beta-catenin plays a critical role in canonical Wnt signaling. We report here that beta-catenin-mediated Wnt signaling does not contribute to tumor progression in Rip1Tag2 mice. E-cadherin downregulates beta-catenin/Tcf-mediated transcriptional activity by sequestrating beta-catenin into E-cadherin cell-adhesion complexes even in the presence of activated Wnt signaling. Upon loss of E-cadherin expression, beta-catenin is degraded and Tcf/beta-catenin-mediated transcriptional activity is not induced. Moreover, forced expression of constitutive-active beta-catenin or genetic ablation of Tcf/beta-catenin transcriptional activity in tumor cells of Rip1Tag2 transgenic mice does not affect tumor progression. Together, the data indicate that signals other than beta-catenin/Tcf-mediated Wnt signaling are induced by the loss of E-cadherin during tumor progression in Rip1Tag2 transgenic mice.

The fission yeast Schizosaccharomyces pombe does not form synaptonemal complexes (SCs) in meiotic prophase nuclei. Instead, thin threads, the so-called linear elements (LEs), are observed at the corresponding stages by electron microscopy. Here, we demonstrate that S. pombe Rec10 is a protein related to the Saccharomyces cerevisiae SC protein Red1 and that it localizes to LEs. Moreover, a homologue to S. cerevisiae Hop1 does exist in S. pombe and we show by in situ immunostaining that it, and the kinase Mek1 (a homologue of which is also known to be associated with SCs), localizes to LEs. These observations indicate the evolutionary relationship of LEs with the lateral elements of SCs and suggest that these structures might exert similar functions in S. cerevisiae and S. pombe.

Obligatory homologous recombination (HR) is required for chiasma formation and chromosome segregation in meiosis I. Meiotic HR is initiated by DNA double-strand breaks (DSBs), generated by Spo11, a homologue of the archaebacterial topoisomerase subunit Top6A. In Saccharomyces cerevisiae, Rad50, Mre11 and Com1/Sae2 are essential to process an intermediate of the cleavage reaction consisting of Spo11 covalently linked to the 5' termini of DNA. While Rad50 and Mre11 also confer genome stability to vegetative cells and are well conserved in evolution, Com1/Sae2 was believed to be fungal-specific. Here, we identify COM1/SAE2 homologues in all eukaryotic kingdoms. Arabidopsis thaliana Com1/Sae2 mutants are sterile, accumulate AtSPO11-1 during meiotic prophase and fail to form AtRAd51 foci despite the presence of unrepaired DSBs. Furthermore, DNA fragmentation in AtCom1 is suppressed by eliminating AtSPO11-1. In addition, AtCOM1 is specifically required for mitomycin C resistance. Interestingly, we identified CtIP, an essential protein interacting with the DNA repair machinery, as the mammalian homologue of Com1/Sae2, with important implications for the molecular role of CtIP.

Analysis of the phosphorylation of mitotic protein complexes suggests that specific members of the complexes relay regulatory signals to these molecular machines.

The conjugation of the small ubiquitin-related modifier, SUMO, to substrate proteins is a reversible and dynamic process, and an important response of plants to environmental challenges. Nevertheless, reliable data have so far been restricted largely to the model plant Arabidopsis thaliana. The increasing availability of genome information for other plant species offers the possibility to identify a core set of indispensable components, and to discover species-specific features of the sumoylation pathway. We analyzed the enzymes responsible for the conjugation of SUMO to substrates for their conservation between dicots and monocots. We thus assembled gene sets that relate the Arabidopsis SUMO conjugation system to that of the dicot species tomato, grapevine and poplar, and to four plant species from the monocot class: rice, Brachypodium distachyon, Sorghum bicolor and maize. We found that a core set of genes with clear assignment in Arabidopsis had highly conserved homologs in all tested plants. However, we also observed a variation in the copy number of homologous genes, and sequence variations that suggested monocot-specific variants. Generally, SUMO ligases and proteases showed the most pronounced differences. Finally, we identified potential SUMO chain-binding ubiquitin ligases, pointing to an in vivo function of SUMO chains as degradation signals in plants.

Abstract The Arabidopsis thaliana genes PROTEIN INHIBITOR OF ACTIVATED STAT LIKE1 (PIAL1) and PIAL2 encode proteins with SP-RING domains, which occur in many ligases of the small ubiquitin-related modifier (SUMO) conjugation pathway. We show that PIAL1 and PIAL2 function as SUMO ligases capable of SUMO chain formation and require the SUMO-modified SUMO-conjugating enzyme SCE1 for optimal activity. Mutant analysis indicates a role for PIAL1 and 2 in salt stress and osmotic stress responses, whereas under standard conditions, the mutants show close to normal growth. Mutations in PIAL1 and 2 also lead to altered sulfur metabolism. We propose that, together with SUMO chain binding ubiquitin ligases, these enzymes establish a pathway for proteolytic removal of sumoylation substrates.

Chromosome segregation depends on sister chromatid cohesion which is established by cohesin during DNA replication. Cohesive cohesin complexes become acetylated to prevent their precocious release by WAPL before cells have reached mitosis. To obtain insight into how DNA replication, cohesion establishment and cohesin acetylation are coordinated, we analysed the interaction partners of 55 human proteins implicated in these processes by mass spectrometry. This proteomic screen revealed that on chromatin the cohesin acetyltransferase ESCO2 associates with the MCM2-7 subcomplex of the replicative Cdc45-MCM-GINS helicase. The analysis of ESCO2 mutants defective in MCM binding indicates that these interactions are required for proper recruitment of ESCO2 to chromatin, cohesin acetylation during DNA replication, and centromeric cohesion. We propose that MCM binding enables ESCO2 to travel with replisomes to acetylate cohesive cohesin complexes in the vicinity of replication forks so that these complexes can be protected from precocious release by WAPL Our results also indicate that ESCO1 and ESCO2 have distinct functions in maintaining cohesion between chromosome arms and centromeres, respectively.

Engagement of macrophages in innate immune responses is directed by type I and type II interferons (IFN-I and IFN-γ, respectively). IFN triggers drastic changes in cellular transcriptomes, executed by JAK-STAT signal transduction and the transcriptional control of interferon-stimulated genes (ISG) by STAT transcription factors. Here, we study the immediate-early nuclear response to IFN-I and IFN-γ in murine macrophages. We show that the mechanism of gene control by both cytokines includes a rapid increase of DNA accessibility and rearrangement of the 3D chromatin contacts particularly between open chromatin of ISG loci. IFN-stimulated gene factor 3 (ISGF3), the major transcriptional regulator of ISG, controlled homeostatic and, most notably, induced-state DNA accessibility at a subset of ISG. Increases in DNA accessibility correlated with the appearance of activating histone marks at surrounding nucleosomes. Collectively our data emphasize changes in the three-dimensional nuclear space and epigenome as an important facet of transcriptional control by the IFN-induced JAK-STAT pathway.

During development of the human cerebral cortex, multipotent neural progenitors generate excitatory neurons and glial cells. Investigations of the transcriptome and epigenome have revealed important gene regulatory networks underlying this crucial developmental event. However, the posttranscriptional control of gene expression and protein abundance during human corticogenesis remains poorly understood. We addressed this issue by using human telencephalic brain organoids grown using a dual reporter cell line to isolate neural progenitors and neurons and performed cell class and developmental stage-specific transcriptome and proteome analysis. Integrating the two datasets revealed modules of gene expression during human corticogenesis. Investigation of one such module uncovered mTOR-mediated regulation of translation of the 5'TOP element-enriched translation machinery in early progenitor cells. We show that in early progenitors partial inhibition of the translation of ribosomal genes prevents precocious translation of differentiation markers. Overall, our multiomics approach proposes novel posttranscriptional regulatory mechanisms crucial for the fidelity of cortical development.

The establishment and maintenance of apical-basal polarity is a fundamental step in brain development, instructing the organization of neural progenitor cells (NPCs) and the developing cerebral cortex. Particularly, basally located extracellular matrix (ECM) is crucial for this process. In vitro, epithelial polarization can be achieved via endogenous ECM production, or exogenous ECM supplementation. While neuroepithelial development is recapitulated in neural organoids, the effects of different ECM sources in tissue morphogenesis remain underexplored. Here, we show that exposure to a solubilized basement membrane matrix substrate, Matrigel, at early neuroepithelial stages causes rapid tissue polarization and rearrangement of neuroepithelial architecture. In cultures exposed to pure ECM components or unexposed to any exogenous ECM, polarity acquisition is slower and driven by endogenous ECM production. After the onset of neurogenesis, tissue architecture and neuronal differentiation are largely independent of the initial ECM source, but Matrigel exposure has long-lasting effects on tissue patterning. These results advance the knowledge on mechanisms of exogenously and endogenously guided morphogenesis, demonstrating the self-sustainability of neuroepithelial cultures by endogenous processes.

We evaluated the evolutionary conservation of glycine myristoylation within eukaryotic sequences. Our large-scale cross-genome analyses, available as MYRbase, show that the functional spectrum of myristoylated proteins is currently largely underestimated. We give experimental evidence for in vitro myristoylation of selected predictions. Furthermore, we classify five membrane-attachment factors that occur most frequently in combination with, or even replacing, myristoyl anchors, as some protein family examples show.

