Jef D. Boeke

The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.

A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed. These strains minimize or eliminate the homology to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. Because the homology between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications. We also report the construction of new members of the pRS400 series of vectors, containing the kanMX, ADE2 and MET15 genes. © 1998 John Wiley & Sons, Ltd.

5-FOA is an extremely useful reagent for the selection of Ura- cells amid a population of Ura+ cells. The selection is effective in transformation and recombination studies where loss of URA3+ is desired. A new plasmid shuffling procedure based on the 5-FOAR selection permits the recovery of conditional lethal mutations in cloned genes that encode vital functions.

Human L1 elements are highly abundant poly(A) (non-LTR) retrotransposons whose second open reading frame (ORF2) encodes a reverse transcriptase (RT). We have identified an endonuclease (EN) domain at the L1 ORF2 N-terminus that is highly conserved among poly(A) retrotransposons and resembles the apurinic/apyrimidinic (AP) endonucleases. Purified L1 EN protein (L1 ENp) makes 5'-PO4, 3'-OH nicks in supercoiled plasmids, shows no preference for AP sites, and preferentially cleaves sequences resembling L1 in vivo target sequences. Mutations in conserved amino acid residues of L1 EN abolish its nicking activity and eliminate L1 retrotransposition. We propose that L1 EN cleaves the target site for L1 insertion and primes reverse transcription.

We previously isolated two human L1 elements (L1.2 and LRE2) as the progenitors of disease-producing insertions. Here, we show these elements can actively retrotranspose in cultured mammalian cells. When stably expressed from an episome in HeLa cells, both elements retrotransposed into a variety of chromosomal locations at a high frequency. The retrotransposed products resembled endogenous L1 insertions, since they were variably 5' truncated, ended in poly(A) tracts, and were flanked by target-site duplications or short deletions. Point mutations in conserved domains of the L1.2-encoded proteins reduced retrotransposition by 100- to 1000-fold. Remarkably, L1.2 also retrotransposed in a mouse cell line, suggesting a potential role for L1-based vectors in random insertional mutagenesis.

We have followed Ty transposition with a donor Ty element, TyH3, whose expression is under the control of the GAL1 promoter. Sequence analysis reveals dramatic structural differences in TyH3 before and after transposition. If the donor TyH3 is marked with an intron-containing fragment, the intron is correctly spliced out of the Ty during transposition, suggesting that the Ty RNA is the intermediate for transposition. Furthermore, the pattern of sequence inheritance in progeny Ty insertions derived from the marked Ty follows the predictions of the model of retroviral reverse transcription. Comparison of marked Ty elements before and after movement shows that transposition is highly mutagenic to the Ty element. These results demonstrate that during transposition, Ty sequence information flows from DNA to RNA to DNA.

Retrotransposable elements are deleterious at many levels, and the failure of host surveillance systems for these elements can thus have negative consequences. However, the contribution of retrotransposon activity to ageing and age-associated diseases is not known. Here we show that during cellular senescence, L1 (also known as LINE-1) retrotransposable elements become transcriptionally derepressed and activate a type-I interferon (IFN-I) response. The IFN-I response is a phenotype of late senescence and contributes to the maintenance of the senescence-associated secretory phenotype. The IFN-I response is triggered by cytoplasmic L1 cDNA, and is antagonized by inhibitors of the L1 reverse transcriptase. Treatment of aged mice with the nucleoside reverse transcriptase inhibitor lamivudine downregulated IFN-I activation and age-associated inflammation (inflammaging) in several tissues. We propose that the activation of retrotransposons is an important component of sterile inflammation that is a hallmark of ageing, and that L1 reverse transcriptase is a relevant target for the treatment of age-associated disorders.

L1 elements are highly repeated mammalian DNA sequences whose structure suggests dispersal by retrotransposition. A consensus L1 element encodes a protein with sequence similarity to known reverse transcriptases. The second open reading frame from the human L1 element L1.2A was expressed as a fusion protein targeted to Ty1 virus-like particles in Saccharomyces cerevisiae and shown to have reverse transcriptase activity. This activity was eliminated by a missense mutation in the highly conserved amino acid motif Y/F-X-D-D. Thus, L1 represents a potential source of the reverse transcriptase activity necessary for dispersion of the many classes of mammalian retroelements.

The yeast Sir2 protein, required for transcriptional silencing, has an NAD(+)-dependent histone deacetylase (HDA) activity. Yeast extracts contain a NAD(+)-dependent HDA activity that is eliminated in a yeast strain from which SIR2 and its four homologs have been deleted. This HDA activity is also displayed by purified yeast Sir2p and homologous Archaeal, eubacterial, and human proteins, and depends completely on NAD(+) in all species tested. The yeast NPT1 gene, encoding an important NAD(+) synthesis enzyme, is required for rDNA and telomeric silencing and contributes to silencing of the HM loci. Null mutants in this gene have significantly reduced intracellular NAD(+) concentrations and have phenotypes similar to sir2 null mutants. Surprisingly, yeast from which all five SIR2 homologs have been deleted have relatively normal bulk histone acetylation levels. The evolutionary conservation of this regulated activity suggests that the Sir2 protein family represents a set of effector proteins in an evolutionarily conserved signal transduction pathway that monitors cellular energy and redox states.

Sirtuins are a family of NAD+-dependent protein deacetylases widely distributed in all phyla of life. Accumulating evidence indicates that sirtuins are important regulators of organism life span. In yeast, these unique enzymes regulate gene silencing by histone deacetylation and via formation of the novel compound 2'-O-acetyl-ADP-ribose. In multicellular organisms, sirtuins deacetylate histones and transcription factors that regulate stress, metabolism, and survival pathways. The chemical mechanism of sirtuins provides novel opportunities for signaling and metabolic regulation of protein deacetylation. The biological, chemical, and structural characteristics of these unusual enzymes are discussed in this review.

Long interspersed nuclear elements (LINEs or L1s) comprise approximately 17% of human DNA; however, only about 60 of the approximately 400,000 L1s are mobile. Using a retrotransposition assay in cultured human cells, we demonstrate that L1-encoded proteins predominantly mobilize the RNA that encodes them. At much lower levels, L1-encoded proteins can act in trans to promote retrotransposition of mutant L1s and other cellular mRNAs, creating processed pseudogenes. Mutant L1 RNAs are mobilized at 0.2 to 0.9% of the retrotransposition frequency of wild-type L1s, whereas cellular RNAs are mobilized at much lower frequencies (ca. 0.01 to 0.05% of wild-type levels). Thus, we conclude that L1-encoded proteins demonstrate a profound cis preference for their encoding RNA. This mechanism could enable L1 to remain retrotransposition competent in the presence of the overwhelming number of nonfunctional L1s present in human DNA.

Genomic silencing is a fundamental mechanism of transcriptional regulation, yet little is known about conserved mechanisms of silencing. We report here the discovery of four Saccharomyces cerevisiae homologs of the SIR2 silencing gene (HSTs), as well as conservation of this gene family from bacteria to mammals. At least three HST genes can function in silencing; HST1 overexpression restores transcriptional silencing to a sir2 mutant and hst3 hst4 double mutants are defective in telomeric silencing. In addition, HST3 and HST4 together contribute to proper cell cycle progression, radiation resistance, and genomic stability, establishing new connections between silencing and these fundamental cellular processes.

Generalized transcriptional repression of large chromosomal regions in Saccharomyces cerevisiae occurs at the silent mating loci and at telomeres and is mediated by the silent information regulator (SIR) genes. We have identified a novel form of transcriptional silencing in S. cerevisiae in the ribosomal DNA (rDNA) tandem array. Ty1 retrotransposons marked with a weakened URA3 gene (Ty1-mURA3) efficiently integrated into rDNA. The mURA3 marker in rDNA was transcriptionally silenced in a SIR2-dependent manner. MET15 and LEU2 were also partially silenced, indicating that rDNA silencing may be quite general. Deletion of SIR4 enhanced mURA3 and MET15 silencing, but deletion of SIR1 or SIR3 did not affect silencing, indicating that the mechanism of silencing differs from that at telomeres and silent mating loci. Deletion of SIR2 resulted in increased psoralen cross-linking of the rDNA in vivo, suggesting that a specific chromatin structure in rDNA down-regulates polymerase II promoters.

This chapter describes the In Vitro mutagenesis and plasmid shuffling in yeast genes. method for generating mutant alleles uses replicating yeast episomes as a means of exchanging the wild-type gene for mutant copies. The basic scheme for the exchange, known as plasmid shuffling In the first step, one copy of the gene of interest is inactivated in a diploid, and a wild-type copy is propagated in the cell on an episome. This allows the generation of a haploid strain with a chromosomal null allele. Mutagenized copies of the gene are then introduced into this cell on a second episome and exchanged (or shuffled) with the wild-type version. Removal of the wild-type gene, YFG in our example, is the key step in any plasmid shuflting scheme. This can be accomplished by taking advantage of two factors. First, even relatively stable YCp episomes are lost from a cell by missegregation or misrepfication at a rate of 10–2 per generation. Second, compounds are available that prevent the growth of cells carrying specific yeast genes, and in the presence of such compounds these genes act as counterselectable markers, allowing one to directly select for cells which have lost this marker. By including one of these counterselectable markers on the same plasmid that contains the wild-type YFG gene, an investigator can select for cells that have lost the entire plasmid.

A network governing DNA integrity was identified in yeast by a global genetic analysis of synthetic fitness or lethality defect (SFL) interactions. Within this network, 16 functional modules or minipathways were defined based on patterns of global SFL interactions. Modules or genes involved in DNA replication, DNA-replication checkpoint (DRC) signaling, and oxidative stress response were identified as the major guardians against lethal spontaneous DNA damage, efficient repair of which requires the functions of the DNA-damage checkpoint signaling and multiple DNA-repair pathways. This genome-wide genetic interaction network also identified novel components (DIA2, NPT1, HST3, HST4, and the CSM1 module) that potentially contribute to mitotic DNA replication and genomic stability and revealed novel functions of well-studied genes (the CTF18 module) in DRC signaling. This network will guide more detailed characterization of mechanisms governing DNA integrity in yeast and other organisms.

The SIR2 (silent information regulator 2) gene family has diverse functions in yeast including gene silencing, DNA repair, cell-cycle progression, and chromosome fidelity in meiosis and aging. Human homologues, termed sirtuins, are highly conserved but are of unknown function. We previously identified a large imprinted gene domain on 11p15.5 and investigated the 11p15.5 sirtuin SIRT3. Although this gene was not imprinted, we found that it is localized to mitochondria, with a mitochondrial targeting signal within a unique N-terminal peptide sequence. The encoded protein was found also to possess NAD(+)-dependent histone deacetylase activity. These results suggest a previously unrecognized organelle for sirtuin function and that the role of SIRT3 in mitochondria involves protein deacetylation.

We conducted a genome-wide survey of Saccharomyces cerevisiae retrotransposons and identified a total of 331 insertions, including 217 Ty1, 34 Ty2, 41 Ty3, 32 Ty4, and 7 Ty5 elements. Eighty-five percent of insertions were solo long terminal repeats (LTRs) or LTR fragments. Overall, retrotransposon sequences constitute >377 kb or 3.1% of the genome. Independent evolution of retrotransposon sequences was evidenced by the identification of a single-base pair insertion/deletion that distinguishes the highly similar Ty1 and Ty2 LTRs and the identification of a distinct Ty1 subfamily (Ty1'). Whereas Ty1, Ty2, and Ty5 LTRs displayed a broad range of sequence diversity (typically ranging from 70%-99% identity), Ty3 and Ty4 LTRs were highly similar within each element family (most sharing >96% nucleotide identity). Therefore, Ty3 and Ty4 may be more recent additions to the S. cerevisiae genome and perhaps entered through horizontal transfer or past polyploidization events. Distribution of Ty elements is distinctly nonrandom: 90% of Ty1, 82% of Ty2, 95% of Ty3, and 88% of Ty4 insertions were found within 750 bases of tRNA genes or other genes transcribed by RNA polymerase III. tRNA genes are the principle determinant of retrotransposon distribution, and there is, on average, 1.2 insertions per tRNA gene. Evidence for recombination was found near many Ty elements, particularly those not associated with tRNA gene targets. For these insertions, 5'- and 3'-flanking sequences were often duplicated and rearranged among multiple chromosomes, indicating that recombination between retrotransposons can influence genome organization. S. cerevisiae offers the first opportunity to view organizational and evolutionary trends among retrotransposons at the genome level, and we hope our compiled data will serve as a starting point for further investigation and for comparison to other, more complex genomes.

