Francesca Palladino

Heritable inactivation of genes occurs in specific chromosomal domains located at the silent mating type loci and at telomeres of S. cerevisiae. The SIR genes (for silent information regulators) are trans-acting factors required for this repression mechanism. We show here that the SIR3 and SIR4 gene products have a sub-nuclear localization similar to the telomere-associated RAP1 protein, which is found primarily in foci at the nuclear periphery of fixed yeast spheroplasts. In strains deficient for either SIR3 or SIR4, telomeres lose their perinuclear localization, as monitored by RAP1 immunofluorescence. The length of the telomeric repeat shortens in sir3 and sir4 mutant strains, and the mitotic stability of chromosome V is reduced. These data suggest that SIR3 and SIR4 are required for both the integrity and subnuclear localization of yeast telomeres, the loss of which correlates with loss of telomere-associated gene repression.

The Silent Information Regulatory proteins, Sir3 and Sir4, and the telomeric repeat-binding protein RAP1 are required for the chromatin-mediated gene repression observed at yeast telomeric regions. All three proteins are localized by immunofluorescence staining to foci near the nuclear periphery suggesting a relationship between subnuclear localization and silencing. We present several lines of immunological and biochemical evidence that Sir3, Sir4, and RAP1 interact in intact yeast cells. First, immunolocalization of Sir3 to foci at the yeast nuclear periphery is lost in rap1 mutants carrying deletions for either the terminal 28 or 165 amino acids of RAP1. Second, the perinuclear localization of both Sir3 and RAP1 is disrupted by overproduction of the COOH terminus of Sir4. Third, overproduction of the Sir4 COOH terminus alters the solubility properties of both Sir3 and full-length Sir4. Finally, we demonstrate that RAP1 and Sir4 coprecipitate in immune complexes using either anti-RAP1 or anti-Sir4 antibodies. We propose that the integrity of a tertiary complex between Sir4, Sir3, and RAP1 is involved in both the maintenance of telomeric repression and the clustering of telomeres in foci near the nuclear periphery.

Abstract The HPR5 gene has been defined by the mutation hpr5-1 that results in an increased rate of gene conversion. This mutation suppresses the UV sensitive phenotype of rad18 mutations in hpr5-1 rad18 double mutants by channeling the aborted repair events into a recombination repair pathway. The HPR5 gene has been cloned and is shown to be allelic to the SRS2/RADH gene, a putative DNA helicase. The HPR5 gene, which is nonessential, is tightly linked to the ARG3 locus chromosome X. The hpr5-1 allele contains missense mutation in the putative ATP binding domain. A comparison of the recombination properties of the hpr5-1 allele and the null allele suggests that recombination events in hpr5 defective strains can be generated by several mechanisms. We propose that the HPR5 gene functions in the RAD6 repair pathway.

Cellular adaptation to environmental stress relies on a wide range of tightly controlled regulatory mechanisms, including transcription. Changes in chromatin structure and organization accompany the transcriptional response to stress, and in some cases, can impart memory of stress exposure to subsequent generations through mechanisms of epigenetic inheritance. In the budding yeast Saccharomyces cerevisiae, histone post-translational modifications, and in particular histone methylation, have been shown to confer transcriptional memory of exposure to environmental stress conditions through mitotic divisions. Recent evidence from Caenorhabditis elegans also implicates histone methylation in transgenerational inheritance of stress responses, suggesting a more widely conserved role in epigenetic memory.

ABSTRACT The Mi-2 protein is the central component of the recently isolated NuRD nucleosome remodelling and histone deacetylase complex. Although the NuRD complex has been the subject of extensive biochemical analyses, little is known about its biological function. Here we show that the two C. elegans Mi-2 homologues, LET-418 and CHD-3, play essential roles during development. The two proteins possess both shared and unique functions during vulval cell fate determination, including antagonism of the Ras signalling pathway required for vulval cell fate induction and the proper execution of the 2° cell fate of vulval precursor cells, a process under the control of LIN-12 Notch signalling.

Proteins of the highly conserved heterochromatin protein 1 (HP1) family have been found to function in the dynamic organization of nuclear architecture and in gene regulation throughout the eukaryotic kingdom. In addition to being key players in heterochromatin-mediated gene silencing, HP1 proteins may also contribute to the transcriptional repression of euchromatic genes via the recruitment to specific promoters. To investigate the role played by these different activities in specific developmental pathways, we identified HP1 homologues in the genome of Caenorhabditis elegans and used RNA-mediated interference to study their function. We show that one of the homologues, HPL-2, is required for the formation of a functional germline and for the development of the vulva by acting in an Rb-related pathway. We suggest that, by acting as repressors of gene expression, HP1 proteins may fulfil specific functions in both somatic and germline differentiation processes throughout development.

The adenovirus tripartite leader is a 200-nucleotide 5' noncoding region that is found on all late viral mRNAs. This segment is required for preferential translation of viral mRNAs at late times during infection. Most tripartite leader-containing mRNAs appear to exhibit little if any requirement for intact cap-binding protein complex, a property previously established only for uncapped poliovirus mRNAs and capped mRNAs with minimal secondary structure. The tripartite leader also permits the translation of mRNAs in poliovirus-infected cells in the apparent absence of active cap-binding protein complex and does not require any adenovirus gene products for this activity. The preferential translation of viral late mRNAs may involve this unusual property.

Methylation of histone H3 lysine 4 (H3K4me), a mark associated with gene activation, is mediated by SET1 and the related mixed lineage leukemia (MLL) histone methyltransferases (HMTs) across species. Mammals contain seven H3K4 HMTs, Set1A, Set1B, and MLL1–MLL5. The activity of SET1 and MLL proteins relies on protein–protein interactions within large multisubunit complexes that include three core components: RbBP5, Ash2L, and WDR5. It remains unclear how the composition and specificity of these complexes varies between cell types and during development. Caenorhabditis elegans contains one SET1 protein, SET-2, one MLL-like protein, SET-16, and single homologs of RbBP5, Ash2L, and WDR5. Here we show that SET-2 is responsible for the majority of bulk H3K4 methylation at all developmental stages. However, SET-2 and absent, small, or homeotic discs 2 (ASH-2) are differentially required for tri- and dimethylation of H3K4 (H3K4me3 and -me2) in embryos and adult germ cells. In embryos, whereas efficient H3K4me3 requires both SET-2 and ASH-2, H3K4me2 relies mostly on ASH-2. In adult germ cells by contrast, SET-2 serves a major role whereas ASH-2 is dispensable for H3K4me3 and most H3K4me2. Loss of SET-2 results in progressive sterility over several generations, suggesting an important function in the maintenance of a functional germ line. This study demonstrates that individual subunits of SET1-related complexes can show tissue specificity and developmental regulation and establishes C. elegans as a model to study SET1-related complexes in a multicellular organism.

Most eukaryotic cells express small regulatory RNAs. The purpose of one class, the somatic endogenous siRNAs (endo-siRNAs), remains unclear. Here, we show that the endo-siRNA pathway promotes odor adaptation in C. elegans AWC olfactory neurons. In adaptation, the nuclear Argonaute NRDE-3, which acts in AWC, is loaded with siRNAs targeting odr-1, a gene whose downregulation is required for adaptation. Concomitant with increased odr-1 siRNA in AWC, we observe increased binding of the HP1 homolog HPL-2 at the odr-1 locus in AWC and reduced odr-1 mRNA in adapted animals. Phosphorylation of HPL-2, an in vitro substrate of the EGL-4 kinase that promotes adaption, is necessary and sufficient for behavioral adaptation. Thus, environmental stimulation amplifies an endo-siRNA negative feedback loop to dynamically repress cognate gene expression and shape behavior. This class of siRNA may act broadly as a rheostat allowing prolonged stimulation to dampen gene expression and promote cellular memory formation.PaperFlickeyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJhOTRjMDJlYThiMjMxZjA5MzEzMGMwYzQwZjcwMmI4ZiIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4NTE1MDg3fQ.Cya2Fmp37CqcwUmxXEgp-PTuycXlkwv9cdGCoAAV5F8j62aotXiioS4nlHEc6b7pZaLBEnnpxhxB56jgbPnW9MsFLT_pdsBQA_IFlI6oxXDexZmG3ZDscysugGTh-dbvBTaG9-e0mtMZjItXs10uybgL50MH3WNHMGE4kIFSRHoL3oJQkrL29_xPpQk_b3GQ--qH6Z9aaMXrR5JAnHF1EM6Hz5iFhmxIEfXWgHW0Y5e090G5szQLi0W3NG-fgo0NLU3_WsVa-N5PJZCCq6Mun3zNSbccZgPzNhpXUl_vn7-i4arfuce5WjK-W4tsfAR4pJPJQQdr4YVQKAsVvLaYCA(mp4, (65.03 MB) Download video

Epigenetics is defined as the study of heritable changes in gene expression that are not accompanied by changes in the DNA sequence. Epigenetic mechanisms include histone post-translational modifications, histone variant incorporation, non-coding RNAs, and nucleosome remodeling and exchange. In addition, the functional compartmentalization of the nucleus also contributes to epigenetic regulation of gene expression. Studies on the molecular mechanisms underlying epigenetic phenomena and their biological function have relied on various model systems, including yeast, plants, flies, and cultured mammalian cells. Here we will expose the reader to the current understanding of epigenetic regulation in the roundworm C. elegans. We will review recent models of nuclear organization and its impact on gene expression, the biological role of enzymes modifying core histones, and the function of chromatin-associated factors, with special emphasis on Polycomb (PcG) and Trithorax (Trx-G) group proteins. We will discuss how the C. elegans model has provided novel insight into mechanisms of epigenetic regulation as well as suggest directions for future research.

The CFP1 CXXC zinc finger protein targets the SET1/COMPASS complex to non-methylated CpG rich promoters to implement tri-methylation of histone H3 Lys4 (H3K4me3). Although H3K4me3 is widely associated with gene expression, the effects of CFP1 loss vary, suggesting additional chromatin factors contribute to context dependent effects. Using a proteomics approach, we identified CFP1 associated proteins and an unexpected direct link between Caenorhabditis elegans CFP-1 and an Rpd3/Sin3 small (SIN3S) histone deacetylase complex. Supporting a functional connection, we find that mutants of COMPASS and SIN3 complex components genetically interact and have similar phenotypic defects including misregulation of common genes. CFP-1 directly binds SIN-3 through a region including the conserved PAH1 domain and recruits SIN-3 and the HDA-1/HDAC subunit to H3K4me3 enriched promoters. Our results reveal a novel role for CFP-1 in mediating interaction between SET1/COMPASS and a Sin3S HDAC complex at promoters.