Article15 November 2007free access A conserved function for a Caenorhabditis elegans Com1/Sae2/CtIP protein homolog in meiotic recombination Alexandra Penkner Alexandra Penkner Department of Chromosome Biology and Max F. Perutz Laboratories, Center for Molecular Biology, University of Vienna, Vienna, Austria Search for more papers by this author Zsuzsanna Portik-Dobos Zsuzsanna Portik-Dobos Department of Chromosome Biology and Max F. Perutz Laboratories, Center for Molecular Biology, University of Vienna, Vienna, Austria Search for more papers by this author Lois Tang Lois Tang Department of Chromosome Biology and Max F. Perutz Laboratories, Center for Molecular Biology, University of Vienna, Vienna, Austria Search for more papers by this author Ralf Schnabel Ralf Schnabel Developmental Genetics, TU Braunschweig, Germany Search for more papers by this author Maria Novatchkova Maria Novatchkova Bioinformatics Group, Research Institute of Molecular Pathology, Vienna, Austria Search for more papers by this author Verena Jantsch Corresponding Author Verena Jantsch Department of Chromosome Biology and Max F. Perutz Laboratories, Center for Molecular Biology, University of Vienna, Vienna, Austria Search for more papers by this author Josef Loidl Corresponding Author Josef Loidl Department of Chromosome Biology and Max F. Perutz Laboratories, Center for Molecular Biology, University of Vienna, Vienna, Austria Search for more papers by this author Alexandra Penkner Alexandra Penkner Department of Chromosome Biology and Max F. Perutz Laboratories, Center for Molecular Biology, University of Vienna, Vienna, Austria Search for more papers by this author Zsuzsanna Portik-Dobos Zsuzsanna Portik-Dobos Department of Chromosome Biology and Max F. Perutz Laboratories, Center for Molecular Biology, University of Vienna, Vienna, Austria Search for more papers by this author Lois Tang Lois Tang Department of Chromosome Biology and Max F. Perutz Laboratories, Center for Molecular Biology, University of Vienna, Vienna, Austria Search for more papers by this author Ralf Schnabel Ralf Schnabel Developmental Genetics, TU Braunschweig, Germany Search for more papers by this author Maria Novatchkova Maria Novatchkova Bioinformatics Group, Research Institute of Molecular Pathology, Vienna, Austria Search for more papers by this author Verena Jantsch Corresponding Author Verena Jantsch Department of Chromosome Biology and Max F. Perutz Laboratories, Center for Molecular Biology, University of Vienna, Vienna, Austria Search for more papers by this author Josef Loidl Corresponding Author Josef Loidl Department of Chromosome Biology and Max F. Perutz Laboratories, Center for Molecular Biology, University of Vienna, Vienna, Austria Search for more papers by this author Author Information Alexandra Penkner1, Zsuzsanna Portik-Dobos1, Lois Tang1, Ralf Schnabel2, Maria Novatchkova3, Verena Jantsch 1 and Josef Loidl 1 1Department of Chromosome Biology and Max F. Perutz Laboratories, Center for Molecular Biology, University of Vienna, Vienna, Austria 2Developmental Genetics, TU Braunschweig, Germany 3Bioinformatics Group, Research Institute of Molecular Pathology, Vienna, Austria *Corresponding authors: Department of Chromosome Biology, Center for Molecular Biology, University of Vienna, Dr Bohr Gasse 1, A-1030, Vienna, Austria. Tel.: +43 1 4277 56200; Fax: +43 1 4277 9562; E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2007)26:5071-5082https://doi.org/10.1038/sj.emboj.7601916 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Genome stability relies on faithful DNA repair both in mitosis and in meiosis. Here, we report on a Caenorhabditis elegans protein that we found to be homologous to the mammalian repair-related protein CtIP and to the budding yeast Com1/Sae2 recombination protein. A com-1 mutant displays normal meiotic chromosome pairing but forms irregular chromatin aggregates instead of diakinesis bivalents. While meiotic DNA double-strand breaks (DSBs) are formed, they appear to persist or undergo improper repair. Despite the presence of DSBs, the recombination protein RAD-51, which is known to associate with single-stranded DNA (ssDNA) flanking DSBs, does not localize to meiotic chromosomes in the com-1 mutant. Exposure of the mutant to γ-radiation, however, induces RAD-51 foci, which suggests that the failure of RAD-51 to load is specific to meiotic (SPO-11-generated) DSBs. These results suggest that C. elegans COM-1 plays a role in the generation of ssDNA tails that can load RAD-51, invade homologous DNA tracts and thereby initiate recombination. Extrapolating from the worm homolog, we expect similar phenotypes for mutations in the mammalian tumor suppressor CtIP. Introduction To compensate for genome duplication upon fertilization, sexually reproducing organisms rely on the haploidization of their chromosome complement during meiosis. This reduction is achieved by a sequence of two divisions without intervening DNA replication. During the first of these divisions (meiosis I), homologous (corresponding paternal and maternal) chromosomes separate, leaving nuclei with a single copy of each chromosome. To ensure the orderly segregation of homologous chromosomes in meiosis I, they form pairs (bivalents), first by being connected by the synaptonemal complex (SC) and after its disassembly, by chiasmata (for a review, see Zickler and Kleckner, 1999). Chiasmata are the consequence of a physical exchange (recombination) between DNA molecules of the homologous chromosomes (crossing over). Crossing over and other forms of meiotic recombination are initiated by double-strand breaks (DSBs) in DNA, which are generated by the meiosis-specific protein SPO-11. SPO-11 cuts DNA by a topoisomerase II-like transesterase reaction by which SPO-11 is covalently bound to the DNA ends at DSBs (Bergerat et al, 1997; Keeney et al, 1997). In the next step, the 5′–3′ half strands of the flanking DNA are resected, creating 3′ single-stranded overhangs on both sides. The loading of the single strands with RAD-51 (and in most organisms also Dmc1) protomers (for a review, see Shinohara and Shinohara, 2004) helps these strands to invade double-stranded homologous DNA regions (with a preference for DNA on the homologous chromosome) and to initiate strand exchange. At this point, a decision is made as to whether the strand exchange will result in a crossover or a non-reciprocal exchange, a conversion (for recent reviews, see Bishop and Zickler, 2004; Hollingsworth and Brill, 2004; Whitby, 2005). In either case, the DSB will be repaired by using the homologous sequence as a template for the synthesis of DNA spanning the DSB. The removal of SPO-11 and single-stranded DNA (ssDNA) resection is essential for the recombinational repair of DSBs. There are several mutations known in budding yeast in which these steps are compromised. Mre11 and Rad50, both of which also have mitotic functions, are required for the formation of DSBs, but certain rad50 and mre11 separation-of-function mutants exist, which cannot remove Spo11 from DSBs (Alani et al, 1990; Baudat and Nicolas, 1997; Nairz and Klein, 1997; Keeney, 2001). In addition, in com1Δ/sae2Δ mutants, Spo11 remains covalently attached to DSB ends, which are left unresected (McKee and Kleckner, 1997; Prinz et al, 1997; Neale et al, 2005; Prieler et al, 2005). COM1/SAE2 was originally discovered independently by two screens in budding yeast for mutants that can only sporulate if meiotic DSBs are not induced due to the absence of Spo11p. Null mutants featured unresected DSBs, absence of recombination, reduced homologous synapsis and a very weak sensitivity to methyl methanesulfonate (MMS) (McKee and Kleckner, 1997; Prinz et al, 1997). Here, we report the discovery of a putative Caenorhabditis elegans homolog of Com1p/Sae2p and the study of its meiotic function by mutant analysis. As recombination is not required for SC formation in C. elegans, pairing functions are not compromised by defects in the recombination pathway in this organism, which therefore facilitates the study of recombination-specific phenotypes elicited by the com-1 mutation. In addition to the homology among the Com1/Sae2 proteins with meiotic roles in C. elegans, Saccharomyces cerevisiae and Arabidopsis (accompanying paper by Uanschou et al), we note their relationship to the mammalian CtIP tumor suppressor protein. Our results, therefore, also predict a potential function of CtIP in meiotic recombination and genome stability in metazoans. Results Identification of the C. elegans com-1 gene and of com-1 mutants Alleles of C. elegans com-1 were first obtained from a mutagenesis screen in the laboratory of RS and characterized as causing maternal-effect embryonic lethality (Gönczy et al, 1999). The screen was designed to recover mutations that are linked to the unc-32 locus and map to the right portion of chromosome III from tra-1 to dpy-1 (see Materials and methods). Two mutants from this screen, carrying alleles let(t1626) and let(t1489) showed hallmarks of meiotic recombination defects (see below). Crosses of animals carrying the two alleles demonstrated that let(t1626) and let(t1489) are non-complementing and represent a single locus. To identify candidate genes within the interval between tra-1 and dpy-1, which could correspond to the mutated locus, we assessed the presence of homologs of known DNA-repair and/or meiotic proteins in this region. With this aim, we extracted all protein sequences from the worm and human genomic databases associated with the gene ontology terms ‘DNA repair’ or ‘meiosis’ (http://wormbase.org; Hammond and Birney, 2004). Approximately 60 genes within the tra-1 to dpy-1 interval could be directly linked to a known meiotic or repair protein based on sequence similarity (Altschul et al, 1997). Among those, the protein encoded by open reading frame (ORF) C44B9.5 stood out as the potential ortholog of the mammalian BRCA1-associating protein CtIP (NP_976036) (Yu et al, 2006 and lit. cit. therein). Similarity searches against the human and worm proteomes using C44B9.5 and CtIP show that these proteins are each other's best and only reciprocal BLAST hits (using full-length sequences with low complexity regions and coiled coils masked off; E-value cut-off 0.001) (Figure 1A). Strikingly, C44B9.5p could be identified as a homolog of S. cerevisiae Com1p/Sae2p (McKee and Kleckner, 1997; Prinz et al, 1997) when subjecting the very C-terminal 100 amino acids (aa) of C44B9.5p (NP_499398) to a PSI-BLAST search against the NCBI non-redundant (nr) database (nr version 02/2007; standard settings; NP_011340 hit in round 7 with an Expect of 3e-05) (Altschul et al, 1997; Marchler-Bauer et al, 2002). The search converged in round 8 with one representative per species, the conserved segment always being situated in the very C-terminus of the gathered proteins (Figure 1A). The collected set of Com1p/Sae2p—C44B9.5p—CtIP homologs (Figure 1B) is in agreement with the protein family identified in rigorous reciprocal searches starting with yeast Com1p/Sae2p (accompanying paper by Uanschou et al). Figure 1.Homologous relationships between C. elegans C44B9.5p and other family members and a map of the C44B9.5 locus. (A) Protein architecture of the homologous sequences CtIP (RBBP8), C44B9.5p (COM-1) and Com1p/Sae2p. The organization of proteins in this family is dominated by intrinsically unstructured regions. Pronounced ordered segments, indicated by gray boxes, are commonly found in the very C-terminus. Reciprocal proteome BLAST searches performed using coils and low-complexity filtered full-length proteins are indicated by black arrows. The gray arrow indicates PSI-BLAST searches started with the C-terminal 100 amino acids (aa) of C. elegans C44B9.5p and leading to S. cerevisiae Com1p/Sae2p in round 7. (B) Multiple sequence alignment of the C-terminal conserved region in Com1/Sae2/C44B9.5/CtIP homologs. Note that the sequenced cDNA reveals a longer C. elegans COM-1 protein sequence due to an additional exon, than predicted by the current WormBase Release WS178. Alignment visualization and gray-scale conservation shading are performed using GeneDoc (Nicholas et al, 1997; http://www.psc.edu/biomed/genedoc/gdfeedb.htm). A sequence consensus is shown beneath the alignment if a relative frequency cut-off (50, 70 or 90%) is met by individual aa or one of the following aa categories: alcoholic (denoted by ‘o’ and including S and T), aliphatic (‘l’ for I, L and V), aromatic (‘a’ for F, H, W and Y), positive (‘+’ for H, K and R), negative (‘−’ for D and E). An indication of selected taxonomic groups is included in front of the species names for nematodes (N), chordates (C) and fungi (F). Genomic sequences as the basis for ‘predicted’ protein sequences were derived from www.genome.wustl.edu for nematodes and www.ensembl.org for chordates. (C) Map of the C44B9.5 (com-1) locus on chromosome III with the positions of the mutations in alleles t1626 and t1489. (D) Amplification of com-1, spo-11 and ubiquitously expressed control lmn-1 cDNAs in wild-type and mutant backgrounds (asterisks: null-template control, M: size marker). Stable com-1 transcripts are recovered from com-1(t1626) and com-1(t1489) mutant hermaphrodites. com-1 but not the meiosis-specific recombination gene spo-11 is mitotically expressed in wild-type embryos. Download figure Download PowerPoint Sequencing of ORF C44B9.5 in the let(t1626) and let(t1489) mutants showed that indeed they carry mutations in C44B9.5. let(t1626) is a C to T transition at base pair (bp) 1809 in the third exon of the ORF, and let(t1489) is a C to T transition at bp 4030 in the sixth exon of the 5357 bp 7-exon ORF. (Sequencing of C44B9.5 cDNA of adult wild-type hermaphrodites revealed a deviation from the annotated mRNA sequence in the current release WS178 of the C. elegans WormBase. It comprises an additional small (57 bp) fifth exon.) Both mutations define a premature stop codon (Figure 1C) and therefore produce proteins that are missing the most conserved and, hence, presumably the functionally most significant C-terminal portion. According to the germline gene expression profile, the expression of C44B9.5 is not enhanced during meiosis (Reinke et al, 2000, 2004; see http://workhorse.stanford.edu/germline/). We confirmed the mitotic expression of ORF C44B9.5 by RT–PCR (Figure 1D). Owing to the protein sequence homology and the similar phenotypes of S. cerevisiae com1/sae2 and C. elegans let(t1626) mutants, we renamed the mutants com-1(t1626) and com-1(t1489) following the designation in budding yeast for Completion Of Meiotic recombination (Prinz et al, 1997), a function the gene is likely to exert in C. elegans as well (see below). com-1(t1626) is considered the strongest allele (phenotypically resembling the knockout by cosuppression, see below) and we used it to further explore the function of the gene. com-1 mutant animals display a normal somatic phenotype but are infertile The constitutive expression of com-1 mRNA (see above) suggests that this gene has a role during somatic growth. However, we found that the development of homozygous com-1 mutant worms originating from heterozygous mothers was not notably affected or retarded compared to unc-32 control animals. On the other hand, anaphase bridges were observed in at least 12% of mitoses (n=47) in embryos produced by these worms (inset in Figure 2). Defective anaphases may be a consequence of the irregular disjunction of chromosomes in the wake of an aberrant meiosis (see below) or could indicate a slight effect of the com-1 mutation on mitosis. Figure 2.Comparison of wild-type (unc-32) and com-1 unc-32 mutant gonads (composite images). The morphology of com-1(t1626) mutant gonads does not notably differ from that of wild-type gonads. The mitotic zone is followed by a distinct transition zone displaying the typical polarized arrangement of meiotic chromosomes. In the pachytene zone, nuclei with DAPI-positive parallel tracks of chromatin are found. The inset shows nuclei of a com-1 embryo with a DNA bridge. DNA is stained with DAPI. Download figure Download PowerPoint As yeast com1Δ/sae2Δ cells are reported to be sensitive to genotoxic stress (see Discussion), we tested whether C. elegans com-1 mutant homozygotes display a somatic growth defect in the presence of MMS. We did not observe an effect of MMS on somatic growth (data not shown), but it is possible that it is prevented by a pool of COM-1 received from the heterozygous mothers. The low fertility of the com-1 mutant (see below) precluded the investigation of a somatic requirement for COM-1 in following generations. The brood size of homozygous com-1 mutants was reduced and the viability of the offspring strongly reduced. Of a total of 1813 fertilized eggs laid by 12 com-1 unc-32 mutant worms (average brood size: 151 fertilized eggs), 12 hatched but died at the L1 or L2 stage, which is a hatch rate of 0.7%. By comparison, of the 1183 eggs laid by 6 unc-32 control animals (average brood size: 197 fertilized eggs), 1177 (99.5%) produced adult progeny. To find out the cause for the sterility of the mutants, we studied gonadal development and meiosis under the microscope. Gonad morphology and meiotic pairing are normal in the com-1 mutant DAPI staining revealed morphologically inconspicuous com-1(t1626) mutant gonads (Figure 2). Like wild-type gonads, they were subdivided into a mitotic plus meiotic entry zone, a transition zone and a pachytene zone (Albertson et al, 1997; Schedl, 1997; Figure 2). To determine whether a normal SC was formed in the pachytene zone, nuclei were immunostained for REC-8 and HIM-3, two components of the lateral elements of the SC. Moreover, the presence and localization of the SC transversal protein SYP-1 was tested by immunostaining. All three proteins showed a normal abundance and distribution in pachytene (Figure 3). Having thus confirmed that extensive SC was formed between chromosomes in the mutant, it was determined whether it connected homologous chromosome regions. For this, homologous chromosome loci were highlighted by HIM-8 staining and FISH. HIM-8 specifically binds to the pairing center of the X chromosome and can be used to mark this locus near the left end of this chromosome (Phillips et al, 2005). Similarly, FISH was used to delineate the 5S rDNA locus on the right arm of chromosome V. Both HIM-8 staining and FISH produced a single signal or two closely associated signals in most pachytene nuclei (Figure 4A), revealing that the corresponding chromosomal regions were homologously paired in com-1(t1626) (Figure 4B). Figure 3.Immunostaining of SC components in the pachytene zone of wild-type and com-1 mutant gonads. Axial element components REC-8 (red) and HIM-3 (green) and the transversal filament component SYP-1 (yellow) appear normal in the mutant indicating normal SC assembly. Chromatin is counterstained with DAPI (blue). Download figure Download PowerPoint Figure 4.Homologous pairing is not affected in the com-1 mutant. (A) FISH of the 5S rDNA locus on chromosome V and immunostaining of the X chromosomal protein HIM-8 both produce a single spot (or two closely adjacent spots) in pachytene nuclei of unc-32 com-1 and of unc-32 control gonads, indicating that the corresponding homologous chromosome regions are paired. Chromatin is counterstained by DAPI (blue). (B) Frequencies of paired chromosomes V and X (as monitored by the association of FISH signals or HIM-8 foci, respectively) along the gonad. Two signals touching each other or merged into a single signal were scored as paired. Gonads were divided into six intervals of equal lengths between the distal tip and the end of the pachytene zone. The lines labelled ‘n=’ indicate the number of nuclei evaluated for the corresponding data point. For the FISH experiment, they were sampled from four and for the HIM-8 experiment from three gonads. Download figure Download PowerPoint No distinct diakinesis bivalents are formed in the com-1 mutant Unlike the wild type where six distinct bivalents can be observed per diakinesis cell, a variable number of diffuse DAPI-stained entities were present in the com-1 mutant (Figure 5A and B). Scoring of 68 late diakineses from 44 gonads revealed a range from 12 chromatin masses to a single large clump. Chromatin masses appeared less condensed than wild-type bivalents at the corresponding stage and they were sometimes connected by thin DAPI-positive threads. Judging from their sizes, some of the free chromatin structures may be bivalents and others, individual chromosomes, whereas yet some others could represent chromosome fragments. A similar phenotype was observed after depletion of COM-1 by cosuppression (Figure 5B). The clumping, stickiness and fragmentation of diakinesis chromosomes possibly indicate inefficient repair of DSBs, as it is frequently observed in mutants in which DSBs are not normally repaired (Rinaldo et al, 2002; Alpi et al, 2003; Takanami et al, 2003; Martin et al, 2005; see Discussion). Figure 5.Appearance and quantification of chromosomal structures in diakinesis nuclei; DAPI staining. (A–E) Diakinesis in the wild type and in mutants (all of the unc-32 background) without and after γ-irradiation. (A) Six bivalents are present in the wild type. (B) Examples of chromatin masses in the com-1 mutant. The mutant displays a variable number of sometimes interconnected chromatin masses ranging from more than six to a single clump plus a few small fragments (arrows). The rightmost image shows a diakinesis from a gonad in a com-1 cosuppression line. (C) Univalent formation by the spo-11Δ mutant (left), and the com-1 spo-11Δ double mutant (right). (D) Depletion of REC-8 by RNAi causes the formation of chromosome fragments (arrows) as an indication of DSBs in the wild type (top) and in the com-1 mutant (middle), but not in the spo-11Δ mutant (bottom). (E) Diakineses after γ-irradiation. Top: dispersed chromatin masses in the com-1 mutant; they do not notably differ from those in unirradiated cells. Middle: irradiation restores bivalent formation in the spo-11Δ mutant. Bivalents are irregularly shaped presumably because the frequency and distribution of crossovers is not wild type. Bottom: irradiation restores chromatin clumping in com-1 spo-11Δ mutant diakineses. (F, G) Frequency distribution of DAPI-positive entities in diakinesis nuclei without (F) and after (G) γ-irradiation. *The frequencies of DAPI-positive entities in the com-1 mutant are based on estimates, as the dispersed and partially interconnected chromatin masses precluded exact counting. Download figure Download PowerPoint To test whether meiotic DSBs are formed in the first place and whether they contribute to the abnormal chromosomal morphology in the com-1 mutant, we constructed a spo-11Δ com-1 double mutant. In the spo-11Δ background, DSBs do not form in the first place (Dernburg et al, 1998) and DSB-dependent clumping and undercondensation should not occur (Figure 5C). Indeed, the com-1 mutant phenotype was largely alleviated by the double mutation (Figure 5C). Of 67 cells in diakinesis, 53 (79%) showed 12 distinct DAPI-positive structures, that is, univalents and another 18% showed 11 entities (Figure 5F). Similarly, the mre-11Δ mutation (which also prevents DSB formation; Chin and Villeneuve, 2001; see below) abolished the clumping phenotype of com-1 and restored normally shaped univalents in a double mutant (data not shown). The absence of clumping in the com-1 spo-11Δ and the com-1 mre-11Δ double mutants suggests that it may be caused by unrepaired or inappropriately processed DSBs in the com-1 mutant. To directly visualize meiotic DSBs in the com-1 mutant, we depleted REC-8 by RNAi in the mutant. In the absence of REC-8, homologous chromosomes are unpaired and sister chromatids are separated which impedes the repair of DSBs by homologous and sister chromatid recombination. Moreover, unrepaired DSBs are exposed as free chromosome fragments which would not be seen if they were attached to their corresponding (intact) sisters in the presence of REC-8 (Pasierbek et al, 2001; Colaiácovo et al, 2003; Figure 5D). Indeed, numerous chromosome fragments were present in diakineses of com-1 mutant animals deprived of REC-8 (Figure 5D), whereas in the spo-11Δ rec-8(RNAi) control no fragments were found (Figure 5D). This observation confirms the occurrence of SPO-11-dependent DSBs in the com-1 mutant. Viability of the com-1 mutant progeny is improved by the absence of meiotic DSB To test whether the high embryonic lethality of the com-1 mutant depends on the induction of meiotic DSBs, we decided to analyze the viability of the progeny in the DSB-less com-1 mre-11Δ double mutant. In this background, viability should be improved to the level permitted by the production of chromosomally balanced gametes and embryos due to the random segregation of univalents. Embryo viability of mre-11Δ hermaphrodites had been found to be 3.1% (Chin and Villeneuve, 2001). (Embryo viability of the spo-11Δ mutant is much lower, which precluded its use for this experiment.) In our hands, the mre-11Δ unc-32 control produced 4.1% viable embryos (n=1763 fertilized eggs laid by 10 animals). The hatch rate of eggs of a com-1 mre-11Δ double mutant was 4.3% (n=1885 eggs from 12 hermaphrodites). Thus, while the hatch rate of com-1 mutant progeny is only 0.7% (see above), the viability of embryos of a com-1 mre-11Δ double mutant is restored to the level of the mre-11Δ single mutant, which is consistent with the interpretation that unrepaired or improperly repaired DSBs are the cause of embryonic lethality in the com-1 mutant. RAD-51 protein foci do not form normally on chromatin in the com-1 mutant The recombination protein RAD-51 associates with 3′ ssDNA overhangs flanking DSBs to form long nucleoprotein filaments (Shinohara et al, 1992). In the wild type, RAD-51 localizes to meiotic chromosomes from late zygotene to mid-pachytene (Alpi et al, 2003; Colaiácovo et al, 2003; Figure 6). This corresponds to the region where recombination is believed to take place. In contrast, in the com-1 mutant, despite the formation and persistence of DSBs, RAD-51 foci were virtually completely missing in the corresponding zone of the gonad (Figure 6; Supplementary Figure 1). As western analysis demonstrated that the RAD-51 protein was expressed in the com-1 mutant (data not shown), the absence of RAD-51 foci must be due to the inability of RAD-51 to associate with DNA flanking the DSBs. Figure 6.Immunostaining of the recombination protein RAD-51 (red) in whole gonads (composite images—left). Enlarged details from the corresponding gonads are shown to the right. While in the wild type abundant RAD-51 foci indicate ongoing recombination in late transition zone to early pachytene, there is no such RAD-51-rich zone in the com-1 mutant gonad. Throughout the gonad, however, stray nuclei show few strong RAD-51 foci. These foci are missing in the spo-11Δ mutant strain but are present in the com-1 spo-11Δ double mutant. (For the frequencies of RAD-51 foci per nucleus and the distribution of RAD-51 foci-bearing nuclei within the gonads see Supplementary Figure 1.) γ-irradiation induces RAD-51 foci formation in all strains, including the com-1 mutant (data not shown) and the com-1 spo-11Δ double mutant. In each gonad, the left arrowhead denotes the border between the mitotic zone and the transition zone and the right arrowhead the border between the transition zone and the pachytene zone. Download figure Download PowerPoint Remarkably, however, immunostaining with RAD-51 antibody produced strong foci in a few nuclei scattered all along the gonad (Figure 6). In the six gonads where these foci were counted, the average number of foci/gonad was 24.5 and the maximum was 41. They occurred frequently in pairs within a nucleus. These sporadic foci did not depend on (untimely expressed) SPO-11, as they also occurred in a spo-11Δ com-1 double mutant (Figure 6; average number 27.3 foci/gonad with a maximum of 37; n=6 gonads). In com-1+/+ controls, such extra foci were virtually missing (unc-32 spo-11Δ strain: ∅ 2.1 foci/gonad, n=8 gonads; unc-32 strain: ∅ 1.9 foci outside the zygotene-mid-pachytene zone, n=11 gonads). Notably, RAD-51 foci, similar in number and distribution to those in the com-1 mutant, were found in an mre-11Δ strain (data not shown). We assume that these foci represent RAD-51-binding repair intermediates at certain DNA lesions that, in the absence of COM-1 and MRE-11, cannot undergo efficient repair. The occurrence of RAD-51 foci in the com-1 mutant also suggests that, while COM-1 is necessary for the loading of RAD-51 at meiotic DSBs, it could play a role subsequent to RAD-51 loading in other repair processes. Recently, spontaneous (SPO-11-independent) RAD-51 foci were also detected in rad-50 mutant germ lines and interpreted as spontaneous DNA breaks that arose during the course of DNA replication (Hayashi et al, 2007). γ-irradiation induces RAD-51 foci in the absence of COM-1 To further test whether the inability to load RAD-51 is specific to masked DNA ends (such as those at meiotic DSBs), we induced DSBs by γ-irradiation. We observed massive RAD-51 foci formation in nuclei along the entire gonad not only in young embryos in the wild type and in the spo-11 mutant but also in the com-1 mutant and the spo-11Δ com-1 double mutant (Figure 6). This demonstrates