Histone protein post-translational modifications (PTMs) are significant for gene expression and DNA repair. Here we report the identification and validation of a new type of PTM in histones, lysine succinylation. The identified lysine succinylated histone peptides were verified by MS/MS of synthetic peptides, HPLC co-elution, and isotopic labeling. We identified 13, 7, 10, and 7 histone lysine succinylation sites in HeLa, mouse embryonic fibroblast, Drosophila S2, and Saccharomyces cerevisiae cells, respectively. We demonstrated that this histone PTM is present in all eukaryotic cells we examined. Mutagenesis of succinylation sites followed by functional assays implied that histone lysine succinylation can cause unique functional consequences. We also identified one and two histone lysine malonylation sites in HeLa and S. cerevisiae cells, respectively. Our results therefore increase potential combinatorial diversity of histone PTMs and suggest possible new connections between histone biology and metabolism. Histone protein post-translational modifications (PTMs) are significant for gene expression and DNA repair. Here we report the identification and validation of a new type of PTM in histones, lysine succinylation. The identified lysine succinylated histone peptides were verified by MS/MS of synthetic peptides, HPLC co-elution, and isotopic labeling. We identified 13, 7, 10, and 7 histone lysine succinylation sites in HeLa, mouse embryonic fibroblast, Drosophila S2, and Saccharomyces cerevisiae cells, respectively. We demonstrated that this histone PTM is present in all eukaryotic cells we examined. Mutagenesis of succinylation sites followed by functional assays implied that histone lysine succinylation can cause unique functional consequences. We also identified one and two histone lysine malonylation sites in HeLa and S. cerevisiae cells, respectively. Our results therefore increase potential combinatorial diversity of histone PTMs and suggest possible new connections between histone biology and metabolism. Histones and p53 are among the proteins that are found to be most frequently modified (1Kouzarides T. Chromatin modifications and their function.Cell. 2007; 128: 693-705Abstract Full Text Full Text PDF PubMed Scopus (8034) Google Scholar, 2Dai C. Gu W. p53 post-translational modification: Deregulated in tumorigenesis.Trends Mol. Med. 2010; 16: 528-536Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). Collective efforts from the research community identified at least 12 types of protein post-translational modifications (PTMs), 1The abbreviation used is:PTMpost-translational modification. 1The abbreviation used is:PTMpost-translational modification. most of which were identified by mass spectrometry (1Kouzarides T. Chromatin modifications and their function.Cell. 2007; 128: 693-705Abstract Full Text Full Text PDF PubMed Scopus (8034) Google Scholar, 3Garcia B.A. Shabanowitz J. Hunt D.F. Characterization of histones and their post-translational modifications by mass spectrometry.Curr. Opin. Chem. Biol. 2007; 11: 66-73Crossref PubMed Scopus (118) Google Scholar, 4Sakabe K. Wang Z. Hart G.W. β-N-acetylglucosamine (O-GlcNAc) is part of the histone code.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 19915-19920Crossref PubMed Scopus (280) Google Scholar, 5Kruse J.P. Gu W. SnapShot: p53 posttranslational modifications.Cell. 2008; 133: 930-30.e1Abstract Full Text PDF PubMed Scopus (124) Google Scholar). In addition, the search for histone PTMs has not been exhausted and, not only novel sites, but also novel types of modifications continue to be discovered. For example, Hart and co-workers (4Sakabe K. Wang Z. Hart G.W. β-N-acetylglucosamine (O-GlcNAc) is part of the histone code.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 19915-19920Crossref PubMed Scopus (280) Google Scholar) recently identified O-GlcNAc modification as a new type of histone PTM. They demonstrated that O-GlcNAcylation is dynamically changed during mitosis and in response to heat shock. post-translational modification. post-translational modification. Mounting evidence suggests that histone PTMs play a crucial regulatory role in diverse biological processes, such as cell differentiation and organismal development, and that aberrant modification of histones contributes to diseases, including cancer (6Ruthenburg A.J. Li H. Patel D.J. Allis C.D. Multivalent engagement of chromatin modifications by linked binding modules.Nat. Rev. Mol. Cell Biol. 2007; 8: 983-994Crossref PubMed Scopus (816) Google Scholar, 7Martin C. Zhang Y. Mechanisms of epigenetic inheritance.Curr. Opin. Cell Biol. 2007; 19: 266-272Crossref PubMed Scopus (179) Google Scholar). Thus, understanding this epigenetic process and its roles in cellular physiology and diseases demands a comprehensive understanding of all possible histone modifications. At least two major mechanisms are thought to be associated with contributions of histone PTMs to dynamic chromatin-templated processes (1Kouzarides T. Chromatin modifications and their function.Cell. 2007; 128: 693-705Abstract Full Text Full Text PDF PubMed Scopus (8034) Google Scholar, 6Ruthenburg A.J. Li H. Patel D.J. Allis C.D. Multivalent engagement of chromatin modifications by linked binding modules.Nat. Rev. Mol. Cell Biol. 2007; 8: 983-994Crossref PubMed Scopus (816) Google Scholar). First, histone PTMs can directly modulate the packaging of chromatin by altering chemical structures of histones or internucleosomal interactions, through a change of the net charge, hydrogen bonding, size, or hydrophobicity in substrate PTM residues. A modified chromatin therefore in turn regulates the access of DNA-binding proteins, such as transcription factors. For example, neutralization of positive charges of lysine residues has been shown to disrupt interactions between positively charged lysine side chain and negatively charged DNA. Second, histone PTMs regulate chromatin structure and function by recruiting PTM-specific binding proteins (also called “readers”), which recognize modified histones via specialized structural folds, such as bromo, chromo, and plant homeo domain (PHD) domains (8Wysocka J. Swigut T. Xiao H. Milne T.A. Kwon S.Y. Landry J. Kauer M. Tackett A.J. Chait B.T. Badenhorst P. Wu C. Allis C.D. A PHD finger of NURF couples histone H3 lysine 4 trimethylation with chromatin remodelling.Nature. 2006; 442: 86-90Crossref PubMed Scopus (875) Google Scholar, 9Wysocka J. Swigut T. Milne T.A. Dou Y. Zhang X. Burlingame A.L. Roeder R.G. Brivanlou A.H. Allis C.D. WDR5 associates with histone H3 methylated at K4 and is essential for H3 K4 methylation and vertebrate development.Cell. 2005; 121: 859-872Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar, 10Zeng L. Zhou M.M. Bromodomain: An acetyl-lysine binding domain.FEBS Lett. 2002; 513: 124-128Crossref PubMed Scopus (552) Google Scholar). Conversely, histone PTMs can also function by inhibiting the interaction of specific binders with chromatin. The remarkable regulatory potential of histone marks has been well illustrated in histone lysine acetylation and lysine methylation. Modifications at different locations (in the residues of histones) are involved in either activation or repression of gene expression. Acetylation versus methylation at the same histone site can be associated with very different transcriptional programs (11Jenuwein T. Allis C.D. Translating the Histone Code.Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7632) Google Scholar). Interestingly, lysine methylation exists in three forms: mono-, di-, and tri-methylation. Subtle chemical differences in these modifications may lead to very different outcomes. Different forms of lysine methylation can be enriched in different parts of chromatin (heterochromatin or euchromatin) (12Martin C. Zhang Y. The diverse functions of histone lysine methylation.Nat. Rev. Mol. Cell Biol. 2005; 6: 838-849Crossref PubMed Scopus (1591) Google Scholar). They can also be associated with different transcriptional regulatory elements of human genome. For example, histone H3K4 monomethylation specifically marks gene promoters, whereas H3K4 trimethylation is primarily associated with enhancers (13Heintzman N.D. Stuart R.K. Hon G. Fu Y. Ching C.W. Hawkins R.D. Barrera L.O. Van Calcar S. Qu C. Ching K.A. Wang W. Weng Z. Green R.D. Crawford G.E. Ren B. Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome.Nat. Genet. 2007; 39: 311-318Crossref PubMed Scopus (2409) Google Scholar). We recently discovered two types of novel PTMs called lysine succinylation and lysine malonylation in non-histone proteins (see Fig. 1A) (14Zhang Z. Tan M. Xie Z. Dai L. Chen Y. Zhao Y. Identification of lysine succinylation as a new post-translational modification.Nat. Chem. Biol. 2011; 7: 58-63Crossref PubMed Scopus (569) Google Scholar, 15Peng C. Lu Z. Xie Z. Cheng Z. Chen Y. Tan M. Luo H. Zhang Y. He W. Yang K. Zwaans B.M. Tishkoff D. Ho L. Lombard D. He T.C. Dai J. Verdin E. Ye Y. Zhao Y. The first identification of lysine malonylation substrates and its regulatory enzyme.Mol. Cell. Proteomics. 2011; 10 (10.1074/mcp.M111.012658)Abstract Full Text Full Text PDF Scopus (511) Google Scholar). In this study, we report that lysine succinylation and lysine malonylation are new types of histone PTMs. Our preliminary studies in Saccharomyces cerevisiae suggest lysine succinylation and malonylation in histones might have functional consequences. All chemicals, unless otherwise indicated, were of the highest purity available or analytical grade purchased from Sigma-Aldrich. 2,2,3,3-D4-succinic acid was purchased from Cambridge Isotope Laboratories (Andover, MA). Dulbecco's modified Eagle's medium and YPD medium were purchased from Fisher. Schneider's Drosophila medium was purchased from Invitrogen. HeLa and mouse embryonic fibroblast cells were obtained from the ATCC (Manassas, VA). All of the synthetic peptides used in this study were synthesized through customer synthesis using N-(9-fluorenyl)methoxycarbonyl-Lys (mono-tert-butyl succinate)-OH. HeLa and mouse embryonic fibroblast cells were grown to 95% confluence in high glucose (4.5 g/liter) Dulbecco's modified Eagle's medium (with glutamine and sodium pyruvate) containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C with 95% air and 5% CO2. Drosophila S2 cells were grown in Schneider's Drosophila medium containing 10% heat-inactivated fetal bovine serum at 26 °C until the cell density reached 1 × 107 cells/ml. Yeast (BY4741) cells were grown in YPD medium at 30 °C for 16 h with shaking at 230–270 rpm until A600 reached 2.4. For isotopic labeling, HeLa cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 1% penicillin-streptomycin, and 50 mm of sodium D4-succinate for 24 h until 95% confluence. Extraction of the histones followed the acid extraction method described previously (16Shechter D. Dormann H.L. Allis C.D. Hake S.B. Extraction, purification and analysis of histones.Nat. Protoc. 2007; 2: 1445-1457Crossref PubMed Scopus (716) Google Scholar). Chemical propionylation of histone extracts was performed using a procedure previously reported with slight modifications (17Garcia B.A. Mollah S. Ueberheide B.M. Busby S.A. Muratore T.L. Shabanowitz J. Hunt D.F. Chemical derivatization of histones for facilitated analysis by mass spectrometry.Nat. Protoc. 2007; 2: 933-938Crossref PubMed Scopus (279) Google Scholar). Briefly, 3 mg of histone extracts were dissolved in 50 μl of 100 mm ammonium bicarbonate buffer (pH 8.0) and added with 300 μl of propionic anhydride in 300 μl of methanol. Ammonium hydroxide was added to adjust the solution pH to ∼8.0. After incubation at 51 °C for 20 min, the mixture was dried in a SpeedVac. In-solution histone digestion was carried out as previously reported (18Kim S.C. Sprung R. Chen Y. Xu Y. Ball H. Pei J. Cheng T. Kho Y. Xiao H. Xiao L. Grishin N.V. White M. Yang X.J. Zhao Y. Substrate and functional diversity of lysine acetylation revealed by a proteomics survey.Mol. Cell. 2006; 23: 607-618Abstract Full Text Full Text PDF PubMed Scopus (1217) Google Scholar). Enrichment of lysine succinylated and malonylated peptides from tryptic digest of histones, with or without in vitro propionylation, by peptide immunoprecipitation with pan anti-succinyllysine and anti-malonyllysine antibodies (PTM Biolabs Inc., Chicago, IL), was carried out as described previously (18Kim S.C. Sprung R. Chen Y. Xu Y. Ball H. Pei J. Cheng T. Kho Y. Xiao H. Xiao L. Grishin N.V. White M. Yang X.J. Zhao Y. Substrate and functional diversity of lysine acetylation revealed by a proteomics survey.Mol. Cell. 2006; 23: 607-618Abstract Full Text Full Text PDF PubMed Scopus (1217) Google Scholar). Peptide samples were analyzed by a NanoLC-1D plus HPLC system (Eksigent Technologies, Dublin, CA) coupled to an LTQ Orbitrap mass spectrometer (ThermoFisher Scientific, San Jose, CA) as described previously (14Zhang Z. Tan M. Xie Z. Dai L. Chen Y. Zhao Y. Identification of lysine succinylation as a new post-translational modification.Nat. Chem. Biol. 2011; 7: 58-63Crossref PubMed Scopus (569) Google Scholar). The peptides were eluted from a home-made capillary Jupiter C12 column (10-cm length × 75-μm inner diameter, 4-μm particle size, 90 Å pore diameter; Phenomenex, St. Torrance, CA) with a 2-h gradient of 2% to 80% HPLC solvent B (0.1% formic acid in acetonitrile, v/v) in solvent A at a flow rate of 200 nl/min. High resolution full scan MS spectra (from m/z 350 to 1800) acquired in the Orbitrap with resolution r = 60,000 at m/z 400 was followed by MS/MS fragmentation of the 20 most intense ions in the linear ion trap analyzer with collisionally activated dissociation energy of 35%. Verification of lysine succinylated peptides by HPLC/MS/MS analysis of synthetic peptides was used the same method as described previously (14Zhang Z. Tan M. Xie Z. Dai L. Chen Y. Zhao Y. Identification of lysine succinylation as a new post-translational modification.Nat. Chem. Biol. 2011; 7: 58-63Crossref PubMed Scopus (569) Google Scholar). Briefly, the affinity-enriched histone succinyllysine peptide, its synthetic counterpart, and their mixture were analyzed by nano-HPLC/MS/MS, respectively. The mass spectrometric data analysis was performed by MASCOT search engine (v2.1; Matrix Science, London, UK). Peak lists were generated by extract_msn.exe software (v5.0; Thermo Scientific). For protein identification, the data from human, mouse, Drosophila, and yeast were searched against International Protein Index (IPI) human protein database (v 3.70, 87069 sequences), IPI mouse protein database (v 3.74,56860 sequences), UniProtKB Drosophila melanogaster protein database (taxonomy: 7227; 17,526 sequences), and Saccharomyces protein database (YeastORF, 6717 sequences), respectively. Significance threshold (p < 0.05) and Ions score cut-off (0) were used for protein identification. The identified proteins from each species were compiled into a new database for PTM analysis. The identified protein lists were included in supplemental Table 1. The search criteria were: mass error for parent ion mass was set as ±10 ppm and for fragment ion as ±0.5 Da. Enzyme was specified as trypsin with six missing cleavages. Methionine oxidation, lysine acetylation, lysine malonylation (K +86.00039 Da), and lysine succinylation (K +100.01604 Da) were specified as variable modifications. For the propionylated histone sample, lysine propionylation was also specified as a variable modification. All of the peptide identifications with Mascot ion score above 20 were manually verified based on their precursor MS and product MS/MS data. The yeast strain used to test silencing phenotypes for histone H2A and H2B is JDY187 (similar to JDY23 and a derivative of GFY167, MATα his3Δ200 leu2Δ1 trp1Δ63 lys2Δ0 ura3-167 met15Δ0 ade2::his RDN1::Ty1-MET15 TELV::ADE2 hta2-htb2::HygMX4 hta1-htb1::NatMX4 pJD78 [CEN URA3 HTA2-HTB2]). All other phenotypes were tested in JDY142 (similar to JDY92 and a derivative of S288C, MATα his3Δ200 leu2Δ0 lys2Δ0 trp1Δ63 ura3Δ0 met15Δ0 hta2-htb2::HygMX4 hta1-htb1::G418 pJD78 [CEN URA3 HTA2-HTB2]). The histone H2A and histone H2B mutants were generated by gene synthesis and integrated at endogenous HTA1-HTB1 locus. The phenotypic assays were described previously (19Dai J. Hyland E.M. Yuan D.S. Huang H. Bader J.S. Boeke J.D. Probing nucleosome function: A highly versatile library of synthetic histone H3 and H4 mutants.Cell. 2008; 134: 1066-1078Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Except for the Glu substitutions, phenotypes for all other histone H3 and H4 mutants were extracted from a previous study (19Dai J. Hyland E.M. Yuan D.S. Huang H. Bader J.S. Boeke J.D. Probing nucleosome function: A highly versatile library of synthetic histone H3 and H4 mutants.Cell. 2008; 134: 1066-1078Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). We hypothesize the existence of histone lysine succinylation in eukaryotic cells based on the fact that all the major PTMs are present in histones, including but not limited to phosphorylation, acetylation, methylation, ubiquitination, and O-GlcNAc modification. To test for the existence of lysine succinylation in core histones, we carried out Western blotting analysis of core histones from four eukaryotic species using an anti-succinyllysine antibody. The experiment detected lysine succinylation signals from the core histones of all four species tested (Fig. 1B). The signals can be efficiently competed away by a succinyllysine peptide library bearing a fixed succinyllysine at the seventh residue but not its corresponding unmodified peptide library. The specificity of the succinyllysine signals was demonstrated by competition experiments using a succinyllysine peptide library and dot-spot assay (14Zhang Z. Tan M. Xie Z. Dai L. Chen Y. Zhao Y. Identification of lysine succinylation as a new post-translational modification.Nat. Chem. Biol. 2011; 7: 58-63Crossref PubMed Scopus (569) Google Scholar). These results indicate that lysine succinylation is found in histones and could represent an evolutionarily conserved histone mark in eukaryotic cells. To identify succinyllysine sites, we extracted core histones from HeLa cells using a procedure described previously (16Shechter D. Dormann H.L. Allis C.D. Hake S.B. Extraction, purification and analysis of histones.Nat. Protoc. 