Abstract The hyper-gene conversion srs2-101 mutation of the SRS2 DNA helicase gene of Saccharomyces cerevisiae has been reported to suppress the UV sensitivity of rad18 mutants. New alleles of SRS2 were recovered using this suppressor phenotype. The alleles have been characterized with respect to suppression of rad18 UV sensitivity, hyperrecombination, reduction of meiotic viability, and definition of the mutational change within the SRS2 gene. Variability in the degree of rad18 suppression and hyperrecombination were found. The alleles that showed the severest effects were found to be missense mutations within the consensus domains of the DNA helicase family of proteins. The effect of mutations in domains I (ATP-binding) and V (proposed DNA binding) are reported. Some alleles of SRS2 reduce spore viability to 50% of wild-type levels. This phenotype is not bypassed by spo13 mutation. Although the srs2 homozygous diploids strains undergo normal commitment to meiotic recombination, this event is delayed by several hours in the mutant strains and the strains appear to stall in the progression from meiosis I to meiosis II.

Histone H3 Lys 4 methylation (H3K4me) is deposited by the conserved SET1/MLL methyltransferases acting in multiprotein complexes, including Ash2 and Wdr5. Although individual subunits contribute to complex activity, how they influence gene expression in specific tissues remains largely unknown. In Caenorhabditis elegans, SET-2/SET1, WDR-5.1, and ASH-2 are differentially required for germline H3K4 methylation. Using expression profiling on germlines from animals lacking set-2, ash-2, or wdr-5.1, we show that these subunits play unique as well as redundant functions in order to promote expression of germline genes and repress somatic genes. Furthermore, we show that in set-2- and wdr-5.1-deficient germlines, somatic gene misexpression is associated with conversion of germ cells into somatic cells and that nuclear RNAi acts in parallel with SET-2 and WDR-5.1 to maintain germline identity. These findings uncover a unique role for SET-2 and WDR-5.1 in preserving germline pluripotency and underline the complexity of the cellular network regulating this process.

Heterochromatin protein 1 (HP1) family proteins have a well-characterized role in heterochromatin packaging and gene regulation. Their function in organismal development, however, is less well understood. Here we used genome-wide expression profiling to assess novel functions of the Caenorhabditis elegans HP1 homolog HPL-2 at specific developmental stages.We show that HPL-2 regulates the expression of germline genes, extracellular matrix components and genes involved in lipid metabolism. Comparison of our expression data with HPL-2 ChIP-on-chip profiles reveals that a significant number of genes up- and down-regulated in the absence of HPL-2 are bound by HPL-2. Germline genes are specifically up-regulated in hpl-2 mutants, consistent with the function of HPL-2 as a repressor of ectopic germ cell fate. In addition, microarray results and phenotypic analysis suggest that HPL-2 regulates the dauer developmental decision, a striking example of phenotypic plasticity in which environmental conditions determine developmental fate. HPL-2 acts in dauer at least partly through modulation of daf-2/IIS and TGF-β signaling pathways, major determinants of the dauer program. hpl-2 mutants also show increased longevity and altered lipid metabolism, hallmarks of the long-lived, stress resistant dauers.Our results suggest that the worm HP1 homologue HPL-2 may coordinately regulate dauer diapause, longevity and lipid metabolism, three processes dependent on developmental input and environmental conditions. Our findings are of general interest as a paradigm of how chromatin factors can both stabilize development by buffering environmental variation, and guide the organism through remodeling events that require plasticity of cell fate regulation.

Cellular identity during metazoan development is maintained by epigenetic modifications of chromatin structure brought about by the activity of specific proteins which mediate histone variant incorporation, histone modifications, and nucleosome remodeling. HP1 proteins directly influence gene expression by modifying chromatin structure. We previously showed that the Caenorhabditis elegans HP1 proteins HPL-1 and HPL-2 are required for several aspects of post-embryonic development. To gain insight into how HPL proteins influence gene expression in a developmental context, we carried out a candidate RNAi screen to identify suppressors of hpl-1 and hpl-2 phenotypes. We identified SET-2, the homologue of yeast and mammalian SET1, as an antagonist of HPL-1 and HPL-2 activity in growth and somatic gonad development. Yeast Set1 and its mammalian counterparts SET1/MLL are H3 lysine 4 (H3K4) histone methyltransferases associated with gene activation as part of large multisubunit complexes. We show that the nematode counterparts of SET1/MLL complex subunits also antagonize HPL function in post-embryonic development. Genetic analysis is consistent with SET1/MLL complex subunits having both shared and unique functions in development. Furthermore, as observed in other species, we find that SET1/MLL complex homologues differentially affect global H3K4 methylation. Our results suggest that HP1 and a SET1/MLL-related complex may play antagonistic roles in the epigenetic regulation of specific developmental programs.

The PTEN tumour suppressor encodes a phosphatase, and its daf-18 orthologue in Caenorhabditis elegans negatively regulates the insulin/IGF-1 DAF-2 receptor pathway that influences lifespan in worms and other species. In order to identify new DAF-18 regulated pathways involved in aging, we initiated a candidate RNAi feeding screen for clones that lengthen lifespan. Here, we report that smg-1 inactivation increases average lifespan in a daf-18 dependent manner. Genetic analysis is consistent with SMG-1 acting at least in part in parallel to the canonical DAF-2 receptor pathway, but converging on the transcription factor DAF-16/FOXO. SMG-1 is a serine-threonine kinase which plays a conserved role in nonsense-mediated mRNA decay (NMD) in worms and mammals. In addition, human SMG-1 has also been implicated in the p53-mediated response to genotoxic stress. The effect of smg-1 inactivation on lifespan appears to be unrelated to its NMD function, but requires the p53 tumour suppressor orthologue cep-1. Furthermore, smg-1 inactivation confers a resistance to oxidative stress in a daf-18-, daf-16- and cep-1-dependent manner. We propose that the role of SMG-1 in lifespan regulation is at least partly dependent on its function in oxidative stress resistance. Taken together, our results unveil a novel role for SMG-1 in lifespan regulation.

Cellular adaptation to environmental changes and stress relies on a wide range of regulatory mechanisms that are tightly controlled at several levels, including transcription. Chromatin structure and chromatin binding proteins are important factors contributing to the transcriptional response to stress. However, it remains largely unknown to what extent specific chromatin factors influence the response to distinct forms of stress in a developmental context. One of the best characterized stress response pathways is the unfolded protein response (UPR), which is activated by accumulation of misfolded proteins in the endoplasmic reticulum (ER). Here, we show that Caenorhabditis elegans heterochromatin protein like-2 (HPL-2), the homolog of heterochromatin protein 1 (HP1), down-regulates the UPR in the intestine. Inactivation of HPL-2 results in an enhanced resistance to ER stress dependent on the X-box binding protein 1 (XBP-1)/inositol requiring enzyme 1 branch of the UPR and the closely related process of autophagy. Increased resistance to ER stress in animals lacking HPL-2 is associated with increased basal levels of XBP-1 activation and ER chaperone expression under physiological conditions, which may in turn activate an adaptive response known as ER hormesis. HPL-2 expression in intestinal cells is sufficient to rescue stress resistance, whereas expression in neuronal cells negatively influenced the ER stress response through a cell-nonautonomous mechanism. We further show that the retinoblastoma protein homolog LIN-35 and the LIN-13 zinc finger protein act in the same pathway as HPL-2 to limit the ER stress response. Altogether, our results point to multiple functions for HP1 in different cell types to maintain ER homeostasis.

Maintaining the integrity of genetic information across generations is essential for both cell survival and reproduction, and requires the timely repair of DNA damage. Histone-modifying enzymes play a central role in the DNA repair process through the deposition and removal of post-translational modifications on the histone tails. Specific histone modification act in the DNA repair process through the recruitment of proteins and complexes with specific enzymatic activities, or by altering the chromatin state at the site of DNA lesions. The conserved SET1/MLL family of histone methyltransferases (HMT) catalyzes methylation of histone H3 on Lysine 4 (H3K4), a histone modification universally associated with actively transcribed genes. Studies have focused on the role of SET1/MLL proteins in epigenetic regulation of gene expression. Much less is known on their role in the DNA repair process in a developmental context. Here we show that SET-2, the Caenorhabditis elegans orthologue of SET1, is required to preserve germline genome integrity over subsequent generations. Animals lacking the SET-2 catalytic subunit show a transgenerational increase in sensitivity to DNA damage-inducing agents that is accompanied by a defect in double-strand break (DSB) repair and chromosome fragmentation. These defects are not due to a failure to activate the DNA damage response (DDR) that allows detection, signaling and repair of DNA lesions, because cell cycle arrest and apoptosis, key components of this pathway, are efficiently induced in set-2 mutant animal. Rather, our results suggest that SET-2 plays a role in the transgenerational maintenance of genome stability by acting in DNA repair downstream of DDR signaling.

HP1 proteins are essential components of heterochromatin and contribute to the transcriptional repression of euchromatic genes via the recruitment to specific promoters by corepressor proteins including TIF1 and Rb. The Caenorhabditis elegans HP1 homologue HPL-2 acts in the "synMuv" (synthetic multivulval) pathway, which defines redundant negative regulators of a Ras signaling cascade required for vulval induction. Several synMuv genes encode for chromatin-associated proteins involved in transcriptional regulation, including Rb and components of the Mi-2/NuRD and TIP60/NuA4 chromatin remodeling complexes. Here, we show that HPL-2 physically interacts in vitro and in vivo with the multiple zinc finger protein LIN-13, another member of the synMuv pathway. A variant of the conserved PXVXL motif found in many HP1-interacting proteins mediates LIN-13 binding to the CSD of HPL-2. We further show by in vivo localization studies that LIN-13 is required for HPL-2 recruitment in nuclear foci. Our data suggest that the LIN-13/HPL-2 complex may physically link a subset of the Rb related synMuv proteins to chromatin.