Crossovers (COs) are essential for the completion of meiosis in most species and lead to new allelic combinations in gametes. Two pathways of meiotic crossover formation have been distinguished. Class I COs, which are the major class of CO in budding yeast, mammals, Caenorhabditis elegans, and Arabidopsis, depend on a group of proteins called ZMM and rely on specific DNA structure intermediates that are processed to form COs. We identified a novel gene, SHOC1, involved in meiosis in Arabidopsis. Shoc1 mutants showed a striking reduction in the number of COs produced, a similar phenotype to the previously described Arabidopsis zmm mutants. The early steps of recombination, revealed by DMC1 foci, and completion of synapsis are not affected in shoc1 mutants. Double mutant analysis showed that SHOC1 acts in the same pathway as AtMSH5, a conserved member of the ZMM group. SHOC1 is thus a novel gene required for class I CO formation in Arabidopsis. Sequence similarity studies detected putative SHOC1 homologs in a large range of eukaryotes including human. SHOC1 appears to be related to the XPF endonuclease protein family, which suggests that it is directly involved in the maturation of DNA intermediates that lead to COs.

Polo-like kinase 1 (PLK1) is a key regulator of mitotic progression and cell division, and small molecule inhibitors of PLK1 are undergoing clinical trials to evaluate their utility in cancer therapy. Despite this importance, current knowledge about the identity of PLK1 substrates is limited. Here we present the results of a proteome-wide analysis of PLK1-regulated phosphorylation sites in mitotic human cells. We compared phosphorylation sites in HeLa cells that were or were not treated with the PLK1-inhibitor BI 4834, by labeling peptides <i>via</i> methyl esterification, fractionation of peptides by strong cation exchange chromatography, and phosphopeptide enrichment <i>via</i> immobilized metal affinity chromatography. Analysis by quantitative mass spectrometry identified 4070 unique mitotic phosphorylation sites on 2069 proteins. Of these, 401 proteins contained one or multiple phosphorylation sites whose abundance was decreased by PLK1 inhibition. These include proteins implicated in PLK1-regulated processes such as DNA damage, mitotic spindle formation, spindle assembly checkpoint signaling, and chromosome segregation, but also numerous proteins that were not suspected to be regulated by PLK1. Analysis of amino acid sequence motifs among phosphorylation sites down-regulated under PLK1 inhibition in this data set identified two potential novel variants of the PLK1 consensus motif.

Significance Local protein synthesis is a highly used mechanism to create functional asymmetries within cells. It underlies diverse biological processes, including the development and function of the nervous and reproductive systems. Cytoplasmic polyadenylation element-binding (CPEB) proteins regulate local translation in early development, synaptic plasticity, and long-term memory. However, their binding specificity is not fully resolved. We used a transcriptome-wide approach and established that Drosophila representatives of two CPEB subfamilies, Orb and Orb2, regulate largely overlapping target mRNAs by binding to CPE-like sequences in their 3′ UTRs, potentially with a shift in specificity for motif variants. Moreover, our data suggest that a subset of these mRNAs is translationally regulated and involved in long-term memory.

PR homology domain-containing member 12 (PRDM12) belongs to a family of conserved transcription factors implicated in cell fate decisions. Here we show that PRDM12 is a key regulator of sensory neuronal specification in Xenopus. Modeling of human PRDM12 mutations that cause hereditary sensory and autonomic neuropathy (HSAN) revealed remarkable conservation of the mutated residues in evolution. Expression of wild-type human PRDM12 in Xenopus induced the expression of sensory neuronal markers, which was reduced using various human PRDM12 mutants. In Drosophila, we identified Hamlet as the functional PRDM12 homolog that controls nociceptive behavior in sensory neurons. Furthermore, expression analysis of human patient fibroblasts with PRDM12 mutations uncovered possible downstream target genes. Knockdown of several of these target genes including thyrotropin-releasing hormone degrading enzyme (TRHDE) in Drosophila sensory neurons resulted in altered cellular morphology and impaired nociception. These data show that PRDM12 and its functional fly homolog Hamlet are evolutionary conserved master regulators of sensory neuronal specification and play a critical role in pain perception. Our data also uncover novel pathways in multiple species that regulate evolutionary conserved nociception.

Significance We show that Bouncer’s homolog in mammals, SPACA4, is required for efficient fertilization in mice. In contrast to fish, in which Bouncer is required for female fertility, SPACA4 is expressed exclusively in the sperm and is required for male fertility. SPACA4 and Bouncer present an intriguing example of homologous proteins that both play key roles in reproduction yet diverged in terms of gene expression pattern and mode of action. Overall, our work identifies SPACA4 as an important sperm protein necessary for zona pellucida penetration during mammalian fertilization. Since human SPACA4 is also expressed exclusively in sperm, we anticipate that our findings in mice will have relevance to human biology.

Nuclear Argonaute proteins, guided by their bound small RNAs to nascent target transcripts, mediate cotranscriptional silencing of transposons and repetitive genomic loci through heterochromatin formation. The molecular mechanisms involved in this process are incompletely understood. Here, we show that the SFiNX complex, a silencing mediator downstream from nuclear Piwi-piRNA complexes in Drosophila , facilitates cotranscriptional silencing as a homodimer. The dynein light chain protein Cut up/LC8 mediates SFiNX dimerization, and its function can be bypassed by a heterologous dimerization domain, arguing for a constitutive SFiNX dimer. Dimeric, but not monomeric SFiNX, is capable of forming molecular condensates in a nucleic acid-stimulated manner. Mutations that prevent SFiNX dimerization result in loss of condensate formation in vitro and the inability of Piwi to initiate heterochromatin formation and silence transposons in vivo. We propose that multivalent SFiNX-nucleic acid interactions are critical for heterochromatin establishment at piRNA target loci in a cotranscriptional manner.

ZNF462 haploinsufficiency is linked to Weiss-Kruszka syndrome, a genetic disorder characterized by neurodevelopmental defects, including autism. Though conserved in vertebrates and essential for embryonic development, the molecular functions of ZNF462 remain unclear. We identified its murine homologue ZFP462 in a screen for mediators of epigenetic gene silencing. Here we show that ZFP462 safeguards neural lineage specification of mouse embryonic stem cells (ESCs) by targeting the H3K9-specific histone methyltransferase complex G9A/GLP to silence meso-endodermal genes. ZFP462 binds to transposable elements that are potential enhancers harbouring pluripotency and meso-endoderm transcription factor binding sites. Recruiting G9A/GLP, ZFP462 seeds heterochromatin, restricting transcription factor binding. Loss of ZFP462 in ESCs results in increased chromatin accessibility at target sites and ectopic expression of meso-endodermal genes. Taken together, ZFP462 confers lineage and locus specificity to the broadly expressed epigenetic regulator G9A/GLP. Our results suggest that aberrant activation of lineage non-specific genes in the neuronal lineage underlies ZNF462-associated neurodevelopmental pathology.