2007; 2: 1445-1457Crossref PubMed Scopus (716) Google Scholar). The core histones were digested in solution, with or without chemical propionylation, and subjected to affinity enrichment using anti-succinyllysine antibody as reported (18Kim S.C. Sprung R. Chen Y. Xu Y. Ball H. Pei J. Cheng T. Kho Y. Xiao H. Xiao L. Grishin N.V. White M. Yang X.J. Zhao Y. Substrate and functional diversity of lysine acetylation revealed by a proteomics survey.Mol. Cell. 2006; 23: 607-618Abstract Full Text Full Text PDF PubMed Scopus (1217) Google Scholar). Lysine succinylated peptides were analyzed by HPLC/MS/MS analysis and protein sequence alignment to identify succinyllysine sites in histones. The succinyllysine residues can be identified based on a mass shift of + 100 Da at the lysine residue. The experiments led to the identification of 13 sites in HeLa core histones (Fig. 2A). Using the same experimental procedure, we also identified 7, 10, and 7 succinylation sites in histones extracted from cells of yeast, Drosophila, and mouse, respectively. The critical MS/MS spectra were manually verified to ensure the high quality of the peptide identification and are included in the text and supplemental materials for readers' reference (Fig. 3, Fig. 4 and supplemental Figs. S4–S6).Fig. 3HPLC and mass spectrometric verification of histone succinylation at H4K31 (DNIQGITK+100.0150 PAIR). A, high resolution MS/MS spectra of H4K31 succinylation peptide (DNIQGITK+100.0150 PAIR) from affinity-enriched HeLa histone extract using anti-succinyllysine pan antibody (top), the synthetic DNIQGITKsuccPAIR (middle), and the mixture of them (bottom). The insets show the precursor ions. The label Δ designates b or y ions with water and/or ammonia loss. B, extracted ion chromatograms of the in vivo derived H4K31 peptide (top), its synthetic counterpart (middle), and their mixture (bottom). C, MS and MS/MS spectrum of D4-succinyllysine peptide.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4HPLC and mass spectrometric verification of histone succinylation at H4K31 (EIAQDFK+100.0152TDLR). A, high resolution MS/MS spectra of in vivo H3K79 succinylation peptide (EIAQDFK+100.0152TDLR) from affinity-enriched HeLa histone extract (top), the synthetic EIAQDFKsuccTDLR (middle), and the mixture of them (bottom). B, extracted ion chromatograms of the in vivo derived H3K79 peptide (top), its synthetic counterpart (middle), and their mixture (bottom). C, MS and MS/MS spectrum of D4-succinyllysine peptide.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We are reasonably confident that the identified mass shift of + 100 Da is caused by lysine succinylation, because the histone succinyllysine peptides were affinity-purified before MS/MS analysis. Because lysine succinylation is a relatively new PTM, it is desirable to confirm the structure of the identified peptides to ensure that the derived mass shifts of + 100 Da is caused by lysine succinylation. MS/MS and HPLC co-elution are gold standards for verifying peptide identification. Toward this goal, we chemically synthesized three representative succinyllysine peptides: DNIQGITKsuccPAIR, EIAQDFKsuccTDLR, and TVTAMDVVYALKsuccR. We then carried out pair-wise MS/MS and co-elution experiments between the synthetic peptides and their in vivo counterparts, respectively. Our result showed that the high resolution MS/MS fragmentation patterns of in vivo DNIQGITK+100.0150PAIR peptide, the synthetic DNIQGITKsuccPAIR peptide, and their mixture were almost identical (Fig. 3A). Furthermore, the mixture of the in vivo DNIQGITK+100.0150PAIR peptide and the synthetic succinyllysine counterpart showed a single co-eluted peak in the HPLC chromatogram, indicating that the detected + 100.0150 mass shift was caused by a succinyl group (Fig. 3B). Using the same method, we also confirmed the peptide identification for EIAQDFKsuccTDLR, TVTAMDVVYALKsuccR (Fig. 4 and supplemental Fig. S1). To further establish the presence of lysine succinylation in core histones, we carried out in vivo isotopic labeling followed by HPLC/MS/MS analysis of histone peptides as described previously (14Zhang Z. Tan M. Xie Z. Dai L. Chen Y. Zhao Y. Identification of lysine succinylation as a new post-translational modification.Nat. Chem. Biol. 2011; 7: 58-63Crossref PubMed Scopus (569) Google Scholar). In this experiment, we labeled HeLa cells with isotopic succinate (sodium 2,2,3,3-D4-succinate) for 24 h. The core histones were extracted and analyzed using the above described procedure. D4-Labeled succinyllysine was identified in 11 histone peptides (Figs. 3C and 4C and supplemental Fig. S3), suggesting that histone lysine succinylation can be labeled in similar fashion to histone lysine acetylation, presumably by using a succinyl coenzyme A precursor (20MacDonald M.J. Fahien L.A. Brown L.J. Hasan N.M. Buss J.D. Kendrick M.A. Perspective: Emerging evidence for signaling roles of mitochondrial anaplerotic products in insulin secretion.Am. J. Physiol. Endocrinol Metab. 2005; 288: E1-E15Crossref PubMed Scopus (201) Google Scholar). To gain some insight into the biological function of lysine succinylation in core histones, we mutated the modified residue to alanine and arginine to prevent succinylation and to glutamic acid to mimic constitutively succinylated lysine. Of the six residues, we found that the Glu substitution, but not Ala or other substitutions on histone H4K31, significantly reduces cell viability (Fig. 5A). Cells bearing Glu substitutions on other sites had no obvious effect on cell growth. After testing under various conditions as described before (19Dai J. Hyland E.M. Yuan D.S. Huang H. Bader J.S. Boeke J.D. Probing nucleosome function: A highly versatile library of synthetic histone H3 and H4 mutants.Cell. 2008; 134: 1066-1078Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar), we found no significant difference for all mutations on histone H2AK13 and H2BK37 compared with wild type. In addition, all of the mutations on histone H3K79 lose silencing at telomeres and rDNA. No distinction was observed among the Glu substitution and other mutations. However, we found that histone H2AK21E, but not K21A or K21R, was sensitive to methyl methanesulfonate (Fig. 5B), suggesting a potentially deleterious effect of succinylation of this residue. In addition, we identified several unique phenotypes of histone H4K77E mutation (Fig. 5C). It causes a loss of silencing at both telomere and rDNA, with a more profound effect on telomeric silencing. On the other hand, there is no silencing defect in a K77R mutant, whereas the K77A mutant loses silencing at telomere but has slightly increased rDNA silencing (19Dai J. Hyland E.M. Yuan D.S. Huang H. Bader J.S. Boeke J.D. Probing nucleosome function: A highly versatile library of synthetic histone H3 and H4 mutants.Cell. 2008; 134: 1066-1078Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Furthermore, the K77E substitution becomes temperature-sensitive at 37 and 39 °C, which is not observed in the other mutants tested. Lysine malonylation has been reported recently in both bacteria and mammalian cells (15Peng C. Lu Z. Xie Z. Cheng Z. Chen Y. Tan M. Luo H. Zhang Y. He W. Yang K. Zwaans B.M. Tishkoff D. Ho L. Lombard D. He T.C. Dai J. Verdin E. Ye Y. Zhao Y. The first identification of lysine malonylation substrates and its regulatory enzyme.Mol. Cell. Proteomics. 2011; 10 (10.1074/mcp.M111.012658)Abstract Full Text Full Text PDF Scopus (511) Google Scholar). However, it is not known whether lysine malonylation exists in histone proteins. By using affinity enrichment with pan anti-malonyllysine antibody and mass spectrometry, we identified one and two malonyllysine sites in histones from HeLa and S. cerevisiae cells, respectively (Fig. 2A). The malonyllysine peptides can be unambiguously identified based upon the characteristic neutral loss of CO2 peaks in their MS/MS spectra (15Peng C. Lu Z. Xie Z. Cheng Z. Chen Y. Tan M. Luo H. Zhang Y. He W. Yang K. Zwaans B.M. Tishkoff D. Ho L. Lombard D. He T.C. Dai J. Verdin E. Ye Y. Zhao Y. The first identification of lysine malonylation substrates and its regulatory enzyme.Mol. Cell. Proteomics. 2011; 10 (10.1074/mcp.M111.012658)Abstract Full Text Full Text PDF Scopus (511) Google Scholar) and their high resolution precursor ion masses (supplemental Fig. S7 and S8). To probe the potential function of lysine malonylation in budding yeast, we mutated both of the Kmal sites (histone H2AK119 and histone H3K56) to glutamic acid to mimic constitutive modification and analyzed the phenotypes of these mutations as described before (19Dai J. Hyland E.M. Yuan D.S. Huang H. Bader J.S. Boeke J.D. Probing nucleosome function: A highly versatile library of synthetic histone H3 and H4 mutants.Cell. 2008; 134: 1066-1078Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). We did not detect significant change of fitness for the H2AK119 mutant compared with wild type. However, the H3K56E mutant reduced cell viability in yeast, as shown in both plasmid shuffling assay and tetrad analyses (supplemental Fig. S9). In this study, seven sites of Ksucc in yeast were identified, of which two sites are on histone H2A, two on histone H2B, two on histone H4, and one on histone H3, respectively. Interestingly, none of these sites are within the N-terminal tail, a region heavily modified in other ways. In fact, two of the succinylated sites (H2AK13 and H2BK37) are located right at the end of its N-terminal tail (Fig. 2B), where the histones make close contact with DNA. Of the six succinylated sites, except H3K79, all of them are somewhat near to the double-stranded DNA. The position of these succinylated residues and the nature of lysine succinylation suggest that it may interfere with the interaction between histones and the negatively charged DNA. Evidently, Glu substitution of histone H4K31, which is located close to the dyad axis of nucleosome, causes a dramatic reduction in cell viability. In addition, it is worth notice that none of the sites are buried within the nucleosome, allowing potential access to “writers” and the “readers.” Among the succinylated lysines, H3K79 is also well known to be methylated. Methylation on H3K79 is carried out by Dot1p in S. cerevisiae and is critical for gene activation (21van Leeuwen F. Gafken P.R. Gottschling D.E. Dot1p modulates silencing in yeast by methylation of the nucleosome core.Cell. 2002; 109: 745-756Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar). Identification of succinylation on this residue raises the possibility that its function may be affected by this “new” modification. H3K56 is known to be acetylated, and acetylation on this residue is important for histone deposition (22Xu F. Zhang K. Grunstein M. Acetylation in histone H3 globular domain regulates gene expression in yeast.Cell. 2005; 121: 375-385Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). We identified H3K56 lysine malonylation in yeast and lysine succinylation in Drosophila, mouse, and human. Substitution of H3K56 with glutamic acid in yeast caused lethality (supplemental Fig. S9), whereas substitution of the site with alanine or arginine did not change yeast cell viability (19Dai J. Hyland E.M. Yuan D.S. Huang H. Bader J.S. Boeke J.D. Probing nucleosome function: A highly versatile library of synthetic histone H3 and H4 mutants.Cell. 2008; 134: 1066-1078Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 23Nakanishi S. Sanderson B.W. Delventhal K.M. Bradford W.D. Staehling-Hampton K. Shilatifard A. A comprehensive library of histone mutants identifies nucleosomal residues required for H3K4 methylation.Nat. Struct. Mol. Biol. 2008; 15: 881-888Crossref PubMed Scopus (142) Google Scholar). Because histone H3K56 localizes near the nucleosome entry site and is proximate to the DNA double helix, the change of charge from positive to negative at the site by malonylation might interfere with its interaction with DNA, causing the loss of cell viability. Although we have not yet performed careful experiments to determine the stoichiometry of histone lysine succinylation and lysine malonylation at specific sites, our initial studies suggest that the overall stoichiometry of these two modifications are less abundant than histone lysine methylation and lysine acetylation, the two most abundant histone PTMs, and are in line with lysine butyrylation. In some cells, some histone lysine succinylation sites can be detected by HPLC/MS/MS, without the enrichment step and are more abundant than some low abundant histone acetyllysine and methyllysine sites (data not shown). Given the fact that histones are so abundant in the cells, low stoichiometry PTMs does not necessarily exclude the possibility that they have important biological functions. As an example, we recently demonstrated that some histone lysine butyrylation mark, a histone PTM with much lower stiochiometry than lysine acetylation, is associated with specific genomic regions, likely having important functions in transcriptional regulation (data not shown). Although we cannot conclusively exclude the possibility that lysine malonylation and lysine succinylation is caused by chemical reaction, originating directly from malonyl- and succinyl-CoA, a few lines of evidence indicate that enzyme-catalyzed lysine succinylation and lysine malonylation exist in cells. First, in this study, we have identified unique Ksucc sites that have not been reported to either have lysine acetylation (H2AK95, H2BK116, and H2BK120) or any other PTMs (H2AK95). Second, half of the Ksucc sites we identified are highly conserved among species. Additionally, most of Ksucc sites are located in the globular domain and the C terminus instead of the N-terminal tails of histones, which are among the residues that easily access other chemicals for chemical reactions. Nevertheless, we argue that both enzyme-catalyzed histone PTMs and chemical reaction-induced histone PTMs may have significant biological consequences, because the removal of these modifications could be a critical event for cellular detoxicification process. Lysine acetylation neutralizes the positive side chain of histone lysine residues, affecting chromatin structure and function by modulating interactions between histones and DNA and recruiting “histone code readers” (e.g. a bromo-domain protein). Within the nucleosome core particle, four lysine residues have direct contact with DNA, and another four of them span the major groove (24Luger K. Richmond T.J. DNA binding within the nucleosome core.Curr. Opin. Struct. Biol. 1998; 8: 33-40Crossref PubMed Scopus (234) Google Scholar), implying a key role in their interactions with DNA through charge-charge interaction. Lysine succinylation and malonylation induce even more significant structural changes than lysine acetylation by changing a positively charged residue to a negative charge. In terms of charge state change, lysine succinylation and malonylation are of the same magnitude as phosphorylation, producing a two-charge shift in the substrate residues. Given the crucial role of histone lysine PTMs (e.g. lysine acetylation and methylation) in DNA-templated processes, such a dramatic structural disturbance is likely to have significant consequences for chromatin structure and function. Therefore, lysine succinylation and malonylation are likely to play important roles in histone structure and function. Download .zip (2.67 MB) Help with zip files