Linker histone (H1) and heterochromatin protein 1 (HP1) are essential components of heterochromatin which contribute to the transcriptional repression of genes. It has been shown that the methylation mark of vertebrate histone H1 is specifically recognized by the chromodomain of HP1. However, the exact biological role of linker histone binding to HP1 has not been determined. Here, we investigate the function of the Caenorhabditis elegans H1 variant HIS-24 and the HP1-like proteins HPL-1 and HPL-2 in the cooperative transcriptional regulation of immune-relevant genes. We provide the first evidence that HPL-1 interacts with HIS-24 monomethylated at lysine 14 (HIS-24K14me1) and associates in vivo with promoters of genes involved in antimicrobial response. We also report an increase in overall cellular levels and alterations in the distribution of HIS-24K14me1 after infection with pathogenic bacteria. HIS-24K14me1 localization changes from being mostly nuclear to both nuclear and cytoplasmic in the intestinal cells of infected animals. Our results highlight an antimicrobial role of HIS-24K14me1 and suggest a functional link between epigenetic regulation by an HP1/H1 complex and the innate immune system in C. elegans.

HP1 proteins are essential components of heterochromatin and contribute to the transcriptional repression of euchromatic genes. Although most species contain more than one HP1 family member which differ in their chromosomal distribution, it is not known to what extent the activity of these different family members is redundant or specific in a developmental context. C. elegans has two HP1 homologues, HPL-1 and HPL-2. While HPL-2 functions in vulval and germline development, no function has so far been attributed to HPL-1. Here we report the characterization of an hpl-1 null allele. We show that while the absence of hpl-1 alone results in no obvious phenotype, hpl-1;hpl-2 double mutants show synthetic, temperature sensitive phenotypes including larval lethality and severe defects in the development of the somatic gonad. Furthermore, we find that hpl-1 has an unexpected role in vulval development by acting redundantly with hpl-2, but not other genes previously implicated in vulval development. Localization studies show that like HPL-2, HPL-1 is a ubiquitously expressed nuclear protein. However, HPL-1 and HPL-2 localization does not completely overlap. Our results show that HPL-1 and HPL-2 play both unique and redundant functions in post-embryonic development.

The precise developmental map of the Caenorhabditis elegans cell lineage, as well as a complete genome sequence and feasibility of genetic manipulation make this nematode species highly attractive to study the role of epigenetics during development. Genetic dissection of phenotypical traits, such as formation of egg-laying organs or starvation-resistant dauer larvae, has illustrated how chromatin modifiers may regulate specific cell-fate decisions and behavioral programs. Moreover, the transparent body of C. elegans facilitates non-invasive microscopy to study tissue-specific accumulation of heterochromatin at the nuclear periphery. We also review here recent findings on how small RNA molecules contribute to epigenetic control of gene expression that can be propagated for several generations and eventually determine longevity.

Auxins are plant growth regulators that influence most aspects of plant development through complex mechanisms. The development of an auxin-inducible degradation (AID) system has enabled rapid, conditional protein depletion in yeast and cultured cells. More recently, the system was successfully adapted to Caenorhabditiselegans to achieve auxin-dependent degradation of targets in all tissues and developmental stages. Whether auxin treatment alone has an impact on nematode physiology is an open question. Here we show that indole-3-acetic acid (IAA), the auxin most commonly used to trigger AID in worms, functions through the conserved IRE-1/XBP-1 branch of the Unfolded Protein Response (UPR) to promote resistance to endoplasmic reticulum (ER) stress. Because the UPR not only plays a central role in restoring ER homeostasis, but also promotes lipid biosynthesis and regulates lifespan, we suggest that extreme caution should be exercised when using the AID system to study these and related processes.

Silent information regulator 3 is an essential component of the Saccharomyces cerevisiae silencing complex that functions at telomeres and the silent mating-type loci, HMR and HML. We show that expression of the N- and C-terminal-encoding halves of SIR3 in transpartially complements the mating defect of the sir3 null allele, suggesting that the two domains have distinct functions. We present here a functional characterization of these domains. The N-terminal domain (Sir3N) increases both the frequency and extent of telomere-proximal silencing when expressed ectopically in SIR+ yeast strains, although we are unable to detect interaction between this domain and any known components of the silencing machinery. In contrast to its effect at telomeres, Sir3N overexpression derepresses transcription of reporter genes inserted in the ribosomal DNA (rDNA) array. Immunolocalization of Sir3N-GFP and Sir2p suggests that Sir3N directly antagonizes nucleolar Sir2p, releasing an rDNA-bound population of Sir2p so that it can enhance repression at telomeres. Overexpression of the C-terminal domain of either Sir3p or Sir4p has a dominant-negative effect on telomeric silencing. In strains overexpressing the C-terminal domain of Sir4p, elevated expression of either full-length Sir3p or Sir3N restores repression and the punctate pattern of Sir3p and Rap1p immunostaining. The similarity of Sir3N and Sir3p overexpression phenotypes suggests that Sir3N acts as an allosteric effector of Sir3p, either enhancing its interactions with other silencing components or liberating the full-length protein from nonfunctional complexes.

In yeast, the study of the teleomere has recently provided new information on the requirements for chromosome stability, on elements influencing nuclear architecture and on position-effect variegation.