Ventral midbrain dopaminergic neurons project to the striatum as well as the cortex and are involved in movement control and reward-related cognition. In Parkinson's disease, nigrostriatal midbrain dopaminergic neurons degenerate and cause typical Parkinson's disease motor-related impairments, while the dysfunction of mesocorticolimbic midbrain dopaminergic neurons is implicated in addiction and neuropsychiatric disorders. Study of the development and selective neurodegeneration of the human dopaminergic system, however, has been limited due to the lack of an appropriate model and access to human material. Here, we have developed a human in vitro model that recapitulates key aspects of dopaminergic innervation of the striatum and cortex. These spatially arranged ventral midbrain-striatum-cortical organoids (MISCOs) can be used to study dopaminergic neuron maturation, innervation and function with implications for cell therapy and addiction research. We detail protocols for growing ventral midbrain, striatal and cortical organoids and describe how they fuse in a linear manner when placed in custom embedding molds. We report the formation of functional long-range dopaminergic connections to striatal and cortical tissues in MISCOs, and show that injected, ventral midbrain-patterned progenitors can mature and innervate the tissue. Using these assembloids, we examine dopaminergic circuit perturbations and show that chronic cocaine treatment causes long-lasting morphological, functional and transcriptional changes that persist upon drug withdrawal. Thus, our method opens new avenues to investigate human dopaminergic cell transplantation and circuitry reconstruction as well as the effect of drugs on the human dopaminergic system.

We have designed the most efficient strategy to knock out genes in fission yeast Schizosaccharomyces pombe on a large scale. Our technique is based on knockout constructs that contain regions homologous to the target gene cloned into vectors carrying dominant drug-resistance markers. Most of the steps are carried out in a 96-well format, allowing simultaneous deletion of 96 genes in one batch. Based on our knockout technique, we designed a strategy for cloning knockout constructs for all predicted fission yeast genes, which is available in a form of a searchable database http://mendel.imp.ac.at/Pombe_deletion/ . We validated this technique in a screen where we identified novel genes required for chromosome segregation during meiosis. Here, we present our protocol with detailed instructions. Using this protocol, one person can knock out 96 S. pombe genes in 8 days.

During meiosis, the micronuclei of the ciliated protist Tetrahymena thermophila elongate dramatically. Within these elongated nuclei, chromosomes are arranged in a bouquet-like fashion and homologous pairing and recombination takes place. We studied meiotic chromosome behavior in Tetrahymena in the absence of two genes, SPO11 and a homolog of HOP2 (HOP2A), which have conserved roles in the formation of meiotic DNA double-strand breaks (DSBs) and their repair, respectively. Single-knockout mutants for each gene display only a moderate reduction in chromosome pairing, but show a complete failure to form chiasmata and exhibit chromosome missegregation. The lack of SPO11 prevents the elongation of meiotic nuclei, but it is restored by the artificial induction of DSBs. In the hop2AΔ mutant, the transient appearance of γ-H2A.X and Rad51p signals indicates the formation and efficient repair of DSBs; but this repair does not occur by interhomolog crossing over. In the absence of HOP2A, the nuclei are elongated, meaning that DSBs but not their conversion to crossovers are required for the development of this meiosis-specific morphology. In addition, by in silico homology searches, we compiled a list of likely Tetrahymena meiotic proteins as the basis for further studies of the unusual synaptonemal complex-less meiosis in this phylogenetically remote model organism.

Lung cancer is the leading cause of cancer deaths. Besides smoking, epidemiological studies have linked female sex hormones to lung cancer in women; however, the underlying mechanisms remain unclear. Here we report that the receptor activator of nuclear factor-kB (RANK), the key regulator of osteoclastogenesis, is frequently expressed in primary lung tumors, an active RANK pathway correlates with decreased survival, and pharmacologic RANK inhibition reduces tumor growth in patient-derived lung cancer xenografts. Clonal genetic inactivation of KRasG12D in mouse lung epithelial cells markedly impairs the progression of KRasG12D -driven lung cancer, resulting in a significant survival advantage. Mechanistically, RANK rewires energy homeostasis in human and murine lung cancer cells and promotes expansion of lung cancer stem-like cells, which is blocked by inhibiting mitochondrial respiration. Our data also indicate survival differences in KRasG12D -driven lung cancer between male and female mice, and we show that female sex hormones can promote lung cancer progression via the RANK pathway. These data uncover a direct role for RANK in lung cancer and may explain why female sex hormones accelerate lung cancer development. Inhibition of RANK using the approved drug denosumab may be a therapeutic drug candidate for primary lung cancer.

Abstract ZMM proteins have been defined in budding yeast as factors that are collectively involved in the formation of interfering crossovers (COs) and synaptonemal complexes (SCs), and they are a hallmark of the predominant meiotic recombination pathway of most organisms. In addition to this so-called class I CO pathway, a minority of crossovers are formed by a class II pathway, which involves the Mus81-Mms4 endonuclease complex. This is the only CO pathway in the SC-less meiosis of the fission yeast. ZMM proteins (including SC components) were always found to be co-occurring and hence have been regarded as functionally linked. Like the fission yeast, the protist Tetrahymena thermophila does not possess a SC, and its COs are dependent on Mus81-Mms4. Here we show that the ZMM proteins Msh4 and Msh5 are required for normal chiasma formation, and we propose that they have a pro-CO function outside a canonical class I pathway in Tetrahymena. Thus, the two-pathway model is not tenable as a general rule.

The cohesion of sister chromatids in the interval between chromosome replication and anaphase is important for preventing the precocious separation, and hence nondisjunction, of chromatids. Cohesion is accomplished by a ring-shaped protein complex, cohesin; and its release at anaphase occurs when separase cleaves the complex's α-kleisin subunit. Cohesin has additional roles in facilitating DNA damage repair from the sister chromatid and in regulating gene expression. We tested the universality of the present model of cohesion by studying cohesin in the evolutionarily distant protist Tetrahymena thermophila. Localization of tagged cohesin components Smc1p and Rec8p (the α-kleisin) showed that cohesin is abundant in mitotic and meiotic nuclei. RNAi knockdown experiments demonstrated that cohesin is crucial for normal chromosome segregation and meiotic DSB repair. Unexpectedly, cohesin does not detach from chromosome arms in anaphase, yet chromosome segregation depends on the activity of separase (Esp1p). When Esp1p is depleted by RNAi, chromosomes become polytenic as they undergo multiple rounds of replication, but fail to separate. The cohesion of such bundles of numerous chromatids suggests that chromatids may be connected by factors in addition to topological linkage by cohesin rings. Although cohesin is not detected in transcriptionally active somatic nuclei, its loss causes a slight defect in their amitotic division. Notably, Tetrahymena uses a single version of α-kleisin for both mitosis and meiosis. Therefore, we propose that the differentiation of mitotic and meiotic cohesins found in most other model systems is not due to the need of a specialized meiotic cohesin, but due to additional roles of mitotic cohesin.

Post-translational modification by Small Ubiquitin-like Modifiers (SUMO) has been implicated in protein targeting, in the maintenance of genomic integrity and in transcriptional control. But the specific molecular effects of SUMO modification on many target proteins remain to be elucidated. Recent findings point at the importance of SUMO-mediated histone NAD-dependent deacetylase (HDAC) recruitment in transcriptional regulation. We describe the RENi family of SUMO-like domain proteins (SDP) with the unique feature of typically containing two carboxy-terminal SUMO-like domains. Using sequence analytic evidence, we collect family members from animals, fungi and plants, most prominent being yeast R ad60, E sc2 and mouse NI P45 http://mendel.imp.univie.ac.at/SEQUENCES/reni/ . Different proteins of the novel family are known to interact directly with histone NAD-dependent deacetylases (HDACs), structural maintenance of chromosomes (SMC) proteins, and transcription factors. In particular, the highly non-trivial designation of the first of the two successive SUMO-domains in non-plant RENi provides a rationale for previously published functionally impaired mutant variants. Till now, SUMO-like proteins have been studied exclusively in the context of their covalent conjugation to target proteins. Here, we present the exciting possibility that SUMO domain proteins, similarly to ubiquitin modifiers, have also evolved in a second line – namely as multi-domain proteins that are non-covalently attached to their target proteins. We suggest that the SUMO stable fusion proteins of the RENi family, which we introduce in this work, might mimic SUMO and share its interaction motifs (in analogy to the way that ubiquitin-like domains mimic ubiquitin). This presumption is supported by parallels in the spectrum of modified or bound proteins e.g. transcription factors and chromatin-associated proteins and in the recruitment of HDAC-activity.

Abstract Tandem affinity purification (TAP) is a method that allows rapid purification of native protein complexes. We developed an improved technique to fuse the fission yeast genes with a TAP tag. Our technique is based on tagging constructs that contain regions homologous to the target gene cloned into vectors carrying a TAP tag. We used this technique to design strategies for TAP‐tagging of predicted Schizosaccharomyces pombe genes ( http://mendel.imp.ac.at/Pombe_tagging/ ). To validate the approach, we purified the proteins, which associated with two evolutionarily conserved proteins Swi5 and Sfr1 as well as three protein kinases Ksg1, Orb6 and Sid1.

HACE1 is an E3 ubiquitin ligase with important roles in tumor biology and tissue homeostasis. Loss or mutation of HACE1 has been associated with the occurrence of a variety of neoplasms, but the underlying mechanisms have not been defined yet. Here, we report that HACE1 is frequently mutated in human lung cancer. In mice, loss of Hace1 led to enhanced progression of KRasG12D -driven lung tumors. Additional ablation of the oncogenic GTPase Rac1 partially reduced progression of Hace1-/- lung tumors. RAC2, a novel ubiquitylation target of HACE1, could compensate for the absence of its homolog RAC1 in Hace1-deficient, but not in HACE1-sufficient tumors. Accordingly, ablation of both Rac1 and Rac2 fully averted the increased progression of KRasG12D -driven lung tumors in Hace1-/- mice. In patients with lung cancer, increased expression of HACE1 correlated with reduced levels of RAC1 and RAC2 and prolonged survival, whereas elevated expression of RAC1 and RAC2 was associated with poor prognosis. This work defines HACE1 as a crucial regulator of the oncogenic activity of RAC-family GTPases in lung cancer development. SIGNIFICANCE: These findings reveal that mutation of the tumor suppressor HACE1 disrupts its role as a regulator of the oncogenic activity of RAC-family GTPases in human and murine lung cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/14/3009/F1.large.jpg.

The HUSH (human silencing hub) complex contains the H3K9me3 binding protein M-phase phosphoprotein 8 (MPP8) and recruits the histone methyltransferase SETDB1 as well as Microrchidia CW-type zinc finger protein 2 (MORC2). Functional and mechanistic studies of the HUSH complex have hitherto been centered around SETDB1 while the in vivo functions of MPP8 and MORC2 remain elusive. Here, we show that genetic inactivation of Mphosph8 or Morc2a in the nervous system of mice leads to increased brain size, altered brain architecture, and behavioral changes. Mechanistically, in both mouse brains and human cerebral organoids, MPP8 and MORC2 suppress the repetitive-like protocadherin gene cluster in an H3K9me3-dependent manner. Our data identify MPP8 and MORC2, previously linked to silencing of repetitive elements via the HUSH complex, as key epigenetic regulators of protocadherin expression in the nervous system and thereby brain development and neuronal individuality in mice and humans.