Rapid advances in DNA synthesis techniques have made it possible to engineer viruses, biochemical pathways and assemble bacterial genomes. Here, we report the synthesis of a functional 272,871-base pair designer eukaryotic chromosome, synIII, which is based on the 316,617-base pair native Saccharomyces cerevisiae chromosome III. Changes to synIII include TAG/TAA stop-codon replacements, deletion of subtelomeric regions, introns, transfer RNAs, transposons, and silent mating loci as well as insertion of loxPsym sites to enable genome scrambling. SynIII is functional in S. cerevisiae. Scrambling of the chromosome in a heterozygous diploid reveals a large increase in a-mater derivatives resulting from loss of the MATα allele on synIII. The complete design and synthesis of synIII establishes S. cerevisiae as the basis for designer eukaryotic genome biology.

We describe complete design of a synthetic eukaryotic genome, Sc2.0, a highly modified Saccharomyces cerevisiae genome reduced in size by nearly 8%, with 1.1 megabases of the synthetic genome deleted, inserted, or altered. Sc2.0 chromosome design was implemented with BioStudio, an open-source framework developed for eukaryotic genome design, which coordinates design modifications from nucleotide to genome scales and enforces version control to systematically track edits. To achieve complete Sc2.0 genome synthesis, individual synthetic chromosomes built by Sc2.0 Consortium teams around the world will be consolidated into a single strain by "endoreduplication intercross." Chemically synthesized genomes like Sc2.0 are fully customizable and allow experimentalists to ask otherwise intractable questions about chromosome structure, function, and evolution with a bottom-up design strategy.

Retrotransposons have shaped eukaryotic genomes for millions of years. To analyze the consequences of human L1 retrotransposition, we developed a genetic system to recover many new L1 insertions in somatic cells. Forty-two de novo integrants were recovered that faithfully mimic many aspects of L1s that accumulated since the primate radiation. Their structures experimentally demonstrate an association between L1 retrotransposition and various forms of genetic instability. Numerous L1 element inversions, extra nucleotide insertions, exon deletions, a chromosomal inversion, and flanking sequence comobilization (called 5' transduction) were identified. In a striking number of integrants, short identical sequences were shared between the donor and the target site's 3' end, suggesting a mechanistic model that helps explain the structure of L1 insertions.

Macromolecular interactions define many biological phenomena. Although genetic methods are available to identify novel protein-protein and DNA-protein interactions, no genetic system has thus far been described to identify molecules or mutations that dissociate known interactions. Herein, we describe genetic systems that detect such events in the yeast Saccharomyces cerevisiae. We have engineered yeast strains in which the interaction of two proteins expressed in the context of the two-hybrid system or the interaction between a DNA-binding protein and its binding site in the context of the one-hybrid system is deleterious to growth. Under these conditions, dissociation of the interaction provides a selective growth advantage, thereby facilitating detection. These methods referred to as the "reverse two-hybrid system" and "reverse one-hybrid system" facilitate the study of the structure-function relationships and regulation of protein-protein and DNA-protein interactions. They should also facilitate the selection of dissociator molecules that could be used as therapeutic agents.

Recent advances in DNA synthesis technology have enabled the construction of novel genetic pathways and genomic elements, furthering our understanding of system-level phenomena. The ability to synthesize large segments of DNA allows the engineering of pathways and genomes according to arbitrary sets of design principles. Here we describe a synthetic yeast genome project, Sc2.0, and the first partially synthetic eukaryotic chromosomes, Saccharomyces cerevisiae chromosome synIXR, and semi-synVIL. We defined three design principles for a synthetic genome as follows: first, it should result in a (near) wild-type phenotype and fitness; second, it should lack destabilizing elements such as tRNA genes or transposons; and third, it should have genetic flexibility to facilitate future studies. The synthetic genome features several systemic modifications complying with the design principles, including an inducible evolution system, SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution). We show the utility of SCRaMbLE as a novel method of combinatorial mutagenesis, capable of generating complex genotypes and a broad variety of phenotypes. When complete, the fully synthetic genome will allow massive restructuring of the yeast genome, and may open the door to a new type of combinatorial genetics based entirely on variations in gene content and copy number.

Metabolic enzymes rarely regulate informational processes like gene expression. Yeast acetyl-CoA synthetases (Acs1p and 2p) are exceptional, as they are important not only for carbon metabolism but also are shown here to supply the acetyl-CoA for histone acetylation by histone acetyltransferases (HATs). acs2-Ts mutants exhibit global histone deacetylation, transcriptional defects, and synthetic growth defects with HAT mutants at high temperatures. In glycerol with ethanol, Acs1p is an alternate acetyl-CoA source for HATs. Rapid deacetylation after Acs2p inactivation suggests nuclear acetyl-CoA synthesis is rate limiting for histone acetylation. Different histone lysines exhibit distinct deacetylation rates, with N-terminal tail lysines deacetylated rapidly and H3 lysine 56 slowly. Yeast mitochondrial and nucleocytosolic acetyl-CoA pools are biochemically isolated. Thus, acetyl-CoA metabolism is directly linked to chromatin regulation and may affect diverse cellular processes in which acetylation and metabolism intersect, such as disease states and aging.

Abstract Homologous integration into the fission yeast Schizosaccharomyces pombe has not been well characterized. In this study, we have examined integration of plasmids carrying the leu1+ and ura4+ genes into their chromosomal loci. Genomic DNA blot analysis demonstrated that the majority of transformants have one or more copies of the plasmid vector integrated via homologous recombination with a much smaller fraction of gene conversion to leu1+ or ura4+. Non-homologous recombination events were not observed for either gene. We describe the construction of generally useful leu1+ and ura4+ plasmids for targeted integration at the leu1-32 and ura4-294 loci of S. pombe.

We characterized two genes, FUS1 and FUS2, which are required for fusion of Saccharomyces cerevisiae cells during conjugation. Mutations in these genes lead to an interruption of the mating process at a point just before cytoplasmic fusion; the partition dividing the mating pair remains undissolved several hours after the cells have initially formed a stable "prezygote." Fusion is only moderately impaired when the two parents together harbor one or two mutant fus genes, and it is severely compromised only when three or all four fus genes are inactivated. Cloning of FUS1 and FUS2 revealed that they share some functional homology; FUS1 on a high-copy number plasmid can partially suppress a fus2 mutant, and vice versa. FUS1 remains essentially unexpressed in vegetative cells, but is strongly induced by incubation of haploid cells with the appropriate mating pheromone. Immunofluorescence microscopy of alpha factor-induced a cells harboring a fus1-LACZ fusion showed the fusion protein to be localized at the cell surface, concentrated at one end of the cell (the shmoo tip). FUS1 maps near HIS4, and the intervening region (including BIK1, a gene required for nuclear fusion) was sequenced along with FUS1. The sequence of FUS1 revealed the presence of three copies of a hexamer (TGAAAC) conserved in the 5' noncoding regions of other pheromone-inducible genes. The deduced FUS1 protein sequence exhibits a striking concentration of serines and threonines at the amino terminus (46%; 33 of 71), followed by a 25-amino acid hydrophobic stretch and a predominantly hydrophilic carboxy terminus, which contains several potential N-glycosylation sites (Asn-X-Ser/Thr). This sequence suggests that FUS1 encodes a membrane-anchored glycoprotein with both N- and O-linked sugars.

Transposons are DNA sequences capable of moving in genomes. Early evidence showed their accumulation in many species and suggested their continued activity in at least isolated organisms. In the past decade, with the development of various genomic technologies, it has become abundantly clear that ongoing activity is the rule rather than the exception. Active transposons of various classes are observed throughout plants and animals, including humans. They continue to create new insertions, have an enormous variety of structural and functional impact on genes and genomes, and play important roles in genome evolution. Transposon activities have been identified and measured by employing various strategies. Here, we summarize evidence of current transposon activity in various plant and animal genomes.

We have found reverse transcriptase activity and virus-like particles only in yeast cells that contain a galactose-promoted Ty element induced on galactose. The cofractionation of reverse transcriptase, genomic-length Ty RNA, and a Ty-specified protein antigen in a particulate fraction and the ability of this complex to synthesize specifically a product that is homologous to the entire Ty suggest that reverse transcription of Ty RNA takes place in the particle. The absence of appreciable levels of reverse transcriptase and particles in uninduced cells despite the presence of at least 35 copies of chromosomal Ty elements suggest that some of these elements may be defective. The numerous virus-like particles visible in thin sections of Ty transposition-induced cells appear not to be infectious. These particles resemble the intracisternal A-type particles of the mouse and copia particles of Drosophila. The results support the idea that Ty elements and retroviruses share a common origin.

Replication des retrotransposons: article de synthese. Presentation des classes principales de retrotransposons. Transcription. Reverse transcription

Study of mutant phenotypes is a fundamental method for understanding gene function. The construction of a near-complete collection of yeast knockouts (YKO) and the unique molecular barcodes (or TAGs) that identify each strain has enabled quantitative functional profiling of Saccharomyces cerevisiae. By using these TAGs and the SGA reporter, MFA1pr-HIS3, which facilitates conversion of heterozygous diploid YKO strains into haploid mutants, we have developed a set of highly efficient microarray-based techniques, collectively referred as dSLAM (diploid-based synthetic lethality analysis on microarrays), to probe genome-wide gene-chemical and gene-gene interactions. Direct comparison revealed that these techniques are more robust than existing methods in functional profiling of the yeast genome. Widespread application of these tools will elucidate a comprehensive yeast genetic network.

Histone acetyltransferases (HATs) and histone deacetylases (HDACs) conduct many critical functions through nonhistone substrates in metazoans, but only chromatin-associated nonhistone substrates are known in Saccharomyces cerevisiae. Using yeast proteome microarrays, we identified and validated many nonchromatin substrates of the essential nucleosome acetyltransferase of H4 (NuA4) complex. Among these, acetylation sites (Lys19 and 514) of phosphoenolpyruvate carboxykinase (Pck1p) were determined by tandem mass spectrometry. Acetylation at Lys514 was crucial for enzymatic activity and the ability of yeast cells to grow on nonfermentable carbon sources. Furthermore, Sir2p deacetylated Pck1p both in vitro and in vivo. Loss of Pck1p activity blocked the extension of yeast chronological life span caused by water starvation. In human hepatocellular carcinoma (HepG2) cells, human Pck1 acetylation and glucose production were dependent on TIP60, the human homolog of ESA1. Our findings demonstrate a regulatory function for the NuA4 complex in glucose metabolism and life span by acetylating a critical metabolic enzyme.

Acetylation of histone H3 lysine 56 (K56Ac) occurs transiently in newly synthesized H3 during passage through S phase and is removed in G2. However, the physiologic roles and effectors of K56Ac turnover are unknown.The sirtuins Hst3p and, to a lesser extent, Hst4p maintain low levels of K56Ac outside of S phase. In hst3 hst4 mutants, K56 hyperacetylation nears 100%. Residues corresponding to the nicotinamide binding pocket of Sir2p are essential for Hst3p function, and H3 K56 deacetylation is inhibited by nicotinamide in vivo. Rapid inactivation of Hst3/Hst4p prior to S phase elevates K56Ac to 50% in G2, suggesting that K56-acetylated nucleosomes are assembled genome-wide during replication. Inducible expression of Hst3p in G1 or G2 triggers deacetylation of mature chromatin. Cells lacking Hst3/Hst4p exhibit many phenotypes: spontaneous DNA damage, chromosome loss, thermosensitivity, and acute sensitivity to genotoxic agents. These phenotypes are suppressed by mutation of histone H3 K56 into a nonacetylatable residue or by loss of K56Ac in cells lacking the histone chaperone Asf1.Our results underscore the critical importance of Hst3/Hst4p in controlling histone H3 K56Ac and thereby maintaining chromosome integrity.

Transposable elements are DNA segments with the unique ability to move about in the genome. This inherent feature can be exploited to harness these elements as gene vectors for genome manipulation. Transposon-based genetic strategies have been established in vertebrate species over the last decade, and current progress in this field suggests that transposable elements will serve as indispensable tools. In particular, transposons can be applied as vectors for somatic and germline transgenesis, and as insertional mutagens in both loss-of-function and gain-of-function forward mutagenesis screens. In addition, transposons will gain importance in future cell-based clinical applications, including nonviral gene transfer into stem cells and the rapidly developing field of induced pluripotent stem cells. Here we provide an overview of transposon-based methods used in vertebrate model organisms with an emphasis on the mouse system and highlight the most important considerations concerning genetic applications of the transposon systems.

Mobile DNAs have had a central role in shaping our genome. More than half of our DNA is comprised of interspersed repeats resulting from replicative copy and paste events of retrotransposons. Although most are fixed, incapable of templating new copies, there are important exceptions to retrotransposon quiescence. De novo insertions cause genetic diseases and cancers, though reliably detecting these occurrences has been difficult. New technologies aimed at uncovering polymorphic insertions reveal that mobile DNAs provide a substantial and dynamic source of structural variation. Key questions going forward include how and how much new transposition events affect human health and disease.

Cancers comprise a heterogeneous group of human diseases. Unifying characteristics include unchecked abilities of tumor cells to proliferate and spread anatomically, and the presence of clonal advantageous genetic changes. However, universal and highly specific tumor markers are unknown. Herein, we report widespread long interspersed element-1 (LINE-1) repeat expression in human cancers. We show that nearly half of all human cancers are immunoreactive for a LINE-1-encoded protein. LINE-1 protein expression is a common feature of many types of high-grade malignant cancers, is rarely detected in early stages of tumorigenesis, and is absent from normal somatic tissues. Studies have shown that LINE-1 contributes to genetic changes in cancers, with somatic LINE-1 insertions seen in selected types of human cancers, particularly colon cancer. We sought to correlate this observation with expression of the LINE-1-encoded protein, open reading frame 1 protein, and found that LINE-1 open reading frame 1 protein is a surprisingly broad, yet highly tumor-specific, antigen.

Ghrelin is a gastric peptide hormone that stimulates weight gain in vertebrates. The biological activities of ghrelin require octanoylation of the peptide on Ser(3), an unusual posttranslational modification that is catalyzed by the enzyme ghrelin O-acyltransferase (GOAT). Here, we describe the design, synthesis, and characterization of GO-CoA-Tat, a peptide-based bisubstrate analog that antagonizes GOAT. GO-CoA-Tat potently inhibits GOAT in vitro, in cultured cells, and in mice. Intraperitoneal administration of GO-CoA-Tat improves glucose tolerance and reduces weight gain in wild-type mice but not in ghrelin-deficient mice, supporting the concept that its beneficial metabolic effects are due specifically to GOAT inhibition. In addition to serving as a research tool for mapping ghrelin actions, GO-CoA-Tat may help pave the way for clinical targeting of GOAT in metabolic diseases.

Characterizing structural variants in the human genome is of great importance, but a genome wide analysis to detect interspersed repeats has not been done. Thus, the degree to which mobile DNAs contribute to genetic diversity, heritable disease, and oncogenesis remains speculative. We perform transposon insertion profiling by microarray (TIP-chip) to map human L1(Ta) retrotransposons (LINE-1 s) genome-wide. This identified numerous novel human L1(Ta) insertional polymorphisms with highly variant allelic frequencies. We also explored TIP-chip's usefulness to identify candidate alleles associated with different phenotypes in clinical cohorts. Our data suggest that the occurrence of new insertions is twice as high as previously estimated, and that these repeats are under-recognized as sources of human genomic and phenotypic diversity. We have just begun to probe the universe of human L1(Ta) polymorphisms, and as TIP-chip is applied to other insertions such as Alu SINEs, it will expand the catalog of genomic variants even further.

Epigenetic mechanisms, including histone post-translational modifications, control longevity in diverse organisms. Relatedly, loss of proper transcriptional regulation on a global scale is an emerging phenomenon of shortened life span, but the specific mechanisms linking these observations remain to be uncovered. Here, we describe a life span screen in Saccharomyces cerevisiae that is designed to identify amino acid residues of histones that regulate yeast replicative aging. Our results reveal that lack of sustained histone H3K36 methylation is commensurate with increased cryptic transcription in a subset of genes in old cells and with shorter life span. In contrast, deletion of the K36me2/3 demethylase Rph1 increases H3K36me3 within these genes, suppresses cryptic transcript initiation, and extends life span. We show that this aging phenomenon is conserved, as cryptic transcription also increases in old worms. We propose that epigenetic misregulation in aging cells leads to loss of transcriptional precision that is detrimental to life span, and, importantly, this acceleration in aging can be reversed by restoring transcriptional fidelity.