Scientific Report4 February 2015free access Genome-wide screen identifies a novel p97/CDC-48-dependent pathway regulating ER-stress-induced gene transcription Esther Marza Esther Marza Team “Endoplasmic Reticulum stress and cancer”, INSERM, UMR1053, Bordeaux, France University of Bordeaux, Bordeaux, France ARNA laboratory, INSERM U869, Bordeaux, France Search for more papers by this author Saïd Taouji Saïd Taouji Team “Endoplasmic Reticulum stress and cancer”, INSERM, UMR1053, Bordeaux, France University of Bordeaux, Bordeaux, France Search for more papers by this author Kim Barroso Kim Barroso Team “Endoplasmic Reticulum stress and cancer”, INSERM, UMR1053, Bordeaux, France University of Bordeaux, Bordeaux, France Search for more papers by this author Anne-Aurélie Raymond Anne-Aurélie Raymond University of Bordeaux, Bordeaux, France “REPTeam”, INSERM, UMR1053, Bordeaux, France Search for more papers by this author Léo Guignard Léo Guignard University of Bordeaux, Bordeaux, France ARNA laboratory, INSERM U869, Bordeaux, France Search for more papers by this author Marc Bonneu Marc Bonneu University of Bordeaux, Bordeaux, France Plateforme Proteome, Bordeaux, France Search for more papers by this author Néstor Pallares-Lupon Néstor Pallares-Lupon Team “Endoplasmic Reticulum stress and cancer”, INSERM, UMR1053, Bordeaux, France University of Bordeaux, Bordeaux, France Search for more papers by this author Jean-William Dupuy Jean-William Dupuy University of Bordeaux, Bordeaux, France Plateforme Proteome, Bordeaux, France Search for more papers by this author Martin E Fernandez-Zapico Martin E Fernandez-Zapico Schulze Center for Novel Therapeutics, Division of Oncology Research, Mayo Clinic, Rochester, MN, USA Search for more papers by this author Jean Rosenbaum Jean Rosenbaum University of Bordeaux, Bordeaux, France “REPTeam”, INSERM, UMR1053, Bordeaux, France Search for more papers by this author Francesca Palladino Francesca Palladino Laboratory of Molecular and Cellular Biology, Ecole Normale Supérieure, CNRS UMR5239, Université de Lyon, Lyon Cedex 07, France Search for more papers by this author Denis Dupuy Denis Dupuy University of Bordeaux, Bordeaux, France ARNA laboratory, INSERM U869, Bordeaux, France Search for more papers by this author Eric Chevet Corresponding Author Eric Chevet Team “Endoplasmic Reticulum stress and cancer”, INSERM, UMR1053, Bordeaux, France University of Bordeaux, Bordeaux, France Centre Régional de Lutte Contre le Cancer Eugène Marquis, Rennes, France Search for more papers by this author Esther Marza Esther Marza Team “Endoplasmic Reticulum stress and cancer”, INSERM, UMR1053, Bordeaux, France University of Bordeaux, Bordeaux, France ARNA laboratory, INSERM U869, Bordeaux, France Search for more papers by this author Saïd Taouji Saïd Taouji Team “Endoplasmic Reticulum stress and cancer”, INSERM, UMR1053, Bordeaux, France University of Bordeaux, Bordeaux, France Search for more papers by this author Kim Barroso Kim Barroso Team “Endoplasmic Reticulum stress and cancer”, INSERM, UMR1053, Bordeaux, France University of Bordeaux, Bordeaux, France Search for more papers by this author Anne-Aurélie Raymond Anne-Aurélie Raymond University of Bordeaux, Bordeaux, France “REPTeam”, INSERM, UMR1053, Bordeaux, France Search for more papers by this author Léo Guignard Léo Guignard University of Bordeaux, Bordeaux, France ARNA laboratory, INSERM U869, Bordeaux, France Search for more papers by this author Marc Bonneu Marc Bonneu University of Bordeaux, Bordeaux, France Plateforme Proteome, Bordeaux, France Search for more papers by this author Néstor Pallares-Lupon Néstor Pallares-Lupon Team “Endoplasmic Reticulum stress and cancer”, INSERM, UMR1053, Bordeaux, France University of Bordeaux, Bordeaux, France Search for more papers by this author Jean-William Dupuy Jean-William Dupuy University of Bordeaux, Bordeaux, France Plateforme Proteome, Bordeaux, France Search for more papers by this author Martin E Fernandez-Zapico Martin E Fernandez-Zapico Schulze Center for Novel Therapeutics, Division of Oncology Research, Mayo Clinic, Rochester, MN, USA Search for more papers by this author Jean Rosenbaum Jean Rosenbaum University of Bordeaux, Bordeaux, France “REPTeam”, INSERM, UMR1053, Bordeaux, France Search for more papers by this author Francesca Palladino Francesca Palladino Laboratory of Molecular and Cellular Biology, Ecole Normale Supérieure, CNRS UMR5239, Université de Lyon, Lyon Cedex 07, France Search for more papers by this author Denis Dupuy Denis Dupuy University of Bordeaux, Bordeaux, France ARNA laboratory, INSERM U869, Bordeaux, France Search for more papers by this author Eric Chevet Corresponding Author Eric Chevet Team “Endoplasmic Reticulum stress and cancer”, INSERM, UMR1053, Bordeaux, France University of Bordeaux, Bordeaux, France Centre Régional de Lutte Contre le Cancer Eugène Marquis, Rennes, France Search for more papers by this author Author Information Esther Marza1,2,3, Saïd Taouji1,2,‡, Kim Barroso1,2,‡, Anne-Aurélie Raymond2,4,‡, Léo Guignard2,3, Marc Bonneu2,5, Néstor Pallares-Lupon1,2, Jean-William Dupuy2,5, Martin E Fernandez-Zapico6, Jean Rosenbaum2,4, Francesca Palladino7, Denis Dupuy2,3 and Eric Chevet 1,2,8 1Team “Endoplasmic Reticulum stress and cancer”, INSERM, UMR1053, Bordeaux, France 2University of Bordeaux, Bordeaux, France 3ARNA laboratory, INSERM U869, Bordeaux, France 4“REPTeam”, INSERM, UMR1053, Bordeaux, France 5Plateforme Proteome, Bordeaux, France 6Schulze Center for Novel Therapeutics, Division of Oncology Research, Mayo Clinic, Rochester, MN, USA 7Laboratory of Molecular and Cellular Biology, Ecole Normale Supérieure, CNRS UMR5239, Université de Lyon, Lyon Cedex 07, France 8Centre Régional de Lutte Contre le Cancer Eugène Marquis, Rennes, France ‡These authors contributed equally to this work *Corresponding author. Tel: +33 557579253; E-mail: [email protected] EMBO Reports (2015)16:332-340https://doi.org/10.15252/embr.201439123 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 The accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the Unfolded Protein Response (UPRER) to restore ER homeostasis. The AAA+ ATPase p97/CDC-48 plays key roles in ER stress by promoting both ER protein degradation and transcription of UPRER genes. Although the mechanisms associated with protein degradation are now well established, the molecular events involved in the regulation of gene transcription by p97/CDC-48 remain unclear. Using a reporter-based genome-wide RNAi screen in combination with quantitative proteomic analysis in Caenorhabditis elegans, we have identified RUVB-2, a AAA+ ATPase, as a novel repressor of a subset of UPRER genes. We show that degradation of RUVB-2 by CDC-48 enhances expression of ER stress response genes through an XBP1-dependent mechanism. The functional interplay between CDC-48 and RUVB-2 in controlling transcription of select UPRER genes appears conserved in human cells. Together, these results describe a novel role for p97/CDC-48, whereby its role in protein degradation is integrated with its role in regulating expression of ER stress response genes. Synopsis During ER stress, p97/CDC-48 mediates reptin degradation thereby enabling both ATF6 activation and XBP1 mRNA splicing. This work uncovers another layer in the regulation of canonical ER stress signaling. p97/CDC-48 induces reptin degradation upon ER stress. Reptin is a repressor of both ATF6 activation and XBP1 mRNA splicing. p97/cdc-48-mediated retpin degradation promotes ER adaptive response to stress. Introduction The endoplasmic reticulum (ER) protein quality control system ensures the correct folding of transmembrane and secretory proteins before their export from this organelle 1. Accumulation of improperly folded proteins in the ER triggers the unfolded protein response (UPRER) to restore ER homeostasis. This is achieved by enhancing ER-Associated Degradation (ERAD), increasing ER protein folding capacity, decreasing protein translation and inducing a defined gene expression profile (UPRER genes) 2. Although most of these molecular events are clearly established, the mechanism leading to the transcriptional regulation of specific genes under ER stress remains poorly understood. Here, using as a model the nematode C. elegans, we identify a novel functional partner for p97/CDC-48, an AAA+ ATPase involved ER stress response, in the regulation of ER-stress-associated UPRER gene transcription. C. elegans expresses two p97/CDC-48 homologs, cdc-48.1 and cdc-48.2, which share similar functions in ERAD. While simultaneous silencing of both cdc-48.1 and cdc-48.2 leads to ER stress, UPRER gene activation and lethality 3. Inactivation of either cdc-48.1 or cdc-48.2 is viable but abolishes the transcriptional activation of UPRER genes in response to ER stress 4. Using a C. elegans strain mutant for the p97/CDC-48 homolog cdc-48.2(−/−), we performed a genome-wide RNAi screen to identify proteins involved in the activation of UPRER genes during ER stress. We found that the AAA+ ATPase RUVB-2 is a regulator of the ER stress response by repressing the transcription of select UPRER genes in non-stressed conditions in both C. elegans and human cells. In response to ER stress, RUVB-2 is degraded in a CDC-48-dependent manner, thereby relieving repression of UPRER genes. Altogether, our results identify a novel mechanism controlling gene expression downstream of p97/CDC-48 and unveil a novel function for RUVB-2 and its human homolog Reptin as a key regulator of the transcriptional response to ER stress. Results and Discussion A genome-wide screen identifies cdc-48 genetic interactors regulating ER-stress-induced gene expression RNAi-mediated knockdown of cdc-48.1 or cdc-48.2 in C. elegans abolishes the ER-stress-induced expression of a set of UPRER genes including ckb-2 4. Using a transcriptional reporter expressing GFP under the control of the ckb-2 promoter, we confirmed the requirement for cdc-48.1 and cdc-48.2 in ER-stress-induced gene transcription (Fig 1A). Mutant cdc-48.1(−/−) and cdc-48.2(−/−) worms failed to respond to the ER-stress inducer tunicamycin while ckb-2p::gfp fluorescence was increased more than threefold in wild-type (WT) worms (Fig 1B). RNAi inactivation of ire-1, the main sensor of ER stress and mediator of UPRER signaling, resulted in a significant decrease in fluorescence intensity in both ckb-2p::gfp and cdc-48.2(−/−); ckb-2p::gfp worms (Fig 1A, Supplementary Table S1). These results confirm that ckb-2p::gfp transcription is IRE1 dependent, as expected of a bona fide UPRER reporter. Figure 1. RNAi screening identifies cdc-48.2 genetics interactors in the ckb-2 transcriptional response to ER stress A. cdc-48.2 is required to activate ckb-2p::gfp transcription in response to tunicamycin. Images of adult worms (left) expressing gfp under the control of the ckb-2 gene promoter in WT (upper panels) and in cdc-48.2(−/−) mutants (lower panels) exposed to tunicamycin (5 μg/ml) or DMSO for 16 h. (Scale bar: 50 μm, obj.: 10×). B. Significant changes in fluorescence intensities were quantified using flow cytometry. L1 larvae (ckb-2p::gfp and cdc-48.2(−/−); ckb-2::pgfp larvae) were fed with bacteria expressing the L4440 empty vector or ire-1 RNAi in liquid culture and exposed to tunicamycin (0.5 μg/ml) or DMSO for 16 h. F0 was defined as the fluorescence intensity obtained in ckb-2p::gfp worms fed with the empty vector and treated with DMSO. (Mean ± s.e.m, N = 8, 200 worms/experiment). P-values were calculated using multiple t-test corrected using the Holm–Sidak method **P < 0.001; *P < 0.01. C. Genome-wide RNAi screen identifies suppressors and enhancers of cdc-48.2(−/−) in ckb-2p::gfp transcription. Volcano plots present results obtained using Caenorhabditis elegans ORFeome library. D. Re-testing of RNAi clones from first round. E, F. Classification of ER stress dependence of the 177 suppressor RNAi clones able to restore ckb-2p::gfp transcription in cdc-48.2(−/−) mutant background. cdc-48.2(−/−); ckb-2p::gfp synchronized L1 larvae were fed with the dsRNA expressing bacteria in liquid culture, treated with tunicamycin (0.5 μg/ml) or DMSO for 16 h, and fluorescence intensities were measured by flow cytometry. (E) Tunicamycin-dependent RNAi clones were defined as those that significantly increased fluorescence ratio following tunicamycin treatment (Tunicamycin/DMSO F/F0 fold change > 1.5). (F) Tunicamycin-independent RNAi clones were defined as those increasing ckb-2p::gfp fluorescence ratio in both conditions (Tunicamycin/DMSO F/F0 fold change < 1.5, P > 0.05). Fluorescence ratios obtained with ruvb-2 RNAi are shown in magenta. Fluorescence ratios obtained with ckb-2p::gfp worms fed with the empty vector and treated with tunicamycin (2.38 ± 0.18) or DMSO (1.05 ± 0.2) are shown in cyan. G–I. F0 was defined as the fluorescence intensity obtained in cdc-48.2(−/−); ckb-2p::gfp worms fed with the empty vector and treated with tunicamycin or DMSO, respectively. (Mean ± s.e.m, N = 5). Identification of ER-stress-dependent RNAi clones targeting genes involved in the same genetic pathway as cdc-48.2 to increase ckb-2p::gfp transcription. Fluorescence ratio were determined on cdc-48.2(−/−); ckb-2::gfp and ckb-2::gfp worms fed with the suppressor RNAi clones and treated with tunicamycin (0.5 μg/ml) for 16 h. Graphs present the RNAi clones whose effect on ckb-2p::gfp fluorescence was higher ((G), (ckb-2::gfp F/F0/cdc-48.2(−/−); ckb-2::gfp F/F0) fold change > 1.4-fold), similar ((H), (ckb-2::gfp F/F0/cdc-48.2(−/−); ckb-2::gfp F/F0) fold change < 1.4, P > 0.05) or lower ((I), (ckb-2::gfp F/F0/cdc-48.2(−/−); ckb-2::gfp F/F0) fold change < 0.75) in ckb-2p::gfp worms compared to cdc-48.2(−/−); ckb-2p::gfp worms. Fluorescence ratios obtained with ruvb-2 RNAi and the two controls empty vector and cdc-48.1 control RNAis are shown in magenta, cyan and brown, respectively. (Mean ± s.e.m, N = 5). Download figure Download PowerPoint Because p97/CDC-48 is involved in protein degradation 5, we reasoned that it might modulate ER-stress-induced ckb-2p transcription by eliminating a transcriptional repressor. To address this hypothesis, we designed an RNAi suppressor screen to identify genes whose knockdown could restore tunicamycin ckb-2p::gfp activation following ER stress in a cdc-48.2(−/−) mutant background (4; Fig 1C). We performed the screen in liquid culture by feeding cdc-48.2(−/−); ckb-2p::gfp synchronized L1 larvae with double-stranded RNA (dsRNA)-expressing E. coli derived from the C. elegans ORFeome library that targets 11,698 open reading frames covering 62% of C. elegans genes 6). We then exposed the worms to a concentration of tunicamycin (0.5 μg/ml for 16 h) leading to maximal ckb-2p::gfp induction in WT worms grown in liquid culture and analyzed them by flow cytometry 7 to measure their length, number and fluorescence intensity. Each RNAi clone was tested in duplicate, and the mean Z-score was calculated. Two-hundred and forty-one RNAi clones synergized with cdc48.2(−/−) to decrease ckb-2p::gfp expression in our primary screen (mean Z-score value less than −1.5, or one of the two independent Z-scores less than −3) (Fig 1C). Of these, 59 clones significantly decreased GFP fluorescence below 0.75-fold (P < 0.05) (Fig 1D, Supplementary Table S2). One-hundred and seventy-seven RNAi clones instead reproducibly increased (average Z-score >1.5 or one of the two individual Z-scores >3) GFP fluorescence 1.5-fold above the fluorescence intensity measured with cdc-48.2(−/−); ckb-2p::gfp worms fed with an empty vector and treated with tunicamycin (P < 0.05). These were classified as potential suppressors of the cdc48.2(−/−) phenotype. To discriminate between ER-stress-dependent and ER-stress-independent activation of ckb-2p::gfp transcription, we measured fluorescence intensity in cdc-48.2(−/−); ckb-2p::gfp worms fed with candidate RNAi clones and treated either with tunicamycin or vehicle (DMSO; Fig 1E). Seventy-seven RNAi clones showing a similar increase in the fluorescence ratios under both conditions were considered ER-stress independent and not further analyzed (Fig 1F, Supplementary Table S3). By contrast, 100 RNAi clones which restored ckb-2p::gfp activation in the cdc48.2(−/−) mutant background specifically under tunicamycin treatment were identified as ER-stress-dependent suppressors of cdc48.2(−/−) (Fig 1E, Supplementary Table S4). We next investigated whether the genes targeted by these RNAi clones could activate gene transcription specifically under ER stress independently of cdc-48.2. If a targeted gene acts exclusively in the same genetic pathway as cdc-48.2, then its knockdown by RNAi should not increase ckb-2p::gfp transcription in a WT background, nor have an additive effect with the cdc-48.2(−/−) mutation on ckb-2p::gfp transcription. We quantified and compared ckb-2p::gfp fluorescence intensities in both WT and cdc-48.2(−/−) mutant worms fed with RNAi and exposed to tunicamycin. Twenty-seven RNAi clones increased fluorescence intensities in WT more than in cdc-48.2(−/−) worms (fold change ≥1.4, Fig 1G). Nine other clones showed higher fluorescence in mutant worms compared to WT (fold change ≥1.4, Fig 1I), similar to cdc-48.1 RNAi. The corresponding 36 genes (27+9) were therefore not considered as strict suppressor of cdc-48.2 and were not further analyzed. We thus identified 64 suppressor RNAi clones that did not show any synthetic enhancement phenotype in cdc-48.2(−/−) relative to WT (fold change < 1.4 and P < 0.05, Fig 1H). Taken together, these results identify genes controlling ckb-2p::gfp expression upon ER stress in a CDC-48 dependent fashion and may provide mechanistic insight for the role of CDC-48 in ER-stress-induced gene expression (Fig 2A). Among these candidates, the AAA+ ATPase Ruvb2 was of particular interest. Figure 2. Identification of RUVB2 as a candidate CDC-48 target List of RNAi clones suppressing the cdc-48.2(−/−) phenotype. Graph representing identified peptide number identified in function of peptide quantity ratio. cdc-48.2(−/−); ckb-2::gfp and ckb-2::gfp synchronized L1 larvae were grown to the L4 stage and exposed to tunicamycin (5 μg/ml) for 16 h on plates. Proteins (60 μg) were separated on a 10% SDS gel. A coomassie blue staining image representative of the SDS gel is shown on the left (1: cdc-48.2(−/−); ckb-2::gfp, 2: ckb-2::gfp). Gel lanes were cut into slices before proteins were in-gel-digested. Peptides were then identified and quantified by label-free LC-MS/MS mass spectrometry. Peptides that were more (magenta) or less (cyan) abundant in the cdc-48.2(−/−); ckb-2::gfp than in ckb-2::gfp worms were defined as those having a ratio above 1.5 or below 0.5, respectively. N = 3. Graph representing peptide quantity ratio ((cdc-48.2(−/−); ckb-2::gfp)/(ckb-2::gfp)) for the 93 proteins that are