EndoGlyx-1, the antigen identified with the monoclonal antibody H572, is a pan-endothelial human cell surface glycoprotein complex composed of four different disulfide-bonded protein species with an apparent molecular mass of approximately 500 kDa. Here, we report the purification and peptide analysis of two EndoGlyx-1 subunits, p125 and p140, and the identification of a common, full-length cDNA with an open reading frame of 2847 base pairs. The EndoGlyx-1 cDNA encodes a protein of 949 amino acids with a predicted molecular mass of 105 kDa, found as an entry for an unnamed protein with unknown function in public data bases. A short sequence tag matching the cDNA of this gene was independently discovered by serial analysis of gene expression profiling as a pan-endothelial marker, PEM87. Bioinformatic evaluation classifies EndoGlyx-1 as an EMILIN-like protein composed of a signal sequence, an N-terminal EMI domain, and a C-terminal C1q-like domain, separated from each other by a central coiled-coil-rich region. Biochemical and carbohydrate analysis revealed that p125, p140, and the two additional EndoGlyx-1 subunits, p110 and p200, are exposed on the cell surface. The three smaller subunits show a similar pattern of N-linked and O-linked carbohydrates, as shown by enzyme digestion. Because the two globular domains of EndoGlyx-1 p125/p140 show structural features shared by EMILIN-1 and Multimerin, two oligomerizing glycoproteins implicated in cell-matrix adhesion and hemostasis, it will be of interest to explore similar functions for EndoGlyx-1 in human vascular endothelium. EndoGlyx-1, the antigen identified with the monoclonal antibody H572, is a pan-endothelial human cell surface glycoprotein complex composed of four different disulfide-bonded protein species with an apparent molecular mass of approximately 500 kDa. Here, we report the purification and peptide analysis of two EndoGlyx-1 subunits, p125 and p140, and the identification of a common, full-length cDNA with an open reading frame of 2847 base pairs. The EndoGlyx-1 cDNA encodes a protein of 949 amino acids with a predicted molecular mass of 105 kDa, found as an entry for an unnamed protein with unknown function in public data bases. A short sequence tag matching the cDNA of this gene was independently discovered by serial analysis of gene expression profiling as a pan-endothelial marker, PEM87. Bioinformatic evaluation classifies EndoGlyx-1 as an EMILIN-like protein composed of a signal sequence, an N-terminal EMI domain, and a C-terminal C1q-like domain, separated from each other by a central coiled-coil-rich region. Biochemical and carbohydrate analysis revealed that p125, p140, and the two additional EndoGlyx-1 subunits, p110 and p200, are exposed on the cell surface. The three smaller subunits show a similar pattern of N-linked and O-linked carbohydrates, as shown by enzyme digestion. Because the two globular domains of EndoGlyx-1 p125/p140 show structural features shared by EMILIN-1 and Multimerin, two oligomerizing glycoproteins implicated in cell-matrix adhesion and hemostasis, it will be of interest to explore similar functions for EndoGlyx-1 in human vascular endothelium. monoclonal antibody base pair human umbilical vein endothelial cell polyacrylamide gel electrophoresis high performance liquid chromatography 4-morpholinepropanesulfonic acid The EndoGlyx-1 antigen was identified in a survey of normal and neoplastic tissues conducted at the Ludwig Institute for Cancer Research in pursuit of new antigenic markers of vascular endothelium. A peculiar feature of the monoclonal antibody (mAb)1 H572, the probe first used to discover EndoGlyx-1 (1Sanz-Moncasi M.P. Garin-Chesa P. Stockert E. Jaffe E.A. Old L.J. Rettig W.J. Lab. Invest. 1994; 71: 366-373PubMed Google Scholar), is its distinctive reactivity with human tissues. In an extensive immunohistochemical survey of normal human fetal and adult tissues as well as human cancer tissues, H572 immunostaining was found exclusively on blood vessel endothelium. Notably, these included capillaries, veins, arterioles, and muscular arteries, but interestingly no immunoreactivity was observed in the sinusoidal endothelial cells of the spleen and liver. In neoplastic tissues, H572 immunostaining was consistently found in tumor capillaries, including “hot spots” of neoangiogenesis in certain tumors (2Belien J.A. van Diest P.J. Baak J.P. J. Pathol. 1999; 189: 309-318Crossref PubMed Scopus (44) Google Scholar). The endothelial staining pattern revealed a uniform cell surface and cytoplasmic distribution of the antigen, in some cases with an accentuated staining at the abluminal side of the endothelial cell layer. All nonendothelial cell types in normal and tumor tissues were unreactive with mAb H572. The expression of the antigen on cultured human tumor cell lines and normal cells in vitro was also studied in detail. Thus, a host of cultured transformed cells of mesenchymal, neuroectodermal, and epithelial derivation as well as normal lymphocytes, hematopoetic cells, and platelets were found to be H572 antigen-negative. Cells of endothelial origin, such as human umbilical vein endothelial cells (HUVEC), microvascular endothelial cells, and the immortalized endothelial hybrid cell line EA.hy926 express high levels of the antigen in tissue culture. Stimulation of HUVECs with a variety of regulatory peptides and mediators that are known to induce expression of endothelial activation antigens found in inflammatory lesions (3Carlos T.M. Harlan J.M. Blood. 1994; 84: 2068-2101Crossref PubMed Google Scholar, 4Lasky L.A. Science. 1992; 258: 964-969Crossref PubMed Scopus (1153) Google Scholar) did not result in an altered expression of the H572 antigen. Radioimmunoprecipitation studies with mAb H572 identified the target antigen on cultured HUVECs as a high molecular mass glycoprotein complex of ∼500 kDa composed of at least four different disulfide-bridged protein subunits migrating at distinct molecular sizes in reducing SDS gels. Direct identification of the epitope-carrying subunit(s) were unsuccessful because of the fact that the epitope was destroyed by the immunoblotting procedure. To reflect the biochemical characteristics and the endothelium-specific expression of the H572 antigen, the molecule was designated EndoGlyx-1. In the absence of any information on the EndoGlyx-1 molecular structure, no clues were available regarding its potential function in endothelial cells. Considering the strong interest in mechanisms that control assembly of endothelial cells into vascular structures and regulate the biological function of established vessels, the present study was designed to characterize the EndoGlyx-1 gene. The results should provide essential information and molecular tools to investigate EndoGlyx-1 function in suitable model systems and to investigate the mode of EndoGlyx-1 regulation in the vascular bed. HUVECs were purchased (BioWhittaker, Walkersville) and cultured on collagen I-coated cell culture plates (Becton Dickinson) in EGM BulletKit medium (BioWhittaker). EA.hy926 were purchased from C. J. Edgell (University of North Carolina at Chapel Hill) and cultured in Dulbecco’s modified Eagle’s medium (BioWhittaker) with 10% fetal calf serum, 1% glutamine, and 1× hypoxanthine/aminopterin/thymidine medium (Life Technologies, Inc.). The anti-EndoGlyx-1 antibody H572 (mouse IgG1) was raised against HUVEC as described (1Sanz-Moncasi M.P. Garin-Chesa P. Stockert E. Jaffe E.A. Old L.J. Rettig W.J. Lab. Invest. 1994; 71: 366-373PubMed Google Scholar). The monoclonal antibody TEA-1/31 to human VE-Cadherin was purchased from BD Biosciences (Heidelberg, Germany). Tissues for immunohistochemistry were obtained from the collection of the Ludwig Institute for Cancer Research, and umbilical cord samples were obtained from the University of Ulm. Immunohistochemistry was carried out using mAb H572, mAb TEA-1/31, or negative control IgG as described previously (5Garin-Chesa P. Old L.J. Rettig W.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7235-7239Crossref PubMed Scopus (519) Google Scholar). For staining endothelium of intact blood vessels, samples of 10 cm in length were generated from freshly obtained, intact human umbilical cord specimens. Before antibody application, the umbilical cord vein was rinsed extensively with cold phosphate-buffered saline, closed at one end by a cable binder, and filled with antibody supernatant at a concentration of 10 μg antibody/ml in Dulbecco’s modified Eagle’s medium, 10% fetal calf serum. Intraluminal application using a 10-ml syringe with a blunt ended needle was followed by an occlusion of the umbilical vein by a second cable binder and subsequent incubation on ice for 1 h before rinsing extensively with cold phosphate-buffered saline. Subsequently, 4–5-mm samples from the central part of the specimens were embedded in OCT (Miles) compound and used for immunohistochemical analysis. For protein purification 3 × 109 EA.hy926 cells were lysed in RIPA buffer (150 mm NaCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate, 10 mm Tris-HCl, pH 7.6, 1 mm phenylmethylsulfonyl fluoride, 1 mmbenzamidine, 0.1 unit/ml α2-macroglobulin (Roche Molecular Biochemicals) at 2 × 108 cells/ml, filtered through a 0.2-μm filter (Gelman/Pall, Ann Arbor, MI), centrifuged for 30 min at 100,000 × g, and precleared with ethanol amine-blocked Affi-Gel 10 (Bio-Rad) for 30 min at 4 °C. EndoGlyx-1 was precipitated from lysates at a ratio of 2 μl of mAb H572 covalently coupled to Affi-Gel 10 (4.9 mg/ml of gel)/108lysed cells. The precipitate was washed three times with IP wash buffer (0.05% Triton X-100, 50 mm Tris-HCl, pH 8.4, 150 mm NaCl, 1 mm CaCl2, 1 mg/ml ovalbumin, 0.02% azid), washed two times with 250 mm NaCl, 25 mm Tris, pH 8.4, and eluted twice for 15 min at 55 °C, using 2 μl of nonreducing Laemmli sample buffer (6Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar)/1 μl of affinity matrix. The eluate was reduced with 50 mmdithiothreitol (Roche Molecular Biochemicals). For peptide analysis, purified protein was separated via SDS-polyacrylamide gel electrophoresis (PAGE) on a 6% gel and visualized by colloidal Coomassie staining (Invitrogen, Groningen, Netherlands). The EndoGlyx-1 bands were excised from the gel, incubated with trypsin solution (Promega, Madison, WI), and eluted peptides were subjected, after HPLC (Waters, Milford, MA) separation, to a 494 cLC ABI-Perkin-Elmer device. The peptides were desalted by ZipTip-procedure and subjected to mass spectroscopy analysis using a matrix-assisted laser disorption ionization-time of flight instrument (Voyager DE-STR, PerSeptive Biosystems/AB, Foster City, CA). The peptide samples were prepared using dihydroxybenzoic acid as matrix (7Christian S. Ahorn H. Koehler A. Eisenhaber F. Rodi H.P. Garin-Chesa P. Park J.E. Rettig W.J. Lenter M.C. J. Biol. Chem. 2001; 276: 7408-7414Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Out of the calibrated MS peptide map, a peak table list of the peptide-mass fingerprint was designed, omitting signals observed in the chemical background spectrum. The peak table list served as input data, and the MS-Fit software program was written by K. R. Clauser and P. Baker (School of Pharmacy, Department of Pharmacology Chemical Mass Spectrometry Facility, UCSF; rafael.ucsf.edu) was used for searching the SWISS Prot and NCBI protein data bases for sequence similarities and thus suggesting the identity of the protein. MS-Fit search results were checked regarding the molecular weight search (MOWSE) score (8Pappin D.J.C. Hojrup P. Bleasby A.J. Curr. Biol. 1993; 3: 327-332Abstract Full Text PDF PubMed Scopus (1420) Google Scholar), molecular mass (Da), pI, species, and percentage of masses matched. Transient transfection of HeLa-S3 cells plated in 60-mm tissue culture dishes (Becton Dickinson) was carried out with the transfection reagent FuGENE 6 (Roche Diagnostics) and expression vector pMH (Roche Diagnostics), containing the complete EndoGlyx-1 p125/p140 coding sequence or as empty vector. The cells were analyzed for antigen expression 48 h after transfection by immunoprecipitation of metabolically labeled cells with mAb H572 or control IgG1 essentially as described (7Christian S. Ahorn H. Koehler A. Eisenhaber F. Rodi H.P. Garin-Chesa P. Park J.E. Rettig W.J. Lenter M.C. J. Biol. Chem. 2001; 276: 7408-7414Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The SV Total RNA Isolation System (Promega) was used for total RNA isolation according to the manufacturer’s instructions. Oligo(dT)-cellulose (Life Technologies, Inc.) was taken for poly(A)+ RNA isolation as described (9Weller A. Isenmann S. Vestweber D. J. Biol. Chem. 1992; 267: 15176-15183Abstract Full Text PDF PubMed Google Scholar). For Northern blots, 1.5 μg of poly(A)+ RNA were electrophoresed on a 0.8% (w/v) agarose gel containing 20 mm MOPS, 5 mm sodium acetate, pH 6.6, and 1.11% formaldehyde. The RNA was blotted in 10× SSC (1.5m NaCl, 0.15 m sodium citrate, pH 7.0) on a Hybond N+ membrane (Amersham Pharmacia Biotech) for 16–20 h and UV-cross-linked. To detect EndoGlyx-1 p125/p140 mRNA, two polymerase chain reaction fragments, a 201-bp 5′ probe comprising positions +174 to +375 or a 290-bp central probe comprising positions +1124 to +1414 of EndoGlyx-1 cDNA, were used. Immunoprecipitation assays were performed as described (9Weller A. Isenmann S. Vestweber D. J. Biol. Chem. 1992; 267: 15176-15183Abstract Full Text PDF PubMed Google Scholar). For cell surface expression analysis, HUVECs (5 × 106 cells) were washed three times with cold phosphate-buffered saline on a cell culture plate and incubated with biotinylation buffer (20 mm HEPES, pH 7.45, 5 mm KCl, 130 mm NaCl, 0.8 mmMgCl2, 1 mm CaCl2, 0.5 mg/ml EZ link Sulfo-NHS-Biotin; Pierce) for 1 h at 4 °C. After removal of the reagent, the cells were washed three times with wash buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 1 mmMgCl2, 1 mm CaCl2) and lysed in 1% Triton X-100 lysis buffer for 1 h. The lysates were centrifuged for 15 min at 15,000 × g. EndoGlyx-1 was immunoprecipitated as described above and eluted with elution buffer (1% SDS, 100 mm NH4HCO3). Eluted proteins were separated by 6% SDS-PAGE and transferred to nitrocellulose (10Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44939) Google Scholar). The nitrocellulose membranes were subsequently incubated, after blocking, with ExtrAvidin-peroxidase conjugates diluted 1:5,000 (Sigma) for 1 h at room temperature. Signals were detected after incubation with ECL reagent and exposure to Hyperfilm ECL (Amersham Pharmacia Biotech). For glycosylation studies, EndoGlyx-1 was immunoprecipitated from cell surface biotinylated HUVECs as described before, using AffiGel 10-bound mAb H572 (4.9 mg/ml). After washing four times with IP wash buffer, the immunoprecipitates were split into four equal aliquots, resuspended in 40 μl of reaction buffer (50 mm sodium phosphate, pH 7.0) and incubated for 16 h either (a) with 1 unit ofN-glycosidase F from Flavobacterium meningosepticum or (b) with 10 milliunits of sialidase from Arthrobacter ureafaciens, 0.5 milliunits ofO-glycosidase from Diplococcus pneumoniae, or (c) with 1 unit of N-glycosidase F, 10 milliunits of sialidase and 0.5 milliunits O-glycosidase (Roche Diagnostics) or (d) in the absence of enzyme (mock treatment). Subsequent washing, elution in Laemmli sample buffer, and immunoblotting followed by ECL detection were performed as described before. Data base mining and sequence analyses were performed as essentially outlined in Ref. 7Christian S. Ahorn H. Koehler A. Eisenhaber F. Rodi H.P. Garin-Chesa P. Park J.E. Rettig W.J. Lenter M.C. J. Biol. Chem. 2001; 276: 7408-7414Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar and are described in great detail at the supplementary World Wide Web pagemendel.imp.univie.ac.at/SEQUENCES/endoglyx/. To compare vascular expression of EndoGlyx-1 with VE-cadherin, a protein complex constituting an antigen specific for the intercellular junctions of endothelium, serial frozen sections obtained from normal human tissues were tested with mAb H572 specific for human EndoGlyx-1 or with mAb TEA-1 specific for human VE-cadherin by the avidin-biotin immunoperoxidase method. The results of these studies showed that VE-cadherin staining concentrated, as described previously (11Navarro P. Ruco L. Dejana E. J. Cell Biol. 1998; 140: 1475-1484Crossref PubMed Scopus (246) Google Scholar), at sites of vascular cell-cell contact (Fig.1, right panel), whereas EndoGlyx-1 showed a diffuse, continuous localization along the endothelial lining of the blood vessels (Fig. 1, left panel). The H572 antigen-positive cell line EA.hy926, obtained by fusion of a human umbilical vein endothelial cell with the transformed tumor cell line A549 (12Edgell C.J. McDonald C.C. Graham J.B. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3734-3737Crossref PubMed Scopus (1360) Google Scholar), was selected as a source for EndoGlyx-1 purification. Protein was purified from detergent extracts of 3 × 109 cells using mAb H572 covalently coupled to Affi-Gel 10 as an immunoaffinity matrix. To analyze the quality of the preparation, an aliquot of the eluted material was separated on a reducing SDS-polyacrylamide gel, and four major protein bands of 110 kDa (p110 subunit), 125 kDa (p125 subunit), 140 kDa (p140 subunit), and 200 kDa (p200 subunit) were visualized by silver staining (Fig.2, arrows). This is in agreement with the EndoGlyx-1 subunit pattern described previously for human umbilical vein endothelial cell cultures (1Sanz-Moncasi M.P. Garin-Chesa P. Stockert E. Jaffe E.A. Old L.J. Rettig W.J. Lab. Invest. 1994; 71: 366-373PubMed Google Scholar). The remaining eluate was separated under reducing conditions on a quantitative SDS-PAGE and yielded 0.1–0.5 pmol of the four specific protein species as determined by colloidal Coomassie staining. Gel fragments containing the EndoGlyx-1 subunits were subjected to a trypsin digest, the eluted peptides were separated by HPLC and analyzed by matrix-assisted laser desorption ionization-time of flight. Subsequent peptide-mass fingerprint analysis gave rise to the molecular masses of nine peptides derived from the p125 subunit (Fig. 3,dashed lines) and fourteen peptides from the p140 subunit (Fig. 3, underlined) matching with the same entry in the public data base of a full-length cDNA (EMBL data base, accession number AK023527) encoding an “unnamed protein” from human placenta. The complete cDNA comprises 3,828 bp and harbors a single open reading frame of 2,847 bp, coding for a protein of 949 amino acids with a predicted molecular mass 104.4 kDa (Fig. 3). The results obtained from peptide analysis of the two remaining EndoGlyx-1 subunits, p110 and p200 (Fig. 2, open arrows), were not interpretable, and their analysis is pending.Figure 3cDNA and deduced amino acid sequence of human EndoGlyx-1 p125/p140. The sequence of full-length cDNA was identified as an entry for an unnamed protein in the public data base using a matrix-assisted laser disorption ionization-time of flight-based peptide mass fingerprint analysis resulting in molecular mass detection of 9 tryptic peptides derived from the p125 protein species (dashed lines) and 14 tryptic fragments derived from the p140 protein species (underlined amino acids). The putative N-terminal signal sequence is boxed, and the potential polyadenylation signal is double underlined. The 11 predicted N-linked glycosylation sites are marked bycircles.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Bioinformatic analysis identifies EndoGlyx-1 p125/p140 as a typical precursor sequence for a eukaryotic protein resulting evolutionarily from exon shuffling (Fig. 4 A). First, there is a putative N-terminal sequence of 23 amino acids similar to a signal leader peptide for translocation into the lumen of the endoplasmatic reticulum. Next there are two major globular segments, an N-terminal EMI domain and a C-terminal C1q-like domain spaced by a central coiled-coil rich region. The EMI domain comprises the amino acids 55–130 including 7 cysteine residues (C1–C7), which potentially form two subdomains (C1–C4 and C5–C7) as determined by sequence similarity searches in data bases by PSI-BLAST tools (13Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60233) Google Scholar). This is followed by a coiled-coil region comprising more than half of the polypeptide (547 amino acids), composed of five subdomains with a strong prediction (amino acids 323–346, 374–481, 532–553, 554–594, and 687–713) and two subdomains with a weak prediction (amino acids 166–188 and 290–313) for coiled-coil formation. These are interrupted by noncoiled regions, allowing structural flexibility. A short cluster (amino acids 779–806) rich in positively charged amino acids separates the coiled-coil region from the C1q-like domain-containing consensus sequences, common in proteins known to interact with glycosaminoglycans such as heparin and heparan sulfate (14Hileman R.E. Fromm J.R. Weiler J.M. Linhardt R.J. Bioessays. 1998; 20: 156-167Crossref PubMed Scopus (519) Google Scholar). There are two well conserved regions in the C1q domain: an aromatic motif (amino acids 844–873) as defined by the Prosite C1q pattern is located within the first part of the domain (amino acids 827–873), the other conserved region is near the extreme C terminus (amino acids 912–947). A C-terminal C1q domain is a common feature of the C1q-family of proteins such as the A, B, and C chains of the complement C1q protein (15Sellar G.C. Blake D.J. Reid K.B. Biochem. J. 1991; 274: 481-490Crossref PubMed Scopus (176) Google Scholar, 16Reid K.B. Immunology. 1985; 55: 185-196PubMed Google Scholar), the type VIII and X collagens (17Muragaki Y. Jacenko O. Apte S. Mattei M.G. Ninomiya Y. Olsen B.R. J. Biol. Chem. 1991; 266: 7721-7727Abstract Full Text PDF PubMed Google Scholar, 18Reichenberger E. Beier F. LuValle P. Olsen B.R. von der Mark K. Bertling W.M. FEBS Lett. 1992; 311: 305-310Crossref PubMed Scopus (47) Google Scholar), the recently identified and broadly expressed extracellular matrix glycoprotein EMILIN (19Doliana R. Mongiat M. Bucciotti F. Giacomello E. Deutzmann R. Volpin D. Bressan G.M. Colombatti A. J. Biol. Chem. 1999; 274: 16773-16781Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), and a large protein of homomultimeric structure called multimerin (20Hayward C.P. Hassell J.A. Denomme G.A. Rachubinski R.A. Brown C. Kelton J.G. J. Biol. Chem. 1995; 270: 18246-18251Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) found in the α-granules of platelets and Waibel-Palade bodies of endothelium. The N-terminal EMI domain of EndoGlyx-1 p125/p140, between amino acids 55 and 130, shares 38% sequence identity with the corresponding region in the precursor protein of human EMILIN-1 (EMBL data base accession number AF088916) and 34% sequence identity with the protein precursor of human multimerin (SwissProt data base,Q13201), respectively (Fig. 4 B). The positions of six (of 7) cysteine residues critical for disulfide bridges are conserved among the three proteins, suggesting a shared three-dimensional structure in this segment. Additionally, a significant level of conservation was found for the C-terminal C1q-like domain, between amino acids 818 and 949, with 26% sequence identity with the respective region in the human EMILIN-1 precursor protein and 32% sequence identity with the human multimerin precursor protein (Fig. 4 B), indicating a shared functional feature provided by this segment, such as the formation of homomultimers, which was demonstrated for several members of the TNF/C1q superfamily (21Mongiat M. Mungiguerra G. Bot S. Mucignat M.T. Giacomello E. Doliana R. Colombatti A. J. Biol. Chem. 2000; 275: 25471-25480Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Additional sequence analysis details can be viewed atmendel.imp.univie.ac.at.SEQUENCES/endoglyx/. Northern blot analysis of poly(A)+ RNA isolated from the EndoGlyx-1-expressing cell line EA.hy926 revealed a single major transcript of about 3,800 bp (Fig. 5) for two distinct radioactive cDNA probes, matching either the 5′ end or the central region of the open reading frame. Thus, p125 and p140 (Fig.2, filled arrows) are encoded by the same mRNA, and detection of a single mRNA transcript by hybridization with two widely spaced probes indicates that the molecular mass difference of the two encoded subunits is more likely due to post-translational events than being mediated by alternative splicing of a common mRNA precursor. To determine the EndoGlyx-1 subunit composition, detergent extracts of metabolically labeled human umbilical vein endothelial cells were purified with mAb H572, and eluted material was separated either by nonreducing or reducing polyacrylamide gel electrophoresis. EndoGlyx-1 was identified by mAb H572 under nonreducing conditions as a single protein species with a molecular mass of ∼500 kDa that barely entered the separating gel (Fig.6 A). Reduction previous to SDS gel electrophoresis resulted in detection of three distinct EndoGlyx-1 species, p125, p140, and p200, indicating a high molecular mass complex cross-linked through intermolecular disulfide bridges (Fig.6 B). To test this, the 500-kDa EndoGlyx-1 protein and a respective control were cut from the nonreduced polyacrylamide gel and re-electrophoresed under reducing conditions on a second gel (Fig. 6,A and B, asterisks), resulting in a protein pattern that appeared to be identical to that of the directly reduced H572 precipitates. This indicates that EndoGlyx-1 is composed of at least three disulfide-bonded protein subunits: p125, p140, and p200 (Fig. 6 B, arrows). In contrast, no major reduction-dependent shift in electrophoretic mobility was observed for proteins in TEA-1 immunoprecipitates, which is in agreement with the reported noncovalent association of VE-cadherin with catenins (22Lampugnani M.G. Corada M. Caveda L. Breviario F. Ayalon O. Geiger B. Dejana E. J. Cell Biol. 1995; 129: 203-217Crossref PubMed Scopus (497) Google Scholar). Transient transfection studies with the EndoGlyx-1-negative cell line HeLa-S3 were used to confirm that the putative cDNA indeed specifies the antigen defined by mAb H572. Unlike the parental or mock transfected cells, which did not react with mAb H572 (data not shown), H572 antigen was immunoprecipitated from detergent lysates of metabolically labeled transfectants generated with an expression vector containing full-length EndoGlyx-1 p125/p140 cDNA, revealing under nonreducing conditions a high molecular mass species of ∼500 kDa, which gave rise after reduction to a major protein band of 125 kDa and to two less prominent species of about 110 and 200 kDa (Fig.6 C, arrowheads). The lack of the p140 species in the transfectants could be due to alternate splicing or differential protein processing. Thus, recombinant EndoGlyx-1 assembles into a disulfide-bonded complex with subunits similar in size to those found in the endogenous EndoGlyx-1 of HUVECs and EA.hy926. To investigate whether the EndoGlyx-1 antigen is expressed on the luminal side of intact blood vessel endothelium, mAb H572 or a corresponding isotype-matched control antibody was infused intraluminally into the umbilical cord vein. To detect bound antibody, frozen sections generated after antibody perfusion were incubated with a biotinylated horse anti-mouse IgG followed by the avidin-biotin complex and diaminobenzidine as substrate for the reaction. Our results show a strong immunoreactivity of mAb H572 with the endothelial lining (Fig.7 A), whereas no staining was detected for the isotype control (data not shown). This indicates that at least a sizable fraction of EndoGlyx-1 is exposed on the luminal surface of human blood vessels. To discriminate between intra- and extracellular distribution of the distinct EndoGlyx-1 subunits, lysates of cell surface biotinylated human umbilical vein endothelial cells were generated and tested by immunoprecipitation with mAb H572 immobilized to Affi-Gel 10. The bound antigen was eluted, separated by reducing SDS-PAGE, and transferred to nitrocellulose, and the subsequent avidin-peroxidase conjugate-based ECL reaction gave rise to the detection of four biotinylated subunits: p110, p125, p140, and p200 (Fig. 7 B). Restriction of biotin label to antigens located on the cell surface was confirmed by the exclusive detection of surface exposed VE-cadherin and not of the intracellular attached catenins, which co-purify in mAb TEA-1 immunoprecipitates (Figs. 7 B and 6 B). This result indicates that EndoGlyx-1 is composed of four cell surface exposed protein subunits. The absence of the p110 species in metabolic studies indicates either a lower content of cysteine/methionine residues or a slower metabolic turnover rate. To investigate the possibility that the different EndoGlyx-1 subunits emerge from a differentially glycosylated core protein, we examined the glycosylation pattern of the endogenous complex. Because direct detection of subunits was hampered by the fact that mAb H572 is not usable in Western blot analysis, we first immunopurified EndoGlyx-1 from lysates of cell surface biotinylated human umbilical vein endothelial cells with immobilized mAb H572. Pretreatment of matrix bound antigen withN-glycosidase F led to a reduction in the size of the three smaller EndoGlyx-1 subunits of ∼15 kDa. This is consistent with the presence of 11 Asn-Xaa-(Ser/Thr) consensus sites in the primary sequence of the identified p125/p140. Additionally, removal ofO-linked carbohydrates by combined treatment withO-glycosidase and sialidase resulted in a collective shift in electrophoretic mobility but less pronounced (∼10 kDa), indicating a similar extent of enzymatically sensitive N- and O-glycosylation of the three smaller EndoGlyx-1 subunits (Fig. 8). This was further supported by the combined removal of N- and O-linked carbohydrates, resulting in an additive (∼25 kDa) and simultaneous shift in the apparent molecular mass of the proteins. The loss in signal intensity after N-glycosidase F treatment could be due to a removal of biotin label immobilized to N-bound sugars. We have cloned the gene coding for the human EndoGlyx-1 subunits p125 and p140 based on two lines of evidence. First, 17 peptide sequences found in the p125 and/or p140 proteins are present in the putative EndoGlyx-1 cDNA. Second, transfection of this cDNA into antigen-negative HeLa-S3 cells results in expression of a protein recognized by cognate mAb H572. The apparent molecular size of transfected recombinant EndoGlyx-1 protein under reducing and nonreducing conditions is in good agreement with the observed size of the endogenous antigen. The present study is not the only evidence linking EndoGlyx-1 expression to the endothelium. Rather, an independent line of investigation, aimed at dissecting comprehensive gene expression profiles for blood vessels in cancer versus normal tissues with the SAGE method, has implicated an EndoGlyx-1-specific tag sequence (23St Croix B. Rago C. Velculescu V. Traverso G. Romans K.E. Montgomery E. Lal A. Riggins G.J. Lengauer C. Vogelstein B. Kinzler K.W. Science. 2000; 289: 1197-1202Crossref PubMed Scopus (1650) Google Scholar). This study identified a SAGE tag sequence designated PEM87 among 93 tagged transcripts elevated at least 20-fold in normal and tumor endothelium compared with other nonendothelial cell types. The PEM87 tag matches the 3′-untranslated region of a full-length cDNA transcript coding for an unnamed protein in human placenta and three oriented expressed sequence tags deposited in the public data base. We show here that PEM87 is the EndoGlyx-1 p125/140 gene. Taking together the EndoGlyx-1 expression data generated with mAb H572 (1Sanz-Moncasi M.P. Garin-Chesa P. Stockert E. Jaffe E.A. Old L.J. Rettig W.J. Lab. Invest. 1994; 71: 366-373PubMed Google Scholar) and the RNA expression data derived from SAGE, a consistent picture of EndoGlyx-1 expression in endothelium emerges. EndoGlyx-1 represents a new member of the unique group of EMILIN-like proteins. This group is shared by two Elastin microfibril interface-located proteins, designated as EMILIN-1 and EMILIN-2, and multimerin, a massive, soluble protein found to be expressed in platelets, megakaryocytes, and vascular endothelium (24Hayward C.P. Song Z. Zheng S. Fung R. Pai M. Masse J.M. Cramer E.M. Blood. 1999; 94: 1337-1347Crossref PubMed Google Scholar). Unlike EndoGlyx-1, EMILINs are expressed in connective tissues of blood vessels, skin, heart, lung, kidney, and cornea (25Colombatti A. Bressan G.M. Volpin D. Castellani I. Coll. Relat. Res. 1985; 5: 181-191Crossref PubMed Scopus (11) Google Scholar, 26Colombatti A. Bressan G.M. Castellani I. Volpin D. J. Cell Biol. 1985; 100: 18-26Crossref PubMed Scopus (40) Google Scholar, 27Colombatti A. Poletti A. Bressan G.M. Carbone A. Volpin D. Coll. Relat. Res. 1987; 7: 259-275Crossref PubMed Scopus (24) Google Scholar). EndoGlyx-1 p125/p140 diverges in domain architecture from EMILIN-1 and multimerin by a short cluster of charged amino acids located in the transition between the coiled-coil and the C1q-like domains. EMILIN-1 shows two leucine zipper motifs followed by a collagenous domain at this position, and multimerin contains an EGF-like domain instead. This region is dominated by basic amino acids (10 of 27 residues) arranged in a sequence similar to consensus motifs found in heparin binding proteins, such as von Willebrand factor (28Sobel M. Soler D.F. Kermode J.C. Harris R.B. J. Biol. Chem. 1992; 267: 8857-8862Abstract Full Text PDF PubMed Google Scholar), which are known to be important for ionic interactions with glucosaminoglycans, such as heparin and heparan sulfate (14Hileman R.E. Fromm J.R. Weiler J.M. Linhardt R.J. Bioessays. 1998; 20: 156-167Crossref PubMed Scopus (519) Google Scholar). Because glucosaminoglycans provide antithrombotic activities to endothelium through activation of the coagulation protease inhibitor ATIII, it will be interesting to test whether EndoGlyx-1 is capable of recruiting glucosaminoglycans or related structures to the endothelial cell surface. The molecular organization of EMILINs and multimerin is highly characteristic; both form homotrimers and larger homomultimers via interchain disulfide bonds, giving rise in the case of EMILINs to high molecular aggregates deposited as a fine network in the extracellular matrix (29Colombatti A. Bonaldo P. Volpin D. Bressan G.M. J. Biol. Chem. 1988; 263: 17534-17540Abstract Full Text PDF PubMed Google Scholar), whereas multimerin oligomers assembled from a precursor protein, prepromultimerin, are stored in secretory granules of platelets and endothelium (24Hayward C.P. Song Z. Zheng S. Fung R. Pai M. Masse J.M. Cramer E.M. Blood. 1999; 94: 1337-1347Crossref PubMed Google Scholar). Exploiting the yeast two-hybrid system with EMILIN-1 indicates that trimerization is initiated by the C1q-like domain followed by a subsequent quarternary assembly mediated by intermolecular disulfide bridges, which was further supported by the fact that a deletion mutant of EMILIN-1, lacking the C1q-like domain, was incapable in multimer formation (21Mongiat M. Mungiguerra G. Bot S. Mucignat M.T. Giacomello E. Doliana R. Colombatti A. J. Biol. Chem. 2000; 275: 25471-25480Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Our analysis shows that the C-terminal portion of EndoGlyx-1 p125/140 consists of an C1q-like domain homologous to EMILIN-1 and multimerin, with 26 and 32% amino acid sequence identity, respectively, which is in accordance with the oligomerization of the different EndoGlyx-1 subunits mediated by intermolecular disulfide bridges. Analysis of tryptic peptides revealed that the p125 and p140 species originate from the same EndoGlyx-1 mRNA precursor. A likely explanation for the difference in molecular mass of the p125 and p140 subunits is proteolytic processing, as has been shown for platelet multimerin, which, expressed as a 170-kDa glycosylated precursor protein form, is proteolytically converted into a stable 155-kDa protein subunit (30Hayward C.P. Warkentin T.E. Horsewood P. Kelton J.G. Blood. 1991; 77: 2556-2560Crossref PubMed Google Scholar, 31Hayward C.P. Bainton D.F. Smith J.W. Horsewood P. Stead R.H. Podor T.J. Warkentin T.E. Kelton J.G. J. Clin. Invest. 1993; 91: 2630-2639Crossref PubMed Scopus (61) Google Scholar). The size difference of the p125 and p140 polypeptide backbone is further supported by enzymatic removal ofN- and O-linked carbohydrates, which resulted in an almost identical reduction in the molecular mass of both species excluding the possibility of a differentially glycosylated common core protein. Because sequence analysis of EndoGlyx-1 p125/p140 cDNA revealed no potential motif for membrane localization, cell surface exposure on endothelium is putatively mediated through association with a plasma membrane-bound and cell surface exposed molecular species. In this context, the as yet unidentified and cell surface exposed subunits p110 and p200 of EndoGlyx-1 are potential candidates for membrane targeting of the complex. However, how the four identified covalently linked subunits contribute to the molecular composition of the individual EndoGlyx-1 complexes awaits elucidation. In conclusion, the surprisingly restricted tissue distribution and unique domain architecture of EndoGlyx-1 implies a potentially important role in vasculogenesis, angiogenesis, or hemostasis. Future investigations focusing on the biological function will benefit from availability of mAb H572 and recombinant EndoGlyx-1 protein. The expert technical assistance of Claudia Eiberle, Anita Fischbach, Petra Lahm, Hans-Peter Rodi, Karin Ruehe, Alexandra Schlegel, and Kai Zuckschwerdt is gratefully acknowledged. We thank Dr. Andreas Koehler and Dr. Karsten Quast for valuable support. We are grateful to Dr. Lloyd. J. Old and Dr. Elisabeth Stockert for contributions and suggestions. We acknowledge the support of Prof. Klaus Pfizenmaier throughout the work.