LINE-1s are active human DNA parasites that are agents of genome dynamics in evolution and disease. These streamlined elements require host factors to complete their life cycles, whereas hosts have developed mechanisms to combat retrotransposition's mutagenic effects. As such, endogenous L1 expression levels are extremely low, creating a roadblock for detailed interactomic analyses. Here, we describe a system to express and purify highly active L1 RNP complexes from human suspension cell culture and characterize the copurified proteome, identifying 37 high-confidence candidate interactors. These data sets include known interactors PABPC1 and MOV10 and, with in-cell imaging studies, suggest existence of at least three types of compositionally and functionally distinct L1 RNPs. Among the findings, UPF1, a key nonsense-mediated decay factor, and PCNA, the polymerase-delta-associated sliding DNA clamp, were identified and validated. PCNA interacts with ORF2p via a PIP box motif; mechanistic studies suggest that this occurs during or immediately after target-primed reverse transcription.

INTRODUCTION The Saccharomyces cerevisiae 2.0 project (Sc2.0) aims to modify the yeast genome with a series of densely spaced designer changes. Both a synthetic yeast chromosome arm (synIXR) and the entirely synthetic chromosome (synIII) function with high fitness in yeast. For designer genome synthesis projects, precise engineering of the physical sequence to match the specified design is important for the systematic evaluation of underlying design principles. Yeast can maintain nuclear chromosomes as rings, occurring by chance at repeated sequences, although the cyclized format is unfavorable in meiosis given the possibility of dicentric chromosome formation from meiotic recombination. Here, we describe the de novo synthesis of synthetic yeast chromosome V (synV) in the “Build-A-Genome China” course, perfectly matching the designer sequence and bearing loxPsym sites, distinguishable watermarks, and all the other features of the synthetic genome. We generated a ring synV derivative with user-specified cyclization coordinates and characterized its performance in mitosis and meiosis. RATIONALE Systematic evaluation of underlying Sc2.0 design principles requires that the final assembled synthetic genome perfectly match the designed sequence. Given the size of yeast chromosomes, synthetic chromosome construction is performed iteratively, and new mutations and unpredictable events may occur during synthesis; even a very small number of unintentional nucleotide changes across the genome could have substantial effects on phenotype. Therefore, precisely matching the physical sequence to the designed sequence is crucial for verification of the design principles in genome synthesis. Ring chromosomes can extend those design principles to provide a model for genomic rearrangement, ring chromosome evolution, and human ring chromosome disorders. RESULTS We chemically synthesized, assembled, and incorporated designer chromosome synV (536,024 base pairs) of S. cerevisiae according to Sc2.0 principles, based on the complete nucleotide sequence of native yeast chromosome V (576,874 base pairs). This work was performed as part of the “Build-A-Genome China” course in Tianjin University. We corrected all mutations found—including duplications, substitutions, and indels—in the initial synV strain by using integrative cotransformation of the precise desired changes and by means of a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)–based method. Altogether, 3331 corrected base pairs were required to match to the designed sequence. We generated a strain that exactly matches all designer sequence changes that displays high fitness under a variety of culture conditions. All corrections were verified with whole-genome sequencing; RNA sequencing revealed only minor changes in gene expression—most notably, decreases in expression of genes relocated near synthetic telomeres as a result of design. We constructed a functional circular synV (ring_synV) derivative in yeast by precisely joining both chromosome ends (telomeres) at specified coordinates. The ring chromosome showed restoration of subtelomeric gene expression levels. The ring_synV strain exhibited fitness comparable with that of the linear synV strain, revealed no change in sporulation frequency, but notably reduced spore viability. In meiosis, heterozygous or homozygous diploid ring_wtV and ring_synV chromosomes behaved similarly, exhibiting substantially higher frequency of the formation of zero-spore tetrads, a type that was not seen in the rod chromosome diploids. Rod synV chromosomes went through meiosis with high spore viability, despite no effort having been made to preserve meiotic competency in the design of synV. CONCLUSION The perfect designer-matched synthetic chromosome V provides strategies to edit sequence variants and correct unpredictable events, such as off-target integration of extra copies of synthetic DNA elsewhere in the genome. We also constructed a ring synthetic chromosome derivative and evaluated its fitness and stability in yeast. Both synV and synVI can be circularized and can power yeast cell growth without affecting fitness when gene content is maintained. These fitness and stability phenotypes of the ring synthetic chromosome in yeast provide a model system with which to probe the mechanism of human ring chromosome disorders. Synthesis, cyclization, and characterization of synV . ( A ) Synthetic chromosome V (synV, 536,024 base pairs) was designed in silico from native chromosome V (wtV, 576,874 base pairs), with extensive genotype modification designed to be phenotypically neutral. ( B ) CRISPR/Cas9 strategy for multiplex repair. ( C ) Colonies of wtV, synV, and ring_synV strains.

Xenografts from genetically modified pigs have become one of the most promising solutions to the dearth of human organs available for transplantation. The challenge in this model has been hyperacute rejection. To avoid this, pigs have been bred with a knockout of the alpha-1,3-galactosyltransferase gene and with subcapsular autologous thymic tissue.We transplanted kidneys from these genetically modified pigs into two brain-dead human recipients whose circulatory and respiratory activity was maintained on ventilators for the duration of the study. We performed serial biopsies and monitored the urine output and kinetic estimated glomerular filtration rate (eGFR) to assess renal function and xenograft rejection.The xenograft in both recipients began to make urine within moments after reperfusion. Over the 54-hour study, the kinetic eGFR increased from 23 ml per minute per 1.73 m2 of body-surface area before transplantation to 62 ml per minute per 1.73 m2 after transplantation in Recipient 1 and from 55 to 109 ml per minute per 1.73 m2 in Recipient 2. In both recipients, the creatinine level, which had been at a steady state, decreased after implantation of the xenograft, from 1.97 to 0.82 mg per deciliter in Recipient 1 and from 1.10 to 0.57 mg per deciliter in Recipient 2. The transplanted kidneys remained pink and well-perfused, continuing to make urine throughout the study. Biopsies that were performed at 6, 24, 48, and 54 hours revealed no signs of hyperacute or antibody-mediated rejection. Hourly urine output with the xenograft was more than double the output with the native kidneys.Genetically modified kidney xenografts from pigs remained viable and functioning in brain-dead human recipients for 54 hours, without signs of hyperacute rejection. (Funded by Lung Biotechnology.).

INTRODUCTION Total synthesis of designer chromosomes and genomes is a new paradigm for the study of genetics and biological systems. The Sc2.0 project is building a designer yeast genome from scratch to test and extend the limits of our biological knowledge. Here we describe the design, rapid assembly, and characterization of synthetic chromosome VI (synVI). Further, we investigate the phenotypic, transcriptomic, and proteomic consequences associated with consolidation of three synthetic chromosomes–synVI, synIII, and synIXR—into a single poly-synthetic strain. RATIONALE A host of Sc2.0 chromosomes, including synVI, have now been constructed in discrete strains. With debugging steps, where the number of bugs scales with chromosome length, all individual synthetic chromosomes have been shown to power yeast cells to near wild-type (WT) fitness. Testing the effects of Sc2.0 chromosome consolidation to uncover possible synthetic genetic interactions and/or perturbations of native cellular networks as the number of designer changes increases is the next major step for the Sc2.0 project. RESULTS SynVI was rapidly assembled using nine sequential steps of SwAP-In (switching auxotrophies progressively by integration), yielding a ~240-kb synthetic chromosome designed to Sc2.0 specifications. We observed partial silencing of the left- and rightmost genes on synVI, each newly positioned subtelomerically relative to their locations on native VI. This result suggests that consensus core X elements of Sc2.0 universal telomere caps are insufficient to fully buffer telomere position effects. The synVI strain displayed a growth defect characterized by an increased frequency of glycerol-negative colonies. The defect mapped to a synVI design feature in the essential PRE4 gene ( YFR050C ), encoding the β7 subunit of the 20 S proteasome. Recoding 10 codons near the 3′ end of the PRE4 open reading frame (ORF) caused a ~twofold reduction in Pre4 protein level without affecting RNA abundance. Reverting the codons to the WT sequence corrected both the Pre4 protein level and the phenotype. We hypothesize that the formation of a stem loop involving recoded codons underlies reduced Pre4 protein level. Sc2.0 chromosomes (synI to synXVI) are constructed individually in discrete strains and consolidated into poly-synthetic (poly-syn) strains by “endoreduplication intercross.” Consolidation of synVI with synthetic chromosomes III (synIII) and IXR (synIXR) yields a triple-synthetic (triple-syn) strain that is ~6% synthetic overall—with almost 70 kb deleted, including 20 tRNAs, and more than 12 kb recoded. Genome sequencing of double-synthetic (synIII synVI, synIII synIXR, synVI synIXR) and triple-syn (synIII synVI synIXR) cells indicates that suppressor mutations are not required to enable coexistence of Sc2.0 chromosomes. Phenotypic analysis revealed a slightly slower growth rate for the triple-syn strain only; the combined effect of tRNA deletions on different chromosomes might underlie this result. Transcriptome and proteome analyses indicate that cellular networks are largely unperturbed by the existence of multiple synthetic chromosomes in a single cell. However, a second bug on synVI was discovered through proteomic analysis and is associated with alteration of the HIS2 transcription start as a consequence of tRNA deletion and loxPsym site insertion. Despite extensive genetic alterations across 6% of the genome, no major global changes were detected in the poly-syn strain “omics” analyses. CONCLUSION Analyses of phenotypes, transcriptomics, and proteomics of synVI and poly-syn strains reveal, in general, WT cell properties and the existence of rare bugs resulting from genome editing. Deletion of subtelomeres can lead to gene silencing, recoding deep within an ORF can yield a translational defect, and deletion of elements such as tRNA genes can lead to a complex transcriptional output. These results underscore the complementarity of transcriptomics and proteomics to identify bugs, the consequences of designer changes in Sc2.0 chromosomes. The consolidation of Sc2.0 designer chromosomes into a single strain appears to be exceptionally well tolerated by yeast. A predictable exception to this is the deletion of tRNAs, which will be restored on a separate neochromosome to avoid synthetic lethal genetic interactions between deleted tRNA genes as additional synthetic chromosomes are introduced. Debugging synVI and characterization of poly-synthetic yeast cells. ( A ) The second Sc2.0 chromosome to be constructed, synVI, encodes a “bug” that causes a variable colony size, dubbed a “glycerol-negative growth-suppression defect.” ( B ) Synonymous changes in the essential PRE4 ORF lead to a reduced protein level, which underlies the growth defect. ( C ) The poly-synthetic strain synIII synVI synIXR directs growth of yeast cells to near WT fitness levels.

We need technology and an ethical framework for genome-scale engineering

INTRODUCTION Design and construction of an extensively modified yeast genome is a direct means to interrogate the integrity, comprehensiveness, and accuracy of the knowledge amassed by the yeast community to date. The international synthetic yeast genome project (Sc2.0) aims to build an entirely designer, synthetic Saccharomyces cerevisiae genome. The synthetic genome is designed to increase genome stability and genetic flexibility while maintaining cell fitness near that of the wild type. A major challenge for a genome synthesis lies in identifying and eliminating fitness-reducing sequence variants referred to as “bugs.” RATIONALE Debugging is imperative for successfully building a fit strain encoding a synthetic genome. However, it is time-consuming and laborious to replace wild-type genes and measure strain fitness systematically. The Sc2.0 PCRTag system, which specifies recoded sequences within open reading frames (ORFs), is designed to distinguish synthetic from wild-type DNA in a simple polymerase chain reaction (PCR) assay. This system provides an opportunity to efficiently map bugs to the related genes by using a pooling strategy and subsequently correct them. Further, as we identify bugs in designer sequences, we will identify gaps in our knowledge and gain a deeper understanding of genome biology, allowing refinement of future design strategies. RESULTS We chemically synthesized yeast chromosome X, synX, designed to be 707,459 base pairs. A high-throughput mapping strategy called pooled PCRTag mapping (PoPM) was developed to identify unexpected bugs during chromosome assembly. With this method, the genotypes of pools of colonies with normal or defective fitness are assessed by PCRTag analysis. The PoPM method exploits the patchwork structure of synthetic and wild-type sequences observed in the majority of putative synthetic DNA integrants or meiotic progeny derived from synthetic/wild-type strain backcross. PCRTag analysis with both synthetic and wild-type specific primers, carried out with genomic DNA extracted from the two pools of clones (normal fitness versus a specific growth defect), can be used to identify regions of synthetic DNA missing from the normal fitness pool and, analogously, sections of wild-type DNA absent from the specific growth-defect pool. In this way, the defect can be efficiently mapped to a very small overlapping region, and subsequent systematic analysis of designed changes in that region can be used to identify the bug. Several bugs were identified and corrected, including a growth defect mapping to a specific synonymously recoded PCRTag sequence in the essential FIP1 ORF and the effect of introducing a loxPsym site that unexpectedly altered the the promoter function of a nearby gene, ATP2. In addition, meiotic crossover was employed to repair the massive duplications and rearrangements in the synthetic chromosome. The debugged synX strain exhibited high fitness under a variety of conditions tested and in competitive growth with the wild-type strain. CONCLUSION Synthetic yeast chromosome X was chemically synthesized from scratch, a rigorous, incremental step toward complete synthesis of the whole yeast genome. Thousands of designer modifications in synX revealed extensive flexibility of the yeast genome. We developed an efficient mapping method, PoPM, to identify bugs during genome synthesis, generalizable to any watermarked synthetic chromosome, and several details of yeast biology were uncovered by debugging. Considering the numerous gene-associated PCRTags available in the synthetic chromosomes, PoPM may represent a powerful tool to map interesting phenotypes of mutated synthetic strains or even mutated wild-type strains to the relevant genes. It may also be useful to study yeast genetic interactions when an unexpected phenotype is generated by alterations in two or more genes, substantially expanding understanding of yeast genomic and cellular functions. The PoPM method is also likely to be useful for mapping phenotype(s) resulting from the genome SCRaMbLE system. Characterization of synX and debugging by pooled PCRTag mapping. ( Top ) Design overview of synthetic chromosome X. ( Bottom ) Flow diagram of pooled PCRTag mapping (PoPM).

The genomes of virtually all organisms contain repetitive sequences that are generated by the activity of transposable elements (transposons). Transposons are mobile genetic elements that can move from one genomic location to another; in this process, they amplify and increase their presence in genomes, sometimes to very high copy numbers. In this Review we discuss new evidence and ideas that the activity of retrotransposons, a major subgroup of transposons overall, influences and even promotes the process of ageing and age-related diseases in complex metazoan organisms, including humans. Retrotransposons have been coevolving with their host genomes since the dawn of life. This relationship has been largely competitive, and transposons have earned epithets such as 'junk DNA' and 'molecular parasites'. Much of our knowledge of the evolution of retrotransposons reflects their activity in the germline and is evident from genome sequence data. Recent research has provided a wealth of information on the activity of retrotransposons in somatic tissues during an individual lifespan, the molecular mechanisms that underlie this activity, and the manner in which these processes intersect with our own physiology, health and well-being.