more abundant in cdc-48.2(−/−) mutant background compared to WT background. (Mean ± s.e.m, N = 3). Download figure Download PowerPoint To confirm the RNAi screen findings, we conducted a quantitative proteomic analysis to identify proteins whose levels are modified in cdc-48.2(−/−); ckb-2p::gfp worms exposed to tunicamycin. We selected proteins represented by at least two peptides and that had a peptide ratio above 2 or below 0.5 between WT and mutant worms exposed to tunicamycin. Ninety-three proteins increased and 15 proteins decreased in abundance in cdc-48.2(−/−) mutants compared to the WT (Fig 2B, Supplementary Table S5). RUVB-2 was the only suppressor identified in our RNAi screen for which an increase in protein abundance could be detected in cdc-48.2(−/−); ckb-2p::gfp compared to ckb-2p::gfp worms (2.5 ± 0.5-fold increase, Fig 2C). Because the quantity of ruvb-2 mRNA was not increased (Supplementary Fig S4A) under these conditions, the increased abundance of RUVB-2 in cdc-48.2(−/−) mutants is likely due to attenuation of protein degradation rather than to increased transcription. Conserved RUVB-2 and CDC-48-dependent regulation of UPRER gene expression RNAi knockdown of ruvb-2 restored ckb-2p::gfp activation both in cdc-48.2(−/−) and cdc-48.1(−/−) mutant worms exposed to tunicamycin (Fig 3A–B). This suggests that, under ER stress, the repressor RUVB-2 is degraded through a CDC-48.1-dependent mechanism to allow full ckb-2p::gfp induction. Moreover, knockdown of xbp-1 reduced ckb-2p::gfp expression in cdc-48.2(−/−); ckb-2p::gfp worms treated with tunicamycin compared to the DMSO-treated ones (Fig 3C). Combined RNAi-mediated knock-down of xbp-1 and ruvb-2 decreased ckb-2p::gfp fluorescence to the same level observed using xbp-1 RNAi alone. This suggests that RUVB-2 is degraded through a CDC-48-dependent mechanism in response to tunicamycin, thus allowing XBP-1s to activate ckb-2 expression. Ruvb-2 inactivation also restored the expression of ER homeostasis regulators (CKB-2, F22E5.6, Y71F9AL.17/COPA-1) observed upon ER stress in WT animals 8 in cdc-48.2(−/−) tunicamycin-treated worms (Fig 3D and Supplementary Fig S4B). These results suggest that RUVB-2 represses the expression of select UPRER target genes. We next tested whether this function was conserved in human cells. To this end, Huh7 cells transfected with the ER stress response element reporter gene (ERSE::tomato 9) were knocked down for Reptin using stable integration of a doxycycline-inducible short hairpin RNA (shRNA 10) (Fig 3E, left). Induction of Reptin shRNA synergized with tunicamycin treatment to activate ERSE::tomato transcription, demonstrating that Reptin can also a repress ER-stress-mediated transcription in human cells. Of note, the silencing of the Reptin homolog Pontin did not affect the transcription of the ERSE::tomato reporter under basal conditions or upon tunicamycin-induced ER stress (Fig 3E, right). We next quantified the mRNA amounts of 4 genes whose products are involved in the control of ER homeostasis (BiP, CHOP, EDEM1, ORP150). This revealed that Reptin silencing significantly increased the expression of BiP, CHOP and EDEM1 while did not affect that of ORP150 (Fig 3F). Moreover, Reptin overexpression led to the significant repression of select genes (CHOP, EDEM1, ORP150) under basal conditions when compared to control transfected cells (Supplementary Fig S5). Altogether, these results suggest the existence of a conserved role for Reptin in repressing expression of ER stress response genes. Figure 3. RUVB-2 is a transcriptional repressor inactivated by CDC-48 upon ER stress Images of cdc-48.2(−/−); ckb-2::gfp adult worms fed with either the L4440 empty vector (upper panel) or ruvb-2 RNAi (lower panel) and treated with tunicamycin (5 μg/ml) or DMSO for 16 h on NGM agar plates. (Scale bar: 50 μm, obj: 10×). Fluorescence was quantified by flow cytometry on ckb-2::gfp, cdc-48.1(−/−); ckb-2::gfp and cdc-48.2(−/−); ckb-2::gfp worms fed with ruvb-2 RNAi or empty vector starting at the L1 stage in liquid culture and exposed to tunicamycin (0.5 μg/ml) or DMSO for 16 h. Fluorescence (F) was normalized to the basal fluorescence obtained with empty vector and DMSO in the WT background (F0). (Mean ± SD, N = 5) ***P < 0.001. Fluorescence (F) was quantified by flow cytometry on ckb-2::gfp and cdc-48.2(−/−); ckb-2::gfp worms fed with either ruvb-2 and empty vector (1:1), xbp-1 and empty vector (1:1), ruvb-2 and xbp-1 RNAi (1:1), or empty vector alone and treated with tunicamycin (0.5 μg/ml) or DMSO for 16 h. Fluorescence (F) was normalized to the basal fluorescence obtained with the empty vector and DMSO in the WT background (F0). (Mean ± s.e.m, N = 3). P-values were calculated using multiple t-test corrected using the Holm-Sidak method. **P < 0.01; ***P < 0.001. RT–qPCR quantification of the relative expression levels of 3 endogenous ER homeostasis genes (ERp19, F22E5.6, Y71F9AL.17/COPA-1), Ckb-2 and Ruvb-2 following tunicamycin treatment in cdc-48.2(−/−) worms subjected or not to ruvb-2 RNAi. Bars represent the mean of three biological replicates. (Mean ± s.e.m, N = 3) **P < 0.01; ***P < 0.001. Fluorescence was quantified in HuH7 cells expressing the ERSE::Tomato construct and either the Reptin shRNA induced with doxycycline (left) or the Pontin shRNA (transient, right). Cells were exposed to tunicamycin (5 μg/ml) for 4 h prior to measurement. Data are presented as mean ± SD of three independent experiments. Note that Reptin levels were decreased upon Tunicamycin treatment (see also Fig 4A). **P < 0.01. RT–qPCR analysis of four ER homeostasis control genes under basal conditions or upon tunicamycin treatment (5 μg/ml, 16 h) in HuH7 cells subjected or not to doxycycline-induced Reptin silencing. Data are presented as mean ± SD of three independent biological triplicates. (Mean ± s.e.m, N = 3) P-value was calculated using multiple t-test corrected using the Holm-Sidak method. *P < 0.05; **P < 0.01. Download figure Download PowerPoint Post-translational control of Reptin expression by p97/CDC-48 impacts on ER stress response in human cells Further, we examined whether p97/CDC-48 could also acted by triggering the degradation of Reptin in response to ER stress as observed in C. elegans (Fig 2). Reptin protein levels were significantly decreased upon tunicamycin treatment, whereas p97/CDC-48, Pontin and Calnexin protein expression remained unaffected (Fig 4A). Conversely, addition of the p97/CDC-48 inhibitor DBeQ stabilized Reptin levels under ER stress (Fig 4B). We then tested whether p97 and Reptin interacted physically using co-immunoprecipitation. These results were confirmed by determining Reptin's half-life upon stress (Supplementary Table S6), and the values obtained under basal conditions were in the range of those determined in S. cerevisiae or S. pombe 11. Reptin immunoprecipitates contained p97/CDC-48, and the interaction was modulated by tunicamycin-induced ER stress, DBeQ or both (Fig 4C). Interestingly, when the reverse experiment was carried out, Reptin was found in the p97/CDC-48 immunoprecipitate as well as a slower migrating Reptin immunoreactive species (Fig 4D, arrow). Sequential immunoprecipitation with p97/CDC-48 and Reptin antibodies suggested that the latter corresponds to an ubiquitylated form of Reptin (Fig 4E). Hence, p97/CDC-48 might control Reptin levels through an ubiquitin-dependent mechanism. Figure 4. p97/CDC-48-mediated degradation of Reptin upon ER stress Reptin, Pontin, calnexin and quantification by immunoblot. Values are expressed as a percentage of the initial protein abundance in total HuH7 cell lysate before addition of tunicamycin (5 μg/ml), (Mean ± SD, N = 5). *P < 0.05; **P < 0.01. Immunoblot analysis of Reptin in total protein extracts from HuH7 cells exposed to tunicamycin (5 μg/ml) for 0–2 h. Protein levels were normalized to Calnexin (mean ± SD, N = 3). *P < 0.05; **P < 0.01. HuH7 cells expressing FLAG tagged Reptin were treated either with the p97/CDC-48 inhibitor DBeQ (20 μM, D), the ER stress inducer tunicamycin (2 μg/ml; T) or both for 4 h. FLAG tagged Reptin was immunoprecipitated from total protein extracts using anti-FLAG antibodies, and p97/CDC-48 association was analyzed by immunoblot. HuH7 cells were treated either with DBeQ (20 μM, D), (2 μg/ml; T) or both for 4 h. P97/CDC-48 was immunoprecipitated from total protein extracts using an antibody specific for p97/CDC-48, and reptin association was analyzed by immunoblotting. HuH7 cells were treated either with DBeQ (20 μM, D), tunicamycin (2 μg/ml; T) or both for 4 h. P97/CDC-48 was immunoprecipitated from total protein extracts using anti-p97/CDC-48 antibodies. P97/CDC-48 immunoprecipitate was disrupted with 50 μl of 1% SDS and heated at 95°C for 5 min. Beads were removed and the supernatant quenched with PBS containing 1% TX-100. Reptin was then sequentially immunoprecipitated and the resulting immunoprecipitate immunoblotted with anti-Ubiquitin or anti-Reptin antibodies. Download figure Download PowerPoint XBP1 mRNA splicing and ATF6 activation are partly regulated by a p97/reptin signaling axis To follow up on the role of Reptin degradation upon ER stress in the expression of ER stress genes, we sought to test whether artificial modulation of Reptin expression also impacted the activation of the three UPR signaling arms. Reptin silencing slightly increased the expression of the ER-stress-upregulated chaperones GRP78 and GRP94, which are canonical targets of ATF6 and XBP1s signaling, under basal conditions (Fig 5A), but did not affect tunicamycin-induced phosphorylation of eIF2α (Fig 5B). ATF6 cleavage activation was increased after Reptin silencing in HuH7 cells (Fig 5C). In accordance with this observation, reptin silencing also enhanced the expression of XBP1u mRNA under basal conditions, as could be expected since XBP1u is a target gene of ATF6 (Fig 5D). Moreover, this occurred without affecting the expression levels of the newly discovered XBP1 mRNA ligase RtcB 12 (Fig 5D). XBP1 mRNA splicing was also increased when Reptin was silenced both in basal conditions and ER stress (Fig 5E). Conversely, DBeQ-mediated p97/CDC-48 inhibition (Fig 5F) or siRNA-mediated p97/CDC-48 silencing (Supplementary Fig S6) and the subsequent stabilization of Reptin led to reduced XBP1 mRNA splicing. Hence, partial stabilization of Reptin has a major impact on XBP1 mRNA splicing, which in turn impacts dramatically on the expression of various UPRER genes. However, we could not detect an interaction between Reptin and XBP1s protein (Fig 5G). Altogether, these results might indicate that Reptin is degraded through ubiquitin and p97/CDC-48-dependent mechanisms under ER stress and further support the role of Reptin in the control of select UPRER genes through repression of XBP1 mRNA splicing and of ATF6 activation. Figure 5. Reptin silencing enhances ATF6 and XBP1s activation GRP78 and GRP94 expression was detected using anti-KDEL antibodies (top blot) in HuH7 cells treated or not with tunicamycin and/or doxycycline (Dox) to induce reptin silencing (bottom blot). Expression of p97 was also monitored (middle blot). eIF2α phosphorylation was monitored using specific antibodies (top blot) and reported to the total expression (bottom blot) in HuH7 cells treated or not with tunicamycin and/or doxycycline (Dox). ATF6 activation was monitored in the same experimental conditions using antibodies against the N-terminal domain of ATF6. Expression of unspliced XBP1 mRNA as determined by RT–PCR and expression of the XBP1s ligase RTCB as determined by immunoblot using anti-RTCB antibodies in HuH7 cells treated or not with tunicamycin and/or doxycycline (Dox). XBP-1 mRNA splicing as determined by RT–PCR under basal conditions or upon tunicamycin treatment (5 μg/ml for 16 h) in HuH7 cells subjected or not to doxycycline-induced Reptin silencing. Three independent experiments were performed, and a representative image is shown. HuH7 cells were treated either with DBeQ (20 μM, D), tunicamycin (2 μg/ml; T) or both for 4 h. XBP-1 mRNA splicing was determined by RT–PCR (Mean ± SD, N = 3). P-values were calculated using multiple t-test corrected using the Holm-Sidak method. *P < 0.05; **P < 0.01. HuH7 cells were treated with tunicamycin (2 μg/ml; T) for 1 h. XBP1s was immunoprecipitated, and the complex was immunoblotted with anti-Reptin antibodies (top blot). Total cell lysate (TCL) was immunoblotted with anti-Reptin (middle blot) or anti-XBP1s (bottom blot). Download figure Download PowerPoint In the present work, we have uncovered a novel regulatory mechanism of UPRER genes expression in response to ER stress conserved throughout metazoan evolution involving two AAA+ ATPases, RUVB-2 (or Reptin) and CDC-48 (or p97). In this model, RUVB-2, which mostly localizes to the cytoplasm and the nucleus, plays an important role in the regulation of XBP1 mRNA splicing by a yet unknown mechanism. Upon ER stress, RUVB-2 is degraded through an ubiquitin and p97/CDC-48-dependent mechanism, thereby allowing the ER-stress-specific transcription factors ATF6 and XBP-1 to activate the transcription of UPRER genes. Beyond unravelling a novel UPRER regulatory network, our data point toward the putative role of Reptin in non-conventional mRNA splicing. Our findings suggest that p97/CDC-48-induced degradation of target proteins plays an important role in the ER homeostasis control both, in the cytoplasm, to influence ERAD and to modulate UPRER gene transcription 13. Materials and Methods RNAi screen The RNAi feeding screen was performed in liquid culture using EM2 animals and carried out as previously described with some modifications 4. RNAi clones from the Worm ORFeome version 1.1 library 6 were grown overnight at 37°C in 96-well plates. Each RNAi plate included a positive control (Y37D8A.10 encoding for a signal peptidase identified in a preliminary screen or BC14636 worms fed with the L4440 empty vector) and a negative control (gfp RNAi). RNAi expression was induced with 1 mM IPTG for 1 h before bacteria were added to the L1 larvae. Adult worms were bleached,and the obtained L1 larvae (200) were added to each well of 96-well plates along with the induced bacteria and S-Medium, 50 μg/ml ampicillin, 1 mM IPTG buffer with a final well volume of 150 μl. The 96-well plates were incubated at 20°C with shaking. Forty-eight hours later, ER stress was induced by tunicamycin (0.5 μg/ml) for 16 h, and measurements were taken using the COPAS Biosort flow cytometer (Union Biometrica, Holliston, MA, USA). Experiments for each 96-well plate from the RNAi library were performed in duplicate. Fluorescence average value for each plate was calculated and used to calculate the individual RNAi fold change. Plates showing no fluorescence induction in the positive control, no fluorescence decrease in negative control, or a high fluorescence mean were discarded and retested. COPAS measurements The COPAS biosort analyzer was purchased from Union Biometrica (Holliston, MA, USA). Photomultiplicator tube control (PMT1) was set up at 600 so that the green fluorescence emission was not saturated in BC14636 worms exposed to tunicamycin (maximum signal) and still detectable in EM2 worms exposed to gfp RNAi (minimum signal). Plates were read through a ReFLx module. Raw data extracted from COPAS included worm axial length (time of flight), worm number (extinction) and fluorescence (green fluorescence emission). Raw data were processed as previously described 14 and used for quantitative analyses. Acknowledgements We thank Dr. R. Pedeux for help with the post-translational modifications of reptin, E Attebi and K Rebora for help with C. elegans screens, Drs E. Snapp and F. Schoenen for the ERSE::Tomato construct and DBeC, respectively, Dr. S. Mitani for the cdc-48.1(tm544) and cdc-48.2(tm659) strains. This work was funded by grants from “Institut National du Cancer” to EC and JR. EM was funded by a scolarship from Association Française contre les Myopathies. Author contributions EM, AAR, ST, KB, EC, JWD and NPL performed experiments. LG and DD developed the bioinformatics tools. MB supervised the proteomics analysis. JR and FP provided tools. EM, DD and EC coordinated the study. EM, MFZ, DD and EC wrote the manuscript. Conflict of interest The authors declare that they have no conflict of interest. Supporting Information Supplementary Figures (PDF document, 5.2 MB) Supplementary Table S1 (Word document, 35 KB) Supplementary Table S2 (Word document, 84 KB) Supplementary Table S3 (Word document, 184 KB) Supplementary Table S4 (Word document, 208.5 KB) Supplementary Table S5 (Word document, 117.5 KB) Supplementary Table S6 (Word document, 29 KB) Review Process File (PDF document, 1.4 MB) References Ellgaard L, Helenius A (2003) Quality control in the endoplasmic reticulum. Nat Rev Mol Cell Biol 4: 181–191CrossrefCASPubMedWeb of Science®Google Scholar Hetz C, Chevet E, Harding HP (2013) Targeting the unfolded protein response in disease. 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Specific nuclear domains are nonrandomly positioned within the nuclear space, and this preferential positioning has been shown to play an important role in genome activity and stability. Well-known examples include the organization of repetitive DNA in telomere clusters or in the chromocenter of Drosophila and mammalian cells, which may provide a means to control the availability of general repressors, such as the heterochromatin protein 1 (HP1). We have specifically characterized the intranuclear positioning of in vivo fluorescence of the Caenorhabditis elegans HP1 homologue HPL-2 as a marker for heterochromatin domains in developing embryos. For this purpose, the wavelet transform modulus maxima (WTMM) segmentation method was generalized and adapted to segment the small embryonic cell nuclei in three dimensions. The implementation of a radial distribution algorithm revealed a preferential perinuclear positioning of HPL-2 fluorescence in wild-type embryos compared with the diffuse and homogeneous nuclear fluorescence observed in the lin-13 mutants. For all other genotypes analyzed, the quantitative analysis highlighted various degrees of preferential HPL-2 positioning at the nuclear periphery, which directly correlates with the number of HPL-2 foci previously counted on 2D projections. Using a probabilistic 3D cell nuclear model, we found that any two nuclei having the same number of foci, but with a different 3D probabilistic positioning scheme, can have significantly different counts in the 2D maximum projection, thus showing the deceptive limitations of using techniques of 2D maximum projection foci counts. By this approach, a strong perinuclear positioning of HPL-2 foci was brought into light upon inactivation of conserved chromatin-associated proteins, including the HAT cofactor TRAPP.