An evolutionarily conserved complex, the Mediator, is a key partner of most transcriptional activators in eukaryotes [1.Lewis B.A. Reinberg D. The mediator coactivator complex: functional and physical roles in transcriptional regulation.J. Cell Sci. 2003; 116: 3667-3675Crossref PubMed Scopus (119) Google Scholar]. Gal11 and ARC105 are Mediator subunits linking specific transcriptional activators to the basal transcription apparatus [2.Kato Y. Habas R. Katsuyama Y. Naar A.M. He X. A component of the ARC/Mediator complex required for TGF beta/Nodal signalling.Nature. 2002; 418: 641-646Crossref PubMed Scopus (129) Google Scholar, 3.Sakurai H. Fukasawa T. Functional connections between mediator components and general transcription factors of Saccharomyces cerevisiae.J. Biol. Chem. 2000; 275: 37251-37256Crossref PubMed Scopus (27) Google Scholar].Gal11 is part of the proposed "Gal11 module", in which it has been suggested to be a direct target of several activators [4.Lee Y.C. Park J.M. Min S. Han S.J. Kim Y.J. An activator binding module of yeast RNA polymerase II holoenzyme.Mol. Cell Biol. 1999; 19: 2967-2976Crossref PubMed Scopus (133) Google Scholar]. However, the existence of mammalian Gal11-like proteins has been debated [5.Lu Z. Ansari A.Z. Lu X. Ogirala A. Ptashne M. A target essential for the activity of a nonacidic yeast transcriptional activator.Proc. Natl. Acad. Sci. USA. 2002; 99: 8591-8596Crossref PubMed Scopus (22) Google Scholar, 6.Boube M. Joulia L. Cribbs D.L. Bourbon H.M. Evidence for a mediator of RNA polymerase II transcriptional regulation conserved from yeast to man.Cell. 2002; 110: 143-151Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar]. Here we show that three families of sequence segments, corresponding to amino-terminal fungal/plant Gal11-like sequences, animal ARC105, and plant CREB binding protein (CBP)-like sequences, are homologous to the KIX-domain of CBP/p300 co-activators (see http://mendel.imp.univie.ac.at/SEQUENCES/GACKIX for details). These segments are members of the new GACKIX-domain superfamily. Sequence analysis suggests that Gal11- and ARC105-like proteins are homologs and have the same function in Mediator complexes.