INTRODUCTION It has long been an interesting question whether a living cell can be constructed from scratch in the lab, a goal that may not be realized anytime soon. Nonetheless, with advances in DNA synthesis technology, the complete genetic material of an organism can now be synthesized chemically. Hitherto, genomes of several organisms including viruses, phages, and bacteria have been designed and constructed. These synthetic genomes are able to direct all normal biological functions, capable of self-replication and production of offspring. Several years ago, a group of scientists worldwide formed an international consortium to reconstruct the genome of budding yeast, Saccharomyces cerevisiae . RATIONALE The synthetic yeast genome, designated Sc2.0, was designed according to a set of arbitrary rules, including the elimination of transposable elements and incorporation of specific DNA elements to facilitate further genome manipulation. Among the 16 S. cerevisiae chromosomes, chromosome XII is unique as one of the longest yeast chromosomes (~1 million base pairs) and additionally encodes the highly repetitive ribosomal DNA locus, which forms the well-organized nucleolus. We report on the design, construction, and characterization of chromosome XII, the physically largest chromosome in S. cerevisiae. RESULTS A 976,067–base pair linear chromosome, synXII, was designed based on the native chromosome XII sequence of S. cerevisiae , and chemically synthesized. SynXII was assembled using a two-step method involving, successive megachunk integration to produce six semisynthetic strains, followed by meiotic recombination–mediated assembly, yielding a full-length functional chromosome in S. cerevisiae. Minor growth defect “bugs” detected in synXII were caused by deletion of tRNA genes and were corrected by introducing an ectopic copy of a single tRNA gene. The ribosomal gene cluster (rDNA) on synXII was left intact during the assembly process and subsequently replaced by a modified rDNA unit. The same synthetic rDNA unit was also used to regenerate rDNA at three distinct chromosomal locations. The rDNA signature sequences of the internal transcribed spacer (ITS), often used to determine species identity by standard DNA barcoding procedures, were swapped to generate a Saccharomyces synXII strain that would be identified as S. bayanus. Remarkably, these substantial DNA changes had no detectable phenotypic consequences under various laboratory conditions. CONCLUSION The rDNA locus of synXII is highly plastic; not only can it be moved to other chromosomal loci, it can also be altered in its ITS region to masquerade as a distinct species as defined by DNA barcoding, used widely in taxonomy. The ability to perform “species morphing” reported here presumably reflects the degree of evolutionary flexibility by which these ITS regions change. However, this barcoding region is clearly not infinitely flexible, as only relatively modest intragenus base changes were tolerated. More severe intergenus differences in ITS sequence did not result in functional rDNAs, probably because of defects in rRNA processing. The ability to design, build, and debug a megabase-sized chromosome, together with the flexibility in rDNA locus position, speaks to the remarkable overall flexibility of the yeast genome. Hierarchical assembly and subsequent restructuring of synXII. SynXII was assembled in two steps: First, six semisynthetic synXII strains were built in which segments of native XII DNA were replaced with the corresponding designer sequences. Next, the semisynthetic strains were combined withmultiple rounds ofmating/sporulation, eventually generating a single strain encoding fulllength synXII.The rDNA repeats were removed, modified, and subsequently regenerated at distinct chromosomal locations for species morphing and genome restructuring.

INTRODUCTION Although much effort has been devoted to studying yeast in the past few decades, our understanding of this model organism is still limited. Rapidly developing DNA synthesis techniques have made a “build-to-understand” approach feasible to reengineer on the genome scale. Here, we report on the completion of a 770-kilobase synthetic yeast chromosome II (synII). SynII was characterized using extensive Trans-Omics tests. Despite considerable sequence alterations, synII is virtually indistinguishable from wild type. However, an up-regulation of translational machinery was observed and can be reversed by restoring the transfer RNA (tRNA) gene copy number. RATIONALE Following the “design-build-test-debug” working loop, synII was successfully designed and constructed in vivo. Extensive Trans-Omics tests were conducted, including phenomics, transcriptomics, proteomics, metabolomics, chromosome segregation, and replication analyses. By both complementation assays and SCRaMbLE (synthetic chromosome rearrangement and modification by loxP -mediated evolution), we targeted and debugged the origin of a growth defect at 37°C in glycerol medium. RESULTS To efficiently construct megabase-long chromosomes, we developed an I- Sce I–mediated strategy, which enables parallel integration of synthetic chromosome arms and reduced the overall integration time by 50% for synII. An I- Sce I site is introduced for generating a double-strand break to promote targeted homologous recombination during mitotic growth. Despite hundreds of modifications introduced, there are still regions sharing substantial sequence similarity that might lead to undesirable meiotic recombinations when intercrossing the two semisynthetic chromosome arm strains. Induction of the I- Sce I–mediated double-strand break is otherwise lethal and thus introduced a strong selective pressure for targeted homologous recombination. Since our strategy is designed to generate a markerless synII and leave the URA3 marker on the wild-type chromosome, we observed a tenfold increase in URA3 -deficient colonies upon I- Sce I induction, meaning that our strategy can greatly bias the crossover events toward the designated regions. By incorporating comprehensive phenotyping approaches at multiple levels, we demonstrated that synII was capable of powering the growth of yeast indistinguishably from wild-type cells (see the figure), showing highly consistent biological processes comparable to the native strain. Meanwhile, we also noticed modest but potentially significant up-regulation of the translational machinery. The main alteration underlying this change in expression is the deletion of 13 tRNA genes. A growth defect was observed in one very specific condition—high temperature (37°C) in medium with glycerol as a carbon source—where colony size was reduced significantly. We targeted and debugged this defect by two distinct approaches. The first approach involved phenotype screening of all intermediate strains followed by a complementation assay with wild-type sequences in the synthetic strain. By doing so, we identified a modification resulting from PCRTag recoding in TSC10 , which is involved in regulation of the yeast high-osmolarity glycerol (HOG) response pathway. After replacement with wild-type TSC10 , the defect was greatly mitigated. The other approach, debugging by SCRaMbLE, showed rearrangements in regions containing HOG regulation genes. Both approaches indicated that the defect is related to HOG response dysregulation. Thus, the phenotypic defect can be pinpointed and debugged through multiple alternative routes in the complex cellular interactome network. CONCLUSION We have demonstrated that synII segregates, replicates, and functions in a highly similar fashion compared with its wild-type counterpart. Furthermore, we believe that the iterative “design-build-test-debug” cycle methodology, established here, will facilitate progression of the Sc2.0 project in the face of the increasing synthetic genome complexity. SynII characterization. ( A ) Cell cycle comparison between synII and BY4741 revealed by the percentage of cells with separated CEN2-GFP dots, metaphase spindles, and anaphase spindles. ( B ) Replication profiling of synII (red) and BY4741 (black) expressed as relative copy number by deep sequencing. ( C ) RNA sequencing analysis revealed that the significant up-regulation of translational machinery in synII is induced by the deletion of tRNA genes in synII.

Retrotransposons are mutagenic units able to move within the genome. Despite many defenses deployed by the host to suppress potentially harmful activities of retrotransposons, these genetic units have found ways to meld with normal cellular functions through processes of exaptation and domestication. The same host mechanisms targeting transposon mobility allow for expansion and rewiring of gene regulatory networks on an evolutionary time scale. Recent works demonstrating retrotransposon activity during development, cell differentiation and neurogenesis shed new light on unexpected activities of transposable elements. Moreover, new technological advances illuminated subtler nuances of the complex relationship between retrotransposons and the host genome, clarifying the role of retroelements in evolution, development and impact on human disease.

The testis expresses the largest number of genes of any mammalian organ, a finding that has long puzzled molecular biologists. Our single-cell transcriptomic data of human and mouse spermatogenesis provide evidence that this widespread transcription maintains DNA sequence integrity in the male germline by correcting DNA damage through a mechanism we term transcriptional scanning. We find that genes expressed during spermatogenesis display lower mutation rates on the transcribed strand and have low diversity in the population. Moreover, this effect is fine-tuned by the level of gene expression during spermatogenesis. The unexpressed genes, which in our model do not benefit from transcriptional scanning, diverge faster over evolutionary timescales and are enriched for sensory and immune-defense functions. Collectively, we propose that transcriptional scanning shapes germline mutation signatures and modulates mutation rates in a gene-specific manner, maintaining DNA sequence integrity for the bulk of genes but allowing for faster evolution in a specific subset.

Cross-talk between histone modifications Histone modifications play pivotal roles within the intricate protein networks that underlie transcription and gene silencing in eukaryotic genomes. The enzymes that deposit them undergo spatiotemporal fine-tuning of their catalytic activity; one example is trans-histone cross-talk, in which one histone modification activates an enzyme responsible for another histone modification. Valencia-Sánchez et al. show that histone H4 lysine 16 acetylation (H4K16ac), a hallmark of decondensed, transcriptionally permissive chromatin, directly stimulates the Dot1 histone H3 lysine 79 methyltransferase. Structural, biochemical, and cellular data explain Dot1's regulation by H4K16ac and show how it coordinates with a second positive regulator of Dot1, histone H2B ubiquitination. Science , this issue p. eabc6663

The Sc2.0 project is building a eukaryotic synthetic genome from scratch. A major milestone has been achieved with all individual Sc2.0 chromosomes assembled. Here, we describe the consolidation of multiple synthetic chromosomes using advanced endoreduplication intercrossing with tRNA expression cassettes to generate a strain with 6.5 synthetic chromosomes. The 3D chromosome organization and transcript isoform profiles were evaluated using Hi-C and long-read direct RNA sequencing. We developed CRISPR Directed Biallelic URA3-assisted Genome Scan, or "CRISPR D-BUGS," to map phenotypic variants caused by specific designer modifications, known as "bugs." We first fine-mapped a bug in synthetic chromosome II (synII) and then discovered a combinatorial interaction associated with synIII and synX, revealing an unexpected genetic interaction that links transcriptional regulation, inositol metabolism, and tRNASerCGA abundance. Finally, to expedite consolidation, we employed chromosome substitution to incorporate the largest chromosome (synIV), thereby consolidating >50% of the Sc2.0 genome in one strain.

<h2>Summary</h2> Here, we report the design, construction, and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, <i>de novo</i> counterpart to the native complement of <i>Saccharomyces cerevisiae</i>. Intending to address one of the central design principles of the Sc2.0 project, the ∼190-kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporates orthogonal genetic elements from non-<i>S. cerevisiae</i> yeast species. Furthermore, the presence of 283 <i>rox</i> recombination sites enables an orthogonal tRNA SCRaMbLE system. Following construction in yeast, we obtained evidence of a potent selective force, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH, and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up opportunities to directly test hypotheses surrounding these essential non-coding RNAs.

Technologies to profoundly engineer biology are becoming increasingly affordable, powerful, and accessible to a widening group of actors. While offering tremendous potential to fuel biological research and the bioeconomy, this development also increases the risk of inadvertent or deliberate creation and dissemination of pathogens. Effective regulatory and technological frameworks need to be developed and deployed to manage these emerging biosafety and biosecurity risks. Here, we review digital and biological approaches of a range of technology readiness levels suited to address these challenges. Digital sequence screening technologies already are used to control access to synthetic DNA of concern. We examine the current state of the art of sequence screening, challenges and future directions, and environmental surveillance for the presence of engineered organisms. As biosafety layer on the organism level, we discuss genetic biocontainment systems that can be used to created host organisms with an intrinsic barrier against unchecked environmental proliferation.

Sox2 expression in mouse embryonic stem cells (mESCs) depends on a distal cluster of DNase I hypersensitive sites (DHSs), but their individual contributions and degree of interdependence remain a mystery. We analyzed the endogenous Sox2 locus using Big-IN to scarlessly integrate large DNA payloads incorporating deletions, rearrangements, and inversions affecting single or multiple DHSs, as well as surgical alterations to transcription factor (TF) recognition sequences. Multiple mESC clones were derived for each payload, sequence-verified, and analyzed for Sox2 expression. We found that two DHSs comprising a handful of key TF recognition sequences were each sufficient for long-range activation of Sox2 expression. By contrast, three nearby DHSs were entirely context dependent, showing no activity alone but dramatically augmenting the activity of the autonomous DHSs. Our results highlight the role of context in modulating genomic regulatory element function, and our synthetic regulatory genomics approach provides a roadmap for the dissection of other genomic loci.

Abstract The loss of the tail is among the most notable anatomical changes to have occurred along the evolutionary lineage leading to humans and to the ‘anthropomorphous apes’ 1–3 , with a proposed role in contributing to human bipedalism 4–6 . Yet, the genetic mechanism that facilitated tail-loss evolution in hominoids remains unknown. Here we present evidence that an individual insertion of an Alu element in the genome of the hominoid ancestor may have contributed to tail-loss evolution. We demonstrate that this Alu element—inserted into an intron of the TBXT gene 7–9 —pairs with a neighbouring ancestral Alu element encoded in the reverse genomic orientation and leads to a hominoid-specific alternative splicing event. To study the effect of this splicing event, we generated multiple mouse models that express both full-length and exon-skipped isoforms of Tbxt , mimicking the expression pattern of its hominoid orthologue TBXT . Mice expressing both Tbxt isoforms exhibit a complete absence of the tail or a shortened tail depending on the relative abundance of Tbxt isoforms expressed at the embryonic tail bud. These results support the notion that the exon-skipped transcript is sufficient to induce a tail-loss phenotype. Moreover, mice expressing the exon-skipped Tbxt isoform develop neural tube defects, a condition that affects approximately 1 in 1,000 neonates in humans 10 . Thus, tail-loss evolution may have been associated with an adaptive cost of the potential for neural tube defects, which continue to affect human health today.

The Sir2 enzyme family is responsible for a newly classified chemical reaction, NAD+-dependent protein deacetylation. New peptide substrates, the reaction mechanism, and the products of the acetyl transfer to NAD+ are described for SIR2. The final products of SIR2 reactions are the deacetylated peptide and the 2‘ and 3‘ regioisomers of O-acetyl ADP ribose (AADPR), formed through an α-1‘-acetyl ADP ribose intermediate and intramolecular transesterification reactions (2‘ → 3‘). The regioisomers, their anomeric forms, the interconversion rates, and the reaction equilibria were characterized by NMR, HPLC, 18O exchange, and MS methods. The mechanism of acetyl transfer to NAD+ includes (1) ADP ribosylation of the peptide acyl oxygen to form a high-energy O-alkyl amidate intermediate, (2) attack of the 2‘-OH group on the amidate to form a 1‘,2‘-acyloxonium species, (3) hydrolysis to 2‘-AADPR by the attack of water on the carbonyl carbon, and (4) an SIR2-independent transesterification equilibrating the 2‘- and 3‘-AADPRs. This mechanism is unprecedented in ADP-ribosyl transferase enzymology. The 2‘- and 3‘-AADPR products are candidate molecules for SIR2-initiated signaling pathways.

Human L1 retrotransposons can produce DNA transduction events in which unique DNA segments downstream of L1 elements are mobilized as part of aberrant retrotransposition events. That L1s are capable of carrying out such a reaction in tissue culture cells was elegantly demonstrated. Using bioinformatic approaches to analyze the structures of L1 element target site duplications and flanking sequence features, we provide evidence suggesting that approximately 15% of full-length L1 elements bear evidence of flanking DNA segment transduction. Extrapolating these findings to the 600,000 copies of L1 in the genome, we predict that the amount of DNA transduced by L1 represents approximately 1% of the genome, a fraction comparable with that occupied by exons.