Chromatin regulators contribute to the maintenance of the germline transcriptional program. In the absence of SET-2, the Caenorhabditis elegans homolog of the SET1/COMPASS H3 Lys4 (H3K4) methyltransferase, animals show transgenerational loss of germline identity, leading to sterility. To identify transcriptional signatures associated with progressive loss of fertility, we performed expression profiling of set-2 mutant germlines across generations. We identify a subset of genes whose misexpression is first observed in early generations, a step we refer to as priming; their misexpression then further progresses in late generations, as animals reach sterility. Analysis of misregulated genes shows that down-regulation of germline genes, expression of somatic transcriptional programs, and desilencing of the X-chromosome are concurrent events leading to loss of germline identity in both early and late generations. Upregulation of transcription factor LIN-15B, the C/EBP homolog CEBP-1, and TGF-β pathway components strongly contribute to loss of fertility, and RNAi inactivation of cebp-1 and TGF-β/Smad signaling delays the onset of sterility, showing they individually contribute to maintenance of germ cell identity. Our approach therefore identifies genes and pathways whose misexpression actively contributes to the loss of germ cell fate. More generally, our data shows how loss of a chromatin regulator in one generation leads to transcriptional changes that are amplified over subsequent generations, ultimately leading to loss of appropriate cell fate.