The ability to perceive noxious stimuli is critical for an animal's survival in the face of environmental danger, and thus pain perception is likely to be under stringent evolutionary pressure. Using a neuronal-specific RNAi knock-down strategy in adult Drosophila, we recently completed a genome-wide functional annotation of heat nociception that allowed us to identify α2δ3 as a novel pain gene. Here we report construction of an evolutionary-conserved, system-level, global molecular pain network map. Our systems map is markedly enriched for multiple genes associated with human pain and predicts a plethora of novel candidate pain pathways. One central node of this pain network is phospholipid signaling, which has been implicated before in pain processing. To further investigate the role of phospholipid signaling in mammalian heat pain perception, we analysed the phenotype of PIP5Kα and PI3Kγ mutant mice. Intriguingly, both of these mice exhibit pronounced hypersensitivity to noxious heat and capsaicin-induced pain, which directly mapped through PI3Kγ kinase-dead knock-in mice to PI3Kγ lipid kinase activity. Using single primary sensory neuron recording, PI3Kγ function was mechanistically linked to a negative regulation of TRPV1 channel transduction. Our data provide a systems map for heat nociception and reinforces the extraordinary conservation of molecular mechanisms of nociception across different species.

Abstract A model of human bastocyst formed from stem cells (blastoid) would support scientific and medical advances. However, its predictive power will depend on the ability to faithfully, efficiently, and timely recapitulate the sequences of blastocyst specification, morphogenesis, and patterning, and form cells reflecting the blastocyst stage. Here we show that naïve human pluripotent stem cells cultured in PXGL conditions and then triply inhibited for the Hippo, transforming growth factor- β (TGF-β), and extracellular signal-regulated kinase (ERK) pathways efficiently form blastoids (&gt;70%). Within 4 days, blastoids sequentially produce analogs of the blastocyst-stage trophoblast and epiblast, followed by the formation of analogs of the primitive endoderm and the polar trophoblasts. This results in the formation of transcriptional analogs of the blastocyst (&gt;96%) and a minority of postimplantation analogs (e.g., amnion, mesoderm). Blastoids efficiently form the embryonic-abembryonic axis marked by the maturation of the polar region (NR2F2+), which acquires the specific potential to directionally attach to hormonally stimulated endometrial cells, as in utero . Such a human blastoid is a scalable, versatile, and ethical model to study human development and implantation in vitro .

Tumor progression is associated with invasiveness and metastatic potential. The special AT-rich binding protein 1 (SATB1) has been identified as a key factor in the progression of breast cancer cells to a malignant phenotype and is associated with progression of human tumors. In normal development, SATB1 coordinates gene expression of progenitor cells by functioning as a genome organizer. In contrast to progenitor and tumor cells, SATB1 expression in nontransformed cells is not compatible with proliferation. Here we show that SATB1 expression in mouse embryonic fibroblasts induces cell cycle arrest and senescence that is associated with elevated p16 protein levels. Deletion of p16 overcomes the SATB1-induced senescence. We further provide evidence for an interaction of SATB1 with the retinoblastoma (RB)/E2F pathway downstream of p16. A combined deletion of the RB proteins, RB, p107 and p130 (triple-mutant; TM), prevents SATB1-induced G1 arrest, which is restored upon the reintroduction of RB into SATB1-expressing TM fibroblasts. SATB1 interacts with the E2F/RB complex and regulates the cyclin E promoter in an E2F-dependent manner. These findings demonstrate that p16 and the RB/E2F pathway are critical for SATB1-induced cell cycle arrest. In the absence of p16, SATB1 causes anchorage-independent growth and invasive phenotype in fibroblasts. Our data illustrate that p16 mutations collaborate with the oncogenic activity of SATB1. Consistent with our finding, a literature survey shows that deletion of p16 is generally associated with SATB1 expressing human cell lines and tumors.

When programmed meiotic DNA double-strand breaks (DSBs) undergo recombinational repair, genetic crossovers (COs) may be formed. A certain level of this is required for the faithful segregation of chromosomes, but the majority of DSBs are processed toward a safer alternative, namely noncrossovers (NCOs), via nonreciprocal DNA exchange. At the crossroads between these two DSB fates is the Msh4-Msh5 (MutSγ) complex, which stabilizes CO-destined recombination intermediates and members of the Zip3/RNF212 family of RING finger proteins, which in turn stabilize MutSγ. These proteins function in the context of the synaptonemal complex (SC) and mainly act on SC-dependent COs. Here we show that in the SC-less ciliate Tetrahymena, Zhp3 (a protein distantly related to Zip3/RNF212), together with MutSγ, is responsible for the majority of COs. This activity of Zhp3 suggests an evolutionarily conserved SC-independent strategy for balancing CO:NCO ratios. Moreover, we report a novel meiosis-specific protein, Sa15, as an interacting partner of Zhp3. Sa15 forms linear structures in meiotic prophase nuclei to which Zhp3 localizes. Sa15 is required for a wild-type level of CO formation. Its linear organization suggests the existence of an underlying chromosomal axis that serves as a scaffold for Zhp3 and other recombination proteins.

Jagunal homolog 1 (JAGN1) has been identified as a critical regulator of neutrophil biology in mutant mice and rare-disease patients carrying JAGN1 mutations. Here, we report that Jagn1 deficiency results in alterations in the endoplasmic reticulum (ER) of antibody-producing cells as well as decreased antibody production and secretion. Consequently, mice lacking Jagn1 in B cells exhibit reduced serum immunoglobulin (Ig) levels at steady state and fail to mount an efficient humoral immune response upon immunization with specific antigens or when challenged with viral infections. We also demonstrate that Jagn1 deficiency in B cells results in aberrant IgG N-glycosylation leading to enhanced Fc receptor binding. Jagn1 deficiency in particular affects fucosylation of IgG subtypes in mice as well as rare-disease patients with loss-of-function mutations in JAGN1. Moreover, we show that ER stress affects antibody glycosylation. Our data uncover a novel and key role for JAGN1 and ER stress in antibody glycosylation and humoral immunity in mice and humans.

Abstract After fertilization, the sperm and egg contribute unequally to the newly formed zygote. While the sperm contributes mainly paternal DNA, the egg provides both maternal DNA and the bulk of the future embryonic cytoplasm. Most embryonic processes (like the onset of zygotic transcription) depend on maternally-provided cytoplasmic components, and it is largely unclear whether paternal components besides the centrosome play a role in the regulation of early embryogenesis. Here we report a reciprocal zebrafish-medaka hybrid system as a powerful tool to investigate paternal vs. maternal influence during early development. By combining expression of zebrafish Bouncer on the medaka egg with artificial egg activation, we demonstrate the in vitro generation of paternal zebrafish x maternal medaka (reripes) hybrids. These hybrids complement the previously established paternal medaka x maternal zebrafish (latio) hybrids (Herberg et al., 2018). As proof of concept, we investigated maternal vs. paternal control of zygotic genome activation (ZGA) timing using this reciprocal hybrid system. RNA-seq analysis of the purebred fish species and hybrids revealed that the onset of ZGA is primarily governed by the egg. Overall, our study establishes the reciprocal zebrafish-medaka hybrid system as a versatile tool to dissect parental control mechanisms during early development.