The gypsy element of Drosophila differs from most LTR retrotransposons in containing a third open reading frame that resembles retroviral env genes. The protein encoded by ORF3 is glycosylated and processed, like all retroviral envelope proteins. The protein is expressed at high levels in fly strains in which gypsy elements are active. In these strains the protein is found primarily in viral particles. When larvae of fly strains in which gypsy is normally inactive are exposed to sucrose gradient fractions containing these particles, a high level of gypsy insertion activity is observed in their progeny. Thus, gypsy has the expected properties of an insect retrovirus.

Piwis are a germline-specific subclass of the Argonaute family of RNA interference (RNAi) effector proteins that are associated with a recently discovered group of small RNAs (piRNAs). Recent studies in Drosophila and zebrafish directly implicate Piwi proteins in piRNA biogenesis to maintain transposon silencing in the germline genome (Brennecke et al., 2007Brennecke J. Aravin A.A. Stark A. Dus M. Kellis M. Sachidanandam R. Hannon G.J. Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila.Cell. 2007; 128: 1098-1103Abstract Full Text Full Text PDF Scopus (1594) Google Scholar, Gunawardane et al., 2007Gunawardane L.S. Saito K. Nishida K.M. Miyoshi K. Kawamura Y. Nagami T. Siomi H. Siomi M.C. A slicer-mediated mechanism for repeat-associated siRNA 5′ end formation in Drosophila.Science. 2007; 315: 1587-1590Crossref PubMed Scopus (814) Google Scholar, Houwing et al., 2007Houwing S. Kamminga L.M. Berezikov E. Cronembold D. Girard A. Moens C.B. Plasterk R.H.A. Hannon G.J. Draper B.W. Ketting R.F. Zebrafish PIWI and piRNAs; Implications for germ cell survival and transposon silencing.Cell. 2007; (this issue)PubMed Google Scholar). This function may be conserved in mice as loss of Miwi2, a mouse Piwi homolog, leads to germline stem cell and meiotic defects correlated with increased transposon activity (Carmell et al., 2007Carmell M.A. Girard A. van de Kant H.J.G. de Rooij D.G. Hannon G.J. Miwi2 is essential for spermatogenesis and repression of transposons in the mouse male germline.Dev. Cell. 2007; (Published online March 29, 2007)https://doi.org/10.1016/j.devcel.2007.03.001Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar). Piwis are a germline-specific subclass of the Argonaute family of RNA interference (RNAi) effector proteins that are associated with a recently discovered group of small RNAs (piRNAs). Recent studies in Drosophila and zebrafish directly implicate Piwi proteins in piRNA biogenesis to maintain transposon silencing in the germline genome (Brennecke et al., 2007Brennecke J. Aravin A.A. Stark A. Dus M. Kellis M. Sachidanandam R. Hannon G.J. Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila.Cell. 2007; 128: 1098-1103Abstract Full Text Full Text PDF Scopus (1594) Google Scholar, Gunawardane et al., 2007Gunawardane L.S. Saito K. Nishida K.M. Miyoshi K. Kawamura Y. Nagami T. Siomi H. Siomi M.C. A slicer-mediated mechanism for repeat-associated siRNA 5′ end formation in Drosophila.Science. 2007; 315: 1587-1590Crossref PubMed Scopus (814) Google Scholar, Houwing et al., 2007Houwing S. Kamminga L.M. Berezikov E. Cronembold D. Girard A. Moens C.B. Plasterk R.H.A. Hannon G.J. Draper B.W. Ketting R.F. Zebrafish PIWI and piRNAs; Implications for germ cell survival and transposon silencing.Cell. 2007; (this issue)PubMed Google Scholar). This function may be conserved in mice as loss of Miwi2, a mouse Piwi homolog, leads to germline stem cell and meiotic defects correlated with increased transposon activity (Carmell et al., 2007Carmell M.A. Girard A. van de Kant H.J.G. de Rooij D.G. Hannon G.J. Miwi2 is essential for spermatogenesis and repression of transposons in the mouse male germline.Dev. Cell. 2007; (Published online March 29, 2007)https://doi.org/10.1016/j.devcel.2007.03.001Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar). Mobile elements can insert themselves at new locations in host genomes to modify gene structure and alter gene expression. Rampant mobility of these elements would endanger both the host and, thereby, the element. Thus a strong selective pressure exists to limit their transposition. Mobile elements are classified into two categories based on the mechanism of their transposition. DNA transposons, such as Drosophila P elements, generally utilize a cut-and-paste mechanism in which the transposon is excised from a donor site and inserted into a new genomic location. Retrotransposons and endogenous retroviruses such as gypsy elements represent a distinct class of mobile genetic sequences that insert into new genomic locations by reverse transcription of an RNA intermediate. Expansion of these selfish elements can occur when novel transposition events are transmitted to subsequent generations after germline hopping; indeed metazoan transposons often show germline-restricted expression. Therefore, it seems likely that metazoan genomes have evolved mechanisms to regulate germline mobilization of transposable elements. DNA methylation is one important mechanism involved in the silencing of transposons in plant, mammalian, and fungal germlines (Yoder et al., 1997Yoder J.A. Walsh C.P. Bestor T.H. Cytosine methylation and the ecology of intragenomic parasites.Trends Genet. 1997; 13: 335-340Abstract Full Text PDF PubMed Scopus (1450) Google Scholar, Martienssen and Colot, 2001Martienssen R.A. Colot V. DNA methylation and epigenetic inheritance in plants and filamentous fungi.Science. 2001; 293: 1070-1074Crossref PubMed Scopus (383) Google Scholar, Selker, 2004Selker E.U. Genome defense and DNA methylation in Neurospora.Cold Spring Harb. Symp. Quant. Biol. 2004; 69: 119-124Crossref PubMed Scopus (22) Google Scholar). Additionally, APOBECs (a class of RNA/DNA-editing enzymes) have been found to be potent genome defense proteins against retroelements (Takaori-Kondo, 2006Takaori-Kondo A. APOBEC family proteins: novel antiviral innate immunity.Int. J. Hematol. 2006; 83: 213-216Crossref PubMed Scopus (20) Google Scholar). RNAi is widely believed to control retrotransposition (Robert et al., 2004Robert V.J. Vastenhouw N.L. Plasterk R.H. RNA interference, transposon silencing, and cosuppression in the Caenorhabditis elegans germ line: similarities and differences.Cold Spring Harb. Symp. Quant. Biol. 2004; 69: 397-402Crossref PubMed Scopus (19) Google Scholar); however, this system has a surprisingly modest effect on silencing mammalian retrotransposons in somatic cells (Yang and Kazazian, 2006Yang N. Kazazian Jr., H.H. L1 retrotransposition is suppressed by endogenously encoded small interfering RNAs in human cultured cells.Nat. Struct. Mol. Biol. 2006; 13: 763-771Crossref PubMed Scopus (297) Google Scholar). With the recent characterization of the molecular function of Piwi (P element-induced wimpy testes) proteins, a novel form of control for mobile elements has emerged involving small RNAs in germ cells. The founding member of this class of proteins, Piwi, was first identified 10 years ago in a genetic screen for mutants that affect asymmetric division of stem cells in the Drosophila germline (Lin and Spradling, 1997Lin H. Spradling A.C. A novel group of pumilio mutations affects the asymmetric division of germline stem cells in the Drosophila ovary.Development. 1997; 124: 2463-2476Crossref PubMed Google Scholar). Early studies demonstrated that Drosophila Piwi is essential for spermatogenesis and is a key regulator of female germline stem cells (Cox et al., 2000Cox D.N. Chao A. Lin H. piwi encodes a nucleoplasmic factor whose activity modulates the number and division rate of germline stem cells.Development. 2000; 127: 503-514PubMed Google Scholar). It was also appreciated that Piwi proteins are an ancient subset of the larger Argonaute protein family (Carmell et al., 2002Carmell M.A. Xuan Z. Zhang M.Q. Hannon G.J. The Argonaute family: tentacles that reach into RNAi, developmental control, stem cell maintenance, and tumorigenesis.Genes Dev. 2002; 16: 2733-2742Crossref PubMed Scopus (668) Google Scholar, Cerutti and Casas-Mollano, 2006Cerutti H. Casas-Mollano J.A. On the origin and functions of RNA-mediated silencing: from protists to man.Curr. Genet. 2006; 50: 81-99Crossref PubMed Scopus (359) Google Scholar), other members of which associate with short-interfering (si)RNAs and micro (mi)RNAs. These small RNAs serve as guides that lead to degradation and/or reduced translation of target mRNAs. Membership in the Argonaute family suggested that Piwi proteins and their associated RNAs might also mediate RNA silencing. The recent identification and characterization of the small Piwi-interacting RNAs (dubbed piRNAs) has indicated that Piwi proteins mediate RNA-mediated silencing of mobile elements, thereby defending the germline genome. The murine Piwi orthologs Miwi and Mili are essential for mammalian spermatogenesis (Deng and Lin, 2002Deng W. Lin H. miwi, a murine homolog of piwi, encodes a cytoplasmic protein essential for spermatogenesis.Dev. Cell. 2002; 2: 819-830Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar, Kuramochi-Miyagawa et al., 2004Kuramochi-Miyagawa S. Kimura T. Ijiri T.W. Isobe T. Asada N. Fujita Y. Ikawa M. Iwai N. Okabe M. Deng W. et al.Mili, a mammalian member of piwi family gene, is essential for spermatogenesis.Development. 2004; 131: 839-849Crossref PubMed Scopus (572) Google Scholar). Mice with targeted mutations in either gene are sterile and have distinct defects in gametogenesis, but unlike the Drosophila piwi mutant, neither loses germline stem cells. To investigate the role of the third mouse Piwi family member in gametogenesis, the gene encoding Miwi2 has now been disrupted. In a report described in Developmental Cell, Carmell et al., 2007Carmell M.A. Girard A. van de Kant H.J.G. de Rooij D.G. Hannon G.J. Miwi2 is essential for spermatogenesis and repression of transposons in the mouse male germline.Dev. Cell. 2007; (Published online March 29, 2007)https://doi.org/10.1016/j.devcel.2007.03.001Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar demonstrate that Miwi2 mutants are unique in their loss of germline stem cells. These observations suggest that the stem cell maintenance functions exhibited by Drosophila Piwi are conserved in mice through the function of Miwi2. After initial characterization of the MILI/MIWI proteins in mice, the next challenge was to identify their small RNA-binding partners. Last year, five independent laboratories reported the identification of mammalian piRNAs from mouse and rat testes (Aravin et al., 2006Aravin A. Gaidatzis D. Pfeffer S. Lagos-Quintana M. Landgraf P. Iovino N. Morris P. Brownstein M.J. Kuramochi-Miyagawa S. Nakano T. et al.A novel class of small RNAs bind to MILI protein in mouse testes.Nature. 2006; 442: 203-207Crossref PubMed Scopus (1010) Google Scholar, Girard et al., 2006Girard A. Sachidanandam R. Hannon G.J. Carmell M.A. A germline-specific class of small RNAs binds mammalian Piwi proteins.Nature. 2006; 442: 199-202Crossref PubMed Scopus (1112) Google Scholar, Grivna et al., 2006Grivna S.T. Beyret E. Wang Z. Lin H. A novel class of small RNAs in mouse spermatogenic cells.Genes Dev. 2006; 20: 1709-1714Crossref PubMed Scopus (602) Google Scholar, Lau et al., 2006Lau N.C. Seto A.G. Kim J. Kuramochi-Miyagawa S. Nakano T. Bartel D.P. Kingston R.E. Characterization of the piRNA complex from rat testes.Science. 2006; 313: 363-367Crossref PubMed Scopus (690) Google Scholar, Watanabe et al., 2006Watanabe T. Takeda A. Tsukiyama T. Mise K. Okuno T. Sasaki H. Minami N. Imai H. Identification and characterization of two novel classes of small RNAs in the mouse germline: retrotransposon-derived siRNAs in oocytes and germline small RNAs in testes.Genes Dev. 2006; 20: 1732-1743Crossref PubMed Scopus (445) Google Scholar). Two of these groups purified ribonucleoprotein complexes with a MILI- or MIWI-specific antibody from adult mouse testes and then cloned and sequenced the associated small RNAs. These MILI- and MIWI-interacting RNAs were termed piRNAs based on their interaction with the mouse Piwi proteins. piRNAs have several interesting characteristics. First, these small RNAs were longer than miRNAs and siRNAs and similar in size to a previously described class of Drosophila RNAs corresponding to repeat sequences, “rasiRNAs” (repeat-associated siRNAs; Aravin et al., 2003Aravin A.A. Lagos-Quintana M. Yalcin A. Zavolan M. Marks D. Snyder B. Gaasterland T. Meyer J. Tuschl T. The small RNA profile during Drosophila melanogaster development.Dev. Cell. 2003; 5: 337-350Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar). Second, the majority of piRNAs mapped to a small number of genomic loci. Individual clusters range between 1 and 100 kb in size and contain between 10 and 4500 piRNAs, demonstrating that thousands of piRNAs may be generated from one particular locus. Third, many of these clusters exhibit remarkable asymmetry, meaning that within a given cluster all piRNAs are derived from the same strand. This asymmetric orientation suggests that piRNAs might be processed from long primary transcripts. When two adjacent clusters were located in close proximity to each other, strand switching was also commonly observed. Aravin et al., 2006Aravin A. Gaidatzis D. Pfeffer S. Lagos-Quintana M. Landgraf P. Iovino N. Morris P. Brownstein M.J. Kuramochi-Miyagawa S. Nakano T. et al.A novel class of small RNAs bind to MILI protein in mouse testes.Nature. 2006; 442: 203-207Crossref PubMed Scopus (1010) Google Scholar postulated that these neighboring clusters with opposite strand polarity might be transcribed divergently from one bidirectional promoter. Sequence analysis of the MILI- and MIWI-associated piRNAs revealed a strong bias for uridine residues at their 5′ termini. This 5′ uridine bias is characteristic of siRNAs and miRNAs processed from double-stranded precursors by RNase III enzymes. However, a computational search for stem loops similar to pre-miRNAs failed to identify any secondary structures in regions flanking piRNAs, suggesting that piRNA processing is distinct from miRNA biogenesis. Finally, ∼17% of mammalian piRNAs mapped to repeat sequences, including LINEs, SINEs, and several classes of DNA transposons. Although this is consistent with a possible role in mobile element defense, considering that ∼40% of the mouse genome is composed of repetitive elements, this is actually less than expected by chance. However, a conserved role for Miwi2 in mobile element control is suggested by the observation of increased L1 retrotransposon expression in the Miwi2 mutant testes (Carmell et al., 2007Carmell M.A. Girard A. van de Kant H.J.G. de Rooij D.G. Hannon G.J. Miwi2 is essential for spermatogenesis and repression of transposons in the mouse male germline.Dev. Cell. 2007; (Published online March 29, 2007)https://doi.org/10.1016/j.devcel.2007.03.001Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar). Interestingly, this increase in L1 transcription was accompanied by decreased L1 DNA methylation, suggesting a possible interplay between Piwi (and perhaps piRNAs) and methylation machinery, reminiscent of the interaction between the siRNA posttranscriptional silencing machinery and chromatin level transcriptional regulation in Schizosaccharomyces pombe (Verdel et al., 2004Verdel A. Jia S. Gerber S. Sugiyama T. Gygi S. Grewal S.I. Moazed D. RNAi-mediated targeting of heterochromatin by the RITS complex.Science. 