In yeast, a position-dependent chromatin-mediated silencing affects several regions of the genome, includingthe silent mating-type loci, telomeric regions, and rDNA.At HM and telomeric loci, repression requires a complexof the silent information regulator proteins (Sir2-Sir4p)that binds nucleosomes via the amino-terminal tails of histones H3 and H4. Nucleation of this complex requiresinteraction with sequence-specific DNA-binding proteins, among which figure Rap1, Abf1, and ORC. Weshow here that both the dosage and balance of Sir proteinsare critical for silencing. Different domains of thesehighly modular proteins are involved in regulating Sirprotein distribution among potential sites of repression.In particular, two factors that bind the amino-terminal271 amino acids of Sir4p (i.e., Sir1p, Sif2p) compete forlimiting pools of Sir factors, antagonizing the formationof repressed chromatin at telomeres. In contrast, the interaction of Ku70/80 with the carboxyl terminus of Sir4pappears to help recruit Sir proteins to telomeres and perhaps under specific conditions to internal sites...

In the yeast Saccharomyces cerevisiae, transcriptional repression at the silent mating-type loci HML and HMR and at telomeres has many of the hallmarks of position-effect variegation in Drosophila, where the condensed higher-order structure of heterochromatin “spreads” into adjacent chromatin, inactivating nearby genes (for review, see Sandell and Zakian 1992). The yeast mating-type genes have provided a genetically tractable system to identify trans-acting factors and cis-acting sequences required for this repression mechanism (for review, see Laurenson and Rine 1992). In particular, deletion studies have identified a cis-acting silencer with a minimal size of 134 bp, which is required for the repression of transcription of the mating-type genes at HMR (Brand et al. 1987). Similar elements have been mapped at HML (Mahoney and Broach 1989). Like enhancers, these silencers function in either orientation and at variable distances from the targeted promoter. A number of trans-acting factors are implicated in the mechanism of...

RNA silencing processes use exogenous or endogenous RNA molecules to specifically and robustly regulate gene expression. In C. elegans, initial mechanistic descriptions of the different silencing processes focused on posttranscriptional regulation. In this review, we discuss recent work showing that, in this model organism, RNA silencing also controls the transcription of target genes by inducing heterochromatin formation. Specifically, it has been shown that ribonucleoprotein complexes containing small RNAs, either processed from exogenous dsRNA or synthesized from the genome itself, and proteins of the Argonaute family, mediate the deposition of repressive histone marks at the targeted loci. Interestingly, the accumulation of repressive marks is required for the inheritance of the silencing effect and the establishment of an epigenetic memory that discriminates self- from non-self-RNAs.

Serine 2 phosphorylation (S2P) within the CTD of RNA polymerase II is considered a Cdk9/Cdk12-dependent mark required for 3'-end processing. However, the relevance of CTD S2P in metazoan development is unknown. We show that cdk-12 lesions or a full-length CTD S2A substitution results in an identical phenotype in Caenorhabditis elegans Embryogenesis occurs in the complete absence of S2P, but the hatched larvae arrest development, mimicking the diapause induced when hatching occurs in the absence of food. Genome-wide analyses indicate that when CTD S2P is inhibited, only a subset of growth-related genes is not properly expressed. These genes correspond to SL2 trans-spliced mRNAs located in position 2 and over within operons. We show that CDK-12 is required for maximal occupancy of cleavage stimulatory factor necessary for SL2 trans-splicing. We propose that CTD S2P functions as a gene-specific signaling mark ensuring the nutritional control of the C. elegans developmental program.

Abstract The SIN3 transcriptional coregulator influences gene expression through multiple interactions that include histone deacetylases (HDACs). Haploinsufficiency and mutations in SIN3 are the underlying cause of Witteveen-Kolk syndrome and related intellectual disability (ID)/autism syndromes, emphasizing its key role in development. However, little is known about the diversity of its interactions and functions in developmental processes. Here we show that loss of SIN-3, the single SIN3 homologue in Caenorhabditis elegans , results in maternal effect sterility associated with deregulation of the germline transcriptome, including desilencing of X-linked genes. We identify at least two distinct SIN3 complexes containing specific HDACs, and show that they differentially contribute to fertility. Single cell smFISH reveals that in sin-3 mutants, the X chromosome becomes re-expressed prematurely and in a stochastic manner in individual germ cells. Furthermore, we identify histone residues whose acetylation increases in the absence of SIN3. Together, this work provides a powerful framework for the in vivo study of SIN3 and associated proteins.

The transcriptional co-regulator SIN3 influences gene expression through multiple interactions that include histone deacetylases. Haploinsufficiency and mutations in SIN3 are the underlying cause of Witteveen-Kolk syndrome and related intellectual disability and autism syndromes, emphasizing its key role in development. However, little is known about the diversity of its interactions and functions in developmental processes. Here, we show that loss of SIN-3, the single SIN3 homolog in Caenorhabditis elegans, results in maternal-effect sterility associated with de-regulation of the germline transcriptome, including de-silencing of X-linked genes. We identify at least two distinct SIN3 complexes containing specific histone deacetylases and show that they differentially contribute to fertility. Single-cell, single-molecule fluorescence in situ hybridization reveals that in sin-3 mutants the X chromosome becomes re-expressed prematurely and in a stochastic manner in individual germ cells, suggesting a role for SIN-3 in its silencing. Furthermore, we identify histone residues whose acetylation increases in the absence of SIN-3. Together, this work provides a powerful framework for the in vivo study of SIN3 and associated proteins.

Changes in histone post-translational modifications are associated with aging through poorly defined mechanisms. Histone 3 lysine 4 (H3K4) methylation at promoters is deposited by SET1 family methyltransferases acting within conserved multiprotein complexes known as COMPASS. Previous work yielded conflicting results about the requirement for H3K4 methylation during aging. Here, we reassessed the role of SET1/COMPASS-dependent H3K4 methylation in Caenorhabditis elegans lifespan and fertility by generating set-2(syb2085) mutant animals that express a catalytically inactive form of SET-2, the C. elegans SET1 homolog. We show that set-2(syb2085) animals retain the ability to form COMPASS, but have a marked global loss of H3K4 di- and trimethylation (H3K4me2/3). Reduced H3K4 methylation was accompanied by loss of fertility, as expected; however, in contrast to earlier studies, set-2(syb2085) mutants displayed a significantly shortened, not extended, lifespan and had normal intestinal fat stores. Other commonly used set-2 mutants were also short-lived, as was a cfp-1 mutant that lacks the SET1/COMPASS chromatin-targeting component. These results challenge previously held views and establish that WT H3K4me2/3 levels are essential for normal lifespan in C. elegans.