Abstract Mutations in ARID1B , a member of the mSWI/SNF complex, cause severe neurodevelopmental phenotypes with elusive mechanisms in humans. The most common structural abnormality in the brain of ARID1B patients is agenesis of the corpus callosum (ACC). This condition is characterized by a partial or complete absence of the corpus callosum (CC), an interhemispheric white matter tract that connects distant cortical regions. Using human neural organoids, we identify a vulnerability of callosal projection neurons (CPNs) to ARID1B haploinsufficiency, resulting in abnormal maturation trajectories and dysregulation of transcriptional programs of CC development. Through a novel in vitro model of the CC tract, we demonstrate that ARID1B mutations reduce the proportion of CPNs capable of forming long-range projections, leading to structural underconnectivity phenotypes. Our study uncovers new functions of the mSWI/SNF during human corticogenesis, identifying cell-autonomous defects in axonogenesis as a cause of ACC in ARID1B patients. Abstract Figure Graphical abstract Human callosal projection neurons are vulnerable to ARID1B haploinsufficiency. (Top) During healthy development, callosal projection neurons (CPNs) project long interhemispheric axons, forming the corpus callosum (CC) tract, which can be modeled in vitro . (Bottom) In ARID1B patients, transcriptional dysregulation of genetic programs of CC development reduces the formation of long-range projections from CPNs, causing CC agenesis in vivo and underconnectivity phenotypes in vitro .

Ascl1 and Ngn2, closely related proneural transcription factors, are able to convert mouse embryonic stem cells into induced neurons. Despite their similarities, these factors elicit only partially overlapping transcriptional programs, and it remains unknown whether cells are converted via distinct mechanisms. Here we show that Ascl1 and Ngn2 induce mutually exclusive side populations by binding and activating distinct lineage drivers. Furthermore, Ascl1 rapidly dismantles the pluripotency network and installs neuronal and trophoblast cell fates, while Ngn2 generates a neural stem cell-like intermediate supported by incomplete shutdown of the pluripotency network. Using CRISPR-Cas9 knockout screening, we find that Ascl1 relies more on factors regulating pluripotency and the cell cycle, such as Tcf7l1. In the absence of Tcf7l1, Ascl1 still represses core pluripotency genes but fails to exit the cell cycle. However, overexpression of Cdkn1c induces cell cycle exit and restores the generation of neurons. These findings highlight that cell type conversion can occur through two distinct mechanistic paths, even when induced by closely related transcription factors.

Mutations in ARID1B, a member of the mSWI/SNF complex, cause severe neurodevelopmental phenotypes with elusive mechanisms in humans. The most common structural abnormality in the brain of ARID1B patients is agenesis of the corpus callosum (ACC), characterized by the absence of an interhemispheric white matter tract that connects distant cortical regions. Here, we find that neurons expressing SATB2, a determinant of callosal projection neuron (CPN) identity, show impaired maturation in ARID1B+/− neural organoids. Molecularly, a reduction in chromatin accessibility of genomic regions targeted by TCF-like, NFI-like, and ARID-like transcription factors drives the differential expression of genes required for corpus callosum (CC) development. Through an in vitro model of the CC tract, we demonstrate that this transcriptional dysregulation impairs the formation of long-range axonal projections, causing structural underconnectivity. Our study uncovers new functions of the mSWI/SNF during human corticogenesis, identifying cell-autonomous axonogenesis defects in SATB2+ neurons as a cause of ACC in ARID1B patients.

BackgroundThe COVID-19 pandemic was largely driven by genetic mutations of SARS-CoV-2, leading in some instances to enhanced infectiousness of the virus or its capacity to evade the host immune system. To closely monitor SARS-CoV-2 evolution and resulting variants at genomic-level, an innovative pipeline termed SARSeq was developed in Austria.AimWe discuss technical aspects of the SARSeq pipeline, describe its performance and present noteworthy results it enabled during the pandemic in Austria.MethodsThe SARSeq pipeline was set up as a collaboration between private and public clinical diagnostic laboratories, a public health agency, and an academic institution. Representative SARS-CoV-2 positive specimens from each of the nine Austrian provinces were obtained from SARS-CoV-2 testing laboratories and processed centrally in an academic setting for S-gene sequencing and analysis.ResultsSARS-CoV-2 sequences from up to 2,880 cases weekly resulted in 222,784 characterised case samples in January 2021-March 2023. Consequently, Austria delivered the fourth densest genomic surveillance worldwide in a very resource-efficient manner. While most SARS-CoV-2 variants during the study showed comparable kinetic behaviour in all of Austria, some, like Beta, had a more focused spread. This highlighted multifaceted aspects of local population-level acquired immunity. The nationwide surveillance system enabled reliable nowcasting. Measured early growth kinetics of variants were predictive of later incidence peaks.ConclusionWith low automation, labour, and cost requirements, SARSeq is adaptable to monitor other pathogens and advantageous even for resource-limited countries. This multiplexed genomic surveillance system has potential as a rapid response tool for future emerging threats.

Summary Mitochondrial ATP production is essential for development, yet the mechanisms underlying the continuous increase in mitochondrial activity during embryogenesis remain elusive. Using zebrafish as a model system for vertebrate development, we comprehensively profile mitochondrial activity, morphology, metabolome, proteome and phospho-proteome as well as respiratory chain enzymatic activity. Our data show that the increase in mitochondrial activity during embryogenesis does not require mitochondrial biogenesis, is not limited by metabolic substrates at early stages, and occurs without an increase in the abundance of respiratory chain complexes or their in vitro activity. Our analyses pinpoint a previously unexplored increase in mitochondrial-ER association during early stages in combination with changes in mitochondrial morphology at later stages as possible contributors to the rise in mitochondrial activity during embryogenesis. Overall, our systematic profiling of the molecular and morphological changes to mitochondria during embryogenesis provides a valuable resource for further studying mitochondrial function during embryogenesis.

The first amino acid of a protein has an important influence on its metabolic stability. A number of ubiquitin ligases contain binding domains for different amino-terminal residues of their substrates, also known as N-degrons, thereby mediating turnover. This review summarizes, in an exemplary way, both older and more recent findings that unveil how destabilizing amino termini are generated. In most cases, a step of proteolytic cleavage is involved. Among the over 500 proteases encoded in the genome of higher eukaryotes, only a few are known to contribute to the generation of N-degrons. It can, therefore, be expected that many processing paths remain to be discovered.

SUMMARY Organoids that self-organize into tissue-like structures have transformed our ability to model human development and disease. To date, all major organs can be mimicked using self-organizing organoids with the notable exception of the human heart. Here, we established self-organizing cardioids from human pluripotent stem cells that intrinsically specify, pattern and morph into chamber-like structures containing a cavity. Cardioid complexity can be controlled by signaling that instructs the separation of cardiomyocyte and endothelial layers, and by directing epicardial spreading, inward migration and differentiation. We find that cavity morphogenesis is governed by a mesodermal WNT-BMP signaling axis and requires its target HAND1, a transcription factor linked to human heart chamber cavity defects. In parallel, a WNT-VEGF axis coordinates myocardial self-organization with endothelial patterning and specification. Human cardioids represent a powerful platform to mechanistically dissect self-organization and congenital heart defects, serving as a foundation for future translational research. Highlights - Cardioids form cardiac-like chambers with inner endothelial lining and interact with epicardium - Cardioid self-organization and lineage complexity can be controlled by signaling - WNT-BMP signaling directs cavity formation in self-organized cardioids via HAND1 - WNT-VEGF coordinate endothelial patterning with myocardial cavity morphogenesis

Abstract New SARS-CoV-2 variants are continuously emerging with critical implications for therapies or vaccinations. All 22 N-glycan sites of SARS-CoV-2 Spike remain highly conserved among the variants B.1.1.7, 501Y.V2 and P.1, opening an avenue for robust therapeutic intervention. Here we used a comprehensive library of mammalian carbohydrate-binding proteins (lectins) to probe critical sugar residues on the full-length trimeric Spike and the receptor binding domain (RBD) of SARS-CoV-2. Two lectins, Clec4g and CD209c, were identified to strongly bind to Spike. Clec4g and CD209c binding to Spike was dissected and visualized in real time and at single molecule resolution using atomic force microscopy. 3D modelling showed that both lectins can bind to a glycan within the RBD-ACE2 interface and thus interferes with Spike binding to cell surfaces. Importantly, Clec4g and CD209c significantly reduced SARS-CoV-2 infections. These data report the first extensive map and 3D structural modelling of lectin-Spike interactions and uncovers candidate receptors involved in Spike binding and SARS-CoV-2 infections. The capacity of CLEC4G and mCD209c lectins to block SARS-CoV-2 viral entry holds promise for pan-variant therapeutic interventions.

Abstract Organoids enable disease modeling in complex and structured human tissue, in vitro . Like most 3D models, they lack sufficient oxygen supply, leading to cellular stress. These negative effects are particularly prominent in complex models, like brain organoids, where they can prevent proper lineage commitment. Here, we analyze brain organoid and fetal single cell RNA sequencing (scRNAseq) data from published and new datasets totaling over 190,000 cells. We describe a unique stress signature found in all organoid samples, but not in fetal samples. We demonstrate that cell stress is limited to a defined organoid cell population, and present Gruffi, an algorithm that uses granular functional filtering to identify and remove stressed cells from any organoid scRNAseq dataset in an unbiased manner. Our data show that adverse effects of cell stress can be corrected by bioinformatic analysis, improving developmental trajectories and resemblance to fetal data.

Abstract Establishment and maintenance of apical-basal polarity is a fundamental step in brain development, instructing the organization of neural progenitor cells (NPCs) and the developing cerebral cortex. Particularly, basally located extracellular matrix (ECM) is crucial for this process. In vitro, epithelial polarization can be achieved via endogenous ECM production, or exogenous ECM supplementation. While neuroepithelial development is recapitulated in cerebral organoids, the effects of different ECM sources in tissue morphogenesis remain unexplored. Here, we show that exposure to exogenous ECM at early neuroepithelial stages causes rapid tissue polarization and complete rearrangement of neuroepithelial architecture within 3 days. In unexposed cultures, endogenous ECM production by NPCs results in gradual polarity acquisition over an extended time. After the onset of neurogenesis, tissue architecture and neuronal differentiation are largely independent of the initial ECM source. These results advance the knowledge on neuroepithelial biology in vitro, with a focus on mechanisms of exogenously- and endogenously-guided morphogenesis. They demonstrate the self-sustainability of neuroepithelial cultures by endogenous processes, prompting an urgent reassessment of indiscriminate use of exogenous ECM in these model systems.

Simian virus 40 large T antigen transforms cells by sequestration and inactivation of the tumor suppressor proteins p53, retinoblastoma gene product (pRb), and the pRb-related proteins p107 and p130. Thus, the absence of functional p53 is expected to promote T antigen-mediated tumorigenesis. However, in a transgenic mouse model of T antigen-mediated beta cell carcinogenesis (Rip1Tag2), tumor volumes are significantly diminished when these mice are intercrossed with p53-deficient mice. Whereas the incidence of beta tumor cell apoptosis is unaffected, their proliferation rate is reduced in p53-deficient beta cell tumors in vivo and in cell lines established from these tumors in vitro. Biochemical analyses reveal higher levels of T antigen in wild-type tumor cells as compared to p53-deficient tumor cells. The data indicate that p53 stabilizes SV40 large T antigen, thereby augmenting its oncogenic potential as manifested by increased proliferation rates in wild-type beta tumor cells as compared to p53-deficient beta tumor cells.

Allergen recognition by IgE antibodies is a key event in allergic inflammation. In this study, the IgE IGHV repertoires of individuals with allergy to the major birch pollen allergen, Bet v 1, were analyzed over a four years period of allergen exposure by RT-PCR and sequencing of cDNA. Approximately half of the IgE transcripts represented non-redundant sequences, which belonged to seventeen different IGHV genes. Most variable regions contained somatic mutations but also non-mutated sequences were identified. There was no evidence for relevant increases of somatic mutations over time of allergen exposure. Highly similar IgE variable regions were found after four years of allergen exposure in the same and in genetically non-related individuals. Our results indicate that allergens select and shape a limited number of similar IgE variable regions in the human IgE repertoire.

A model of the human blastocyst formed from stem cells (blastoid) would support scientific and medical advances. However, its predictive power will depend on its ability to efficiently, timely, and faithfully recapitulate the sequences of blastocyst development (morphogenesis, specification, patterning), and to form cells reflecting the blastocyst stage. Here we show that naïve human pluripotent stem cells cultured in PXGL conditions and then triply inhibited for the Hippo, transforming growth factor- β, and extracellular signal-regulated kinase pathways efficiently undergo morphogenesis to form blastoids (>70%). Matching with developmental timing (~4 days), blastoids unroll the blastocyst sequence of specification by producing analogs of the trophoblast and epiblast, followed by the formation of analogs of the primitive endoderm and the polar trophoblasts. This results in the formation of cells transcriptionally similar to the blastocyst (>96%) and a minority of post-implantation analogs. Blastoids efficiently pattern by forming the embryonic-abembryonic axis marked by the maturation of the polar region (NR2F2+), which acquires the specific potential to directionally attach to hormonally stimulated endometrial cells, as in utero. Such a human blastoid is a scalable, versatile, and ethical model to study human development and implantation in vitro.

Abstract Nuclear mitotic apparatus protein (NuMA) is an essential vertebrate component in organizing microtubule ends at spindle poles. The NuMA‐dynactin/dynein motor multiprotein complex not only explains the transport of NuMA along spindle fibers but also is linked to the process of microtubule focusing. The interaction sites of NuMA to dynein/dynactin have not been mapped. In the yet functionally uncharacterized N terminus of NuMA, we predict a calponin‐homology (CH) domain, a motif with binding activity for actin‐like molecules. We substantiate the primary sequence analysis‐based prediction with secondary structure and fold recognition analysis, and we propose the N‐terminal CH domain of NuMA as a likely interaction site for actin‐related protein 1 (Arp1) protein of the dynactin/dynein complex.