Deposition of histone H3 lysine 4 (H3K4) methylation at promoters is catalyzed by the SET1/COMPASS complex and is associated with context-dependent effects on gene expression and local changes in chromatin organization. The role of SET1/COMPASS in shaping chromosome architecture has not been investigated. Here we used Caenorhabditis elegans to address this question through a live imaging approach and genetic analysis. Using quantitative FRET (Förster resonance energy transfer)-based fluorescence lifetime imaging microscopy (FLIM) on germ cells expressing histones eGFP-H2B and mCherry-H2B, we find that SET1/COMPASS influences meiotic chromosome organization, with marked effects on the close proximity between nucleosomes. We further show that inactivation of set-2, encoding the C. elegans SET1 homologue, or CFP-1, encoding the chromatin targeting subunit of COMPASS, enhances germline chromosome organization defects and sterility of condensin-II depleted animals. set-2 loss also aggravates germline defects resulting from conditional inactivation of topoisomerase II, another structural component of chromosomes. Expression profiling of set-2 mutant germlines revealed only minor transcriptional changes, suggesting that the observed effects are at least partly independent of transcription. Altogether, our results are consistent with a role for SET1/COMPASS in shaping meiotic chromosomes in C. elegans, together with the non-histone proteins condensin-II and topoisomerase. Given the high degree of conservation, our findings expand the range of functions attributed to COMPASS and suggest a broader role in genome organization in different species.

Abstract A current model for Caenorhabditis elegans vulval cell fate specification is that SynMuv genes act redundantly in the hyp7 hypodermal syncytium to repress the LIN-3/EGF inducer and prevent ectopic vulval induction of vulva precursor cells (VPCs). Here we show that the SynMuv gene hpl-2/HP1 has an additional function in VPCs, where it may act through target genes including LIN-39/Hox.

Abstract The CFP1 CXXC zinc finger protein targets the SET1/COMPASS complex to non-methylated CpG rich promoters to implement tri-methylation of histone H3 Ly4 (H3K4me3). Although H3K4me3 is widely associated with gene expression, the effects of CFP1 loss depend on chromatin context, so it is important to understand the relationship between CFP1 and other chromatin factors. Using a proteomics approach, we identified an unexpected link between C. elegans CFP-1 and a Rpd3/Sin3 histone deacetylase complex. We find that mutants of CFP-1, SIN-3, and the catalytic subunit SET-2/SET1 have similar phenotypes and misregulate common genes. CFP-1 directly binds SIN-3 through a region including the conserved PAH1 domain and recruits SIN-3 and the HDA-1/HDAC subunit to H3K4me3 enriched promoters. Our results reveal a novel role for CFP-1 in mediating interaction between SET1/COMPASS and a Sin3 HDAC complex at promoters and uncover coordinate regulation of gene expression by chromatin complexes having distinct activities.

A correction to this paper has been published: https://doi.org/10.1007/s11306-021-01784-5

Abstract Mitochondrial function relies on the coordinated transcription of mitochondrial and nuclear genomes to assemble respiratory chain complexes. Across species, the SIN3 coregulator influences mitochondrial functions, but how its loss impacts mitochondrial homeostasis and metabolism in the context of a whole organism is unknown. Exploring this link is important because SIN3 haploinsufficiency causes intellectual disability/autism syndromes and SIN3 plays an important role in tumor biology. Here we show that loss of C. elegans SIN-3 results in transcriptional deregulation of mitochondrial- and nuclear encoded mitochondrial genes, potentially leading to mito-nuclear imbalance. Consistent with impaired mitochondrial function, sin-3 mutants show extensive mitochondrial fragmentation by transmission electron microscopy (TEM) and in vivo imaging, and altered oxygen consumption. Metabolomic analysis of sin-3 mutant animals identifies a signature of mitochondria stress, and deregulation of methionine flux resulting in decreased S-adenosyl methionine (SAM), and increased polyamine levels. Our results identify SIN3 as a key regulator of mitochondrial dynamics and metabolic flux, with important implications for human pathologies.

Abstract Post-translational modification of histones, particularly lysine methylation, are thought to play a crucial role in the aging process. Histone 3 lysine 4 (H3K4) methylation, a modification associated with active chromatin, is mediated by a family of SET1 methyltransferases acting within conserved multiprotein complexes known as COMPASS. Previous work in model organisms with partial or complete deletion of COMPASS subunits has yielded conflicting results about the requirement for H3K4 methylation during aging. Here, we reassessed the role of SET1/COMPASS-dependent H3K4 methylation in Caenorhabditis elegans lifespan regulation and fertility by generating set-2(syb2085) mutant animals that express a catalytically inactive form of SET-2, the C. elegans homolog of SET1. We show that animals bearing catalytically inactive SET-2 retain the ability to form COMPASS complexes but have a marked global loss of H3K4 dimethylation and trimethylation. Consistent with previous work, reduced H3K4 methylation was accompanied by loss of fertility; however, in striking contrast to earlier studies, set-2(syb2085) mutants displayed a significantly shortened, not extended, lifespan and had normal intestinal fat stores. Furthermore, other commonly used set-2 mutants were also short-lived, as was a cfp-1 mutant that lacks a non-catalytic SET1/COMPASS component and displays reduced H3K4 methylation. These results challenge previously held views and establish that wild-type H3K4 methylation levels are necessary to achieve a normal lifespan in C. elegans .

Abstract Chromatin factors contribute to germline maintenance by preserving a germline-appropriate transcriptional program. In the absence of the conserved histone H3 Lys4 (H3K4) methyltransferase SET-2, C. elegans germ cells progressively lose their identity over generations, leading to sterility. How this transgenerational loss of fertility results from the absence of SET-2 is unknown. Here we performed expression profiling across generations on germlines from mutant animals lacking SET-2 activity. We found that gene deregulation occurred in 2 steps: a priming step in early generations progressing to loss of fertility in later generations. By performing Within-Class Analysis (WCA), a derivative of Principal Component Analysis, we identified transcriptional signatures associated with SET-2 inactivation, both at the priming step and later on during loss of fertility. Further analysis showed that repression of germline genes, derepression of somatic programs, and X-chromosome desilencing through interference with PRC2-dependent repression, are priming events driving loss of germline identity in the absence of SET-2. Decreasing expression of identified priming genes, including the C/EBP homologue cebp-1 and TGF-β pathway components, was sufficient to delay the onset of sterility, suggesting that they individually contribute to the loss of germ cell fate. Altogether, our findings illustrate how the loss of a chromatin regulator at one generation can progressively deregulate multiple transcriptional and signaling programs, ultimately leading to loss of appropriate cell fate.

Abstract Auxins are plant growth regulators that influence most aspects of plant development through complex mechanisms. The development of an auxin-inducible degradation (AID) system has enabled rapid, conditional protein depletion in yeast and cultured cells. More recently, the system was successfully adapted to C. elegans to achieve auxin-dependent degradation of targets in all tissues and developmental stages. Whether auxin treatment alone has an impact on nematode physiology is an open question. Here we show that indole-3-acetic acid (IAA), the auxin most commonly used to trigger AID in worms, functions through the conserved IRE-1/XBP-1 branch of the Unfolded Protein Response (UPR) to promote resistance to Endoplasmic Reticulum (ER) stress. Because of the central function played by the UPR in protein folding, lipid biosynthesis and lifespan regulation, these results suggest that extreme caution should be exercised when using the AID system to study these and related processes.

The PTEN tumour suppressor encodes a phosphatase, and its daf-18 orthologue in Caenorhabditis elegans negatively regulates the insulin/IGF-1 DAF-2 receptor pathway that influences lifespan in worms and other species.In order to identify new DAF-18 regulated pathways involved in aging, we initiated a candidate RNAi feeding screen for clones that lengthen lifespan.Here, we report that smg-1 inactivation increases average lifespan in a daf-18 dependent manner.Genetic analysis is consistent with SMG-1 acting at least in part in parallel to the canonical DAF-2 receptor pathway, but converging on the transcription factor DAF-16/FOXO.SMG-1 is a serine-threonine kinase which plays a conserved role in nonsense-mediated mRNA decay (NMD) in worms and mammals.In addition, human SMG-1 has also been implicated in the p53-mediated response to genotoxic stress.The effect of smg-1 inactivation on lifespan appears to be unrelated to its NMD function, but requires the p53 tumour suppressor orthologue cep-1.Furthermore, smg-1 inactivation confers a resistance to oxidative stress in a daf-18-, daf-16-and cep-1-dependent manner.We propose that the role of SMG-1 in lifespan regulation is at least partly dependent on its function in oxidative stress resistance.Taken together, our results unveil a novel role for SMG-1 in lifespan regulation.

Abstract Background Histone-modifying activities play important roles in gene expression and influence higher-order genome organization. SET1/COMPASS (Complex Proteins Associated with Set1) deposits h istone H3 lysine 4 (H3K4) methylation at promoter regions and is associated with context-dependent effects on gene expression. Whether it also contributes to higher-order chromosome organization has not been explored. Results Using a quantitative FRET (Förster resonance energy transfer)-based fluorescence lifetime imaging microscopy (FLIM) approach to assay nanometer scale chromatin compaction in live animals, we reveal a novel role for SET1/COMPASS in structuring meiotic chromosomes in the C. elegans germline . Inactivation of SET-2, the C. elegans homologue of SET1, strongly enhanced chromosome organization defects and loss of fertility resulting from depletion of condensin-II, and aggravated defects in chromosome morphology resulting from inactivation of topoisomerase II, another major structural component of chromosomes. Loss of CFP-1, the chromatin targeting subunit of COMPASS, similarly affected germline chromatin compaction measured by FLIM-FRET and enhanced condensin-II knock-down phenotypes. Conclusions The data presented here are consistent with a role of SET1/ COMPASS in shaping meiotic chromosomes in the C. elegans germline. This new insight has important implications for how c hromatin-modifying complexes and histone modifications may cooperate with non histone-proteins to achieve proper chromosome organization, not only in meiosis, but also in mitosis.

Abstract Deposition of histone H3 lysine 4 (H3K4) methylation at promoters by SET1/COMPASS is associated with context-dependent effects on gene expression and local changes in chromatin organization. Whether SET1/COMPASS also contributes to higher-order chromosome structure has not been investigated. Here, we address this question by quantitative FRET (Förster resonance energy transfer)-based fluorescence lifetime imaging microscopy (FLIM) on C. elegans germ cells expressing histones H2B-eGFP and H2B-mCherry. We find that SET1/COMPASS subunits strongly influence meiotic chromosome organization, with marked effects on the close proximity between nucleosomes. We further show that inactivation of SET-2, the C. elegans homologue of SET1, or CFP-1, the chromatin targeting subunit of COMPASS, strongly enhance chromosome organization defects and loss of fertility resulting from depletion of condensin-II. Defects in chromosome morphology resulting from conditional inactivation of topoisomerase II, another structural component of chromosomes, were also aggravated in the absence of SET-2. Combined, our in vivo findings suggest a model in which the SET1/COMPASS histone methyltransferase complex plays a role in shaping meiotic chromosome in cooperation with the non-histone proteins condensin-II and topoisomerase.