2004; 303: 672-676Crossref PubMed Scopus (911) Google Scholar). However, this analogy notwithstanding, it is important to note that no Miwi2-specific piRNAs have yet been described, so it is formally possible that this pathway is piRNA independent. This raises the question, how pervasive is the Piwi-piRNA-genome defense association? In several of the earlier piRNA sequence studies, the majority of piRNAs were identified only once, suggesting a high degree of complexity in piRNA populations. Comparative genomics further revealed that the piRNA loci, but not their sequences, are conserved throughout evolution. As Girard et al., 2006Girard A. Sachidanandam R. Hannon G.J. Carmell M.A. A germline-specific class of small RNAs binds mammalian Piwi proteins.Nature. 2006; 442: 199-202Crossref PubMed Scopus (1112) Google Scholar point out, this may indicate that the sequence of a piRNA does not necessarily specify its function. Rather, its true function may be determined by the abundance of piRNAs produced from any individual locus. Despite these interesting and confounding discoveries, several important questions remained. Do piRNAs exist in invertebrates and other vertebrate species? What are their mRNA targets? Are piRNAs similar to Drosophila rasiRNAs? Is there more compelling evidence that piRNAs provide defense against genome intruders like mobile elements? Two new papers, one in this issue of Cell (Brennecke et al., 2007Brennecke J. Aravin A.A. Stark A. Dus M. Kellis M. Sachidanandam R. Hannon G.J. Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila.Cell. 2007; 128: 1098-1103Abstract Full Text Full Text PDF Scopus (1594) Google Scholar, Houwing et al., 2007Houwing S. Kamminga L.M. Berezikov E. Cronembold D. Girard A. Moens C.B. Plasterk R.H.A. Hannon G.J. Draper B.W. Ketting R.F. Zebrafish PIWI and piRNAs; Implications for germ cell survival and transposon silencing.Cell. 2007; (this issue)PubMed Google Scholar), shed light on some of these questions and provide us with more food for thought. Although piRNAs were first identified in mammals, analogous studies in flies revealed that this class of small RNAs also exists in invertebrates. Recently, two Piwi family members in Drosophila, Aubergine and Piwi, were found to bind small RNAs (Saito et al., 2006Saito K. Nishida K.M. Mori T. Kawamura Y. Miyoshi K. Nagami T. Siomi H. Siomi M.C. Specific association of Piwi with rasiRNAs derived from retrotransposon and heterochromatic regions in the Drosophila genome.Genes Dev. 2006; 20: 2214-2222Crossref PubMed Scopus (459) Google Scholar, Vagin et al., 2006Vagin V.V. Sigova A. Li C. Seitz H. Gvozdev V. Zamore P.D. A distinct small RNA pathway silences selfish genetic elements in the germline.Science. 2006; 313: 320-324Crossref PubMed Scopus (900) Google Scholar). In a study reporting a few hundred piRNA sequences, Saito et al. demonstrated that Piwi complex immunopurified from Drosophila ovaries contained a class of small RNAs distinct in size from siRNAs and miRNAs. Sequencing revealed that most of these piRNAs corresponded to repetitive elements and heterochromatic genome regions. Tuschl and colleagues had previously identified about 4000 Drosophila germline rasiRNAs (Aravin et al., 2003Aravin A.A. Lagos-Quintana M. Yalcin A. Zavolan M. Marks D. Snyder B. Gaasterland T. Meyer J. Tuschl T. The small RNA profile during Drosophila melanogaster development.Dev. Cell. 2003; 5: 337-350Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar), which also corresponded to repetitive elements, suggesting that they might regulate chromatin structure and transposon activity. Based on current evidence, it appears that most rasiRNAs in flies are simply a (very important) subclass of piRNAs. In recent years, Piwi proteins were recognized as having potential anti-mobile element activity. Transposition of telomeric retroelements and P elements is enhanced in aubergine mutants whereas piwi mutants mobilized the endogenous retrovirus gypsy (Sarot et al., 2004Sarot E. Payen-Groschene G. Bucheton A. Pelisson A. Evidence for a piwi-dependent RNA silencing of the gypsy endogenous retrovirus by the Drosophila melanogaster flamenco gene.Genetics. 2004; 166: 1313-1321Crossref PubMed Scopus (187) Google Scholar) and showed increased expression of copia and mdg1 elements (Kalmykova et al., 2005Kalmykova A.I. Klenov M.S. Gvozdev V.A. Argonaute protein PIWI controls mobilization of retrotransposons in the Drosophila male germline.Nucleic Acids Res. 2005; 33: 2052-2059Crossref PubMed Scopus (168) Google Scholar). Vagin et al., 2006Vagin V.V. Sigova A. Li C. Seitz H. Gvozdev V. Zamore P.D. A distinct small RNA pathway silences selfish genetic elements in the germline.Science. 2006; 313: 320-324Crossref PubMed Scopus (900) Google Scholar also demonstrated that expression of retrotransposons was derepressed in the germline of piwi and aubergine mutants. Importantly, silencing of these retroelements did not require RNAi or miRNA proteins. These findings suggested that Piwi proteins and their associated small RNAs might silence mobile elements in the germline. In a recent issue of Cell, Brennecke et al., 2007Brennecke J. Aravin A.A. Stark A. Dus M. Kellis M. Sachidanandam R. Hannon G.J. Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila.Cell. 2007; 128: 1098-1103Abstract Full Text Full Text PDF Scopus (1594) Google Scholar investigate the small RNA-binding partners of Piwi, Aubergine, and Ago3 in the Drosophila female germline at high resolution. After purifying RNP complexes using antibodies specific to each of the three proteins, cDNA libraries were prepared from each of the piRNA populations. 454 sequencing yielded more than 60,000 piRNA reads, providing a much larger sequence population to analyze than in the earlier fly studies. Similar to mammalian piRNAs, Drosophila piRNAs are longer than miRNAs and siRNAs and map to discrete genomic clusters. For example, the largest 15 clusters account for 70% of all piRNAs, suggesting that a limited number of master piRNA loci might control germline mobile element activity. Unlike their mammalian counterparts, most piRNAs in flies (∼80%) are present in pericentromeric and telomeric heterochromatin and correspond to truncated or defective repeat elements. How do these findings square with earlier studies of transposon control mechanisms? One model of transposon control proposes that transposon resistance is due to discrete genomic loci and is supported by studies of the gypsy element, the first endogenous retrovirus discovered in invertebrates. The mobility of gypsy and two other retroelements, Idefix and ZAM, is controlled by flamenco, a specific heterochromatic locus in the X chromosome (Bucheton, 1995Bucheton A. The relationship between the flamenco gene and gypsy in Drosophila: how to tame a retrovirus.Trends Genet. 1995; 11: 349-353Abstract Full Text PDF PubMed Scopus (73) Google Scholar). Despite intensive study of flamenco, no “transposon repressor locus” could be identified in the sequence. Rather, it contained a jumble of different types of transposable elements, but exactly how these elements might be involved in a transposon defense system remained unclear. Sarot et al., 2004Sarot E. Payen-Groschene G. Bucheton A. Pelisson A. Evidence for a piwi-dependent RNA silencing of the gypsy endogenous retrovirus by the Drosophila melanogaster flamenco gene.Genetics. 2004; 166: 1313-1321Crossref PubMed Scopus (187) Google Scholar provided one connection by showing that flamenco-mediated silencing of gypsy depends on Piwi. Now, Brennecke et al., 2007Brennecke J. Aravin A.A. Stark A. Dus M. Kellis M. Sachidanandam R. Hannon G.J. Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila.Cell. 2007; 128: 1098-1103Abstract Full Text Full Text PDF Scopus (1594) Google Scholar provide direct sequence evidence that a large piRNA locus spanning more than 150 kb corresponds to the flamenco locus. The depth of the sequencing allowed them to find many instances of mobile element-derived piRNAs mapping uniquely to flamenco. Further supporting the notion that piRNA clusters are control loci that regulate transposon activity through the Piwi pathway, Brennecke et al. performed several functional tests using flamenco mutants. In agreement with their hypothesis, mature piRNA expression levels decreased in flamenco mutants whereas gypsy mRNA expression increased. Brennecke et al. also demonstrate that the subtelomeric TAS repeat on the X chromosome (X-TAS) corresponds to yet another piRNA cluster. Previous studies have linked specific alleles of this locus, here designated X-TASP, to the global control of P elements (see references in Brennecke et al., 2007Brennecke J. Aravin A.A. Stark A. Dus M. Kellis M. Sachidanandam R. Hannon G.J. Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila.Cell. 2007; 128: 1098-1103Abstract Full Text Full Text PDF Scopus (1594) Google Scholar). Those alleles are distinguished by containing P element insertions in X-TAS. The sites from which piRNAs (not complementary to P elements) emanate in the Oregon R fly strain analyzed by Brennecke et al., 2007Brennecke J. Aravin A.A. Stark A. Dus M. Kellis M. Sachidanandam R. Hannon G.J. Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila.Cell. 2007; 128: 1098-1103Abstract Full Text Full Text PDF Scopus (1594) Google Scholar correspond to the insertion positions of three P elements found in a series of X-TASP strains. The Oregon R fly strain does not contain P sequences at X-TAS. Thus it seems likely that the X-TASP loci will produce P element-derived piRNAs. This is truly remarkable because P elements invaded the D. melanogaster genome only within the last 50 years, presumably sweeping in through contact with a sibling species (Kidwell, 1983Kidwell M.G. Evolution of hybrid dysgenesis determinants in Drosophila melanogaster.Proc. Natl. Acad. Sci. USA. 1983; 80: 1655-1659Crossref PubMed Scopus (212) Google Scholar). The implication is that the resistance locus was born when P elements inserted into X-TAS, within very recent history, showing how dynamic the interplay between host and genome parasite can be even on a short time scale. A model for piRNA-mediated suppression of transposons is shown in Figure 1. Using flamenco and X-TAS as examples, these heterochromatic loci generate hundreds of distinct piRNAs that correspond to transposon repeats dispersed throughout the Drosophila genome. These piRNAs associate with Piwi proteins and serve as guides that lead to cleavage of expressed transposon targets. By examining the strand bias of piRNAs derived from each of the three Piwi complexes, these authors, as well as Gunawardane et al., 2007Gunawardane L.S. Saito K. Nishida K.M. Miyoshi K. Kawamura Y. Nagami T. Siomi H. Siomi M.C. A slicer-mediated mechanism for repeat-associated siRNA 5′ end formation in Drosophila.Science. 2007; 315: 1587-1590Crossref PubMed Scopus (814) Google Scholar who performed a smaller piRNA sequencing study, made several other important observations consistent with a genome defense mechanism. Piwi and Aubergine preferentially bind piRNAs corresponding to the antisense strand of transposons. In contrast, Ago3 complexes are biased for the sense strand of transposons. Perhaps one of the most intriguing findings is the observation of a unique complementary relationship between these sense and antisense piRNAs. Assuming that piRNAs are ∼25 nucleotides long, one would expect corresponding sense and antisense piRNAs to overlap by 23 nucleotides with a 2 nucleotide 3′ overhang at each end if processed in an siRNA- or miRNA-like manner. In fact, this was not observed with complementary piRNAs. Instead, the 5′ ends of complementary piRNAs were separated by 10 nucleotides, with the strongest complementarity observed between Ago3- and Aubergine-associated piRNAs. Yet another surprise was the enrichment of 5′-terminal uridine residues in Piwi- and Aubergine-bound piRNAs, which correspond to the antisense strand of transposons. As might be expected for sense strand piRNAs bound to Ago3, these show a dramatic enrichment for adenine at position 10, and they complement the 5′-terminal uridine of an antisense piRNA bound to Piwi or Aubergine. Notably, the same strand bias was observed for piRNAs bound to Drosophila Piwi proteins (Gunawardane et al., 2007Gunawardane L.S. Saito K. Nishida K.M. Miyoshi K. Kawamura Y. Nagami T. Siomi H. Siomi M.C. A slicer-mediated mechanism for repeat-associated siRNA 5′ end formation in Drosophila.Science. 2007; 315: 1587-1590Crossref PubMed Scopus (814) Google Scholar). These findings suggest that Piwi-mediated cleavage events generate new piRNAs. In light of these findings, both groups propose a self-reinforcing amplification cycle for piRNA generation that may be analogous to secondary siRNA generation by RNA-dependent RNA polymerases (Figure 2). According to this model, initiation of the cycle begins with processing of primary piRNAs, which are derived from defective transposon copies in regions of heterochromatin. These piRNAs are antisense to expressed transposons and bind either Piwi or Aubergine. Together, the Piwi/Aubergine-piRNA complexes identify and cleave their active transposon targets, generating new sense piRNAs that bind Ago3. Next, a sense piRNA-Ago3 complex directs another cleavage event of a piRNA cluster transcript, creating a new antisense piRNA capable of binding to Piwi or Aubergine. Amplification of the response is dependent on the interaction between piRNA sequences in different clusters. As long as secondary antisense piRNA complexes are able to recognize and silence their target transposable elements, the cycle is reinforced through the production of additional sense piRNAs. Although several aspects of the model have yet to be validated, this amplification loop has important implications for mobile element control in the germline. The proposed model raises several important questions. How is the amplification cycle initiated with primary antisense piRNAs loaded into Piwi or Aubergine? Although it is logical that Ago3-bound piRNAs would be in the sense orientation if they were generated solely by piRNA-mediated cleavage of transposon sequences, the origin of the strict antisense strand bias of Piwi- and Aubergine-bound piRNAs is not intuitive. Brennecke et al., 2007Brennecke J. Aravin A.A. Stark A. Dus M. Kellis M. Sachidanandam R. Hannon G.J. Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila.Cell. 2007; 128: 1098-1103Abstract Full Text Full Text PDF Scopus (1594) Google Scholar demonstrate that there are special loci such as flamenco from which piRNAs are generated from only one strand and specificall

L1 elements are polyA retrotransposons which inhabit the human genome. Recent work has defined an endonuclease (L1 EN) encoded by the L1 element required for retrotransposition. We report the sequence specificity of this nicking endonuclease and the physical basis of its DNA recognition. L1 endonuclease is specific for the unusual DNA structural features found at the TpA junction of 5'(dTn-dAn) x 5'(dTn-dAn) tracts. Within the context of this sequence, substitutions which generate a pyrimidine-purine junction are tolerated, whereas purine-pyrimidine junctions greatly reduce or eliminate nicking activity. The A-tract conformation of the DNA substrate 5' of the nicked site is required for L1 EN nicking. Chemical or physical unwinding of the DNA helix enhances L1 endonuclease activity, while disruption of the adenine mobility associated with TpA junctions reduces it. Akin to the protein-DNA interactions of DNase I, L1 endonuclease DNA recognition is likely mediated by minor groove interactions. Unlike several of its homologues, however, L1 EN exhibits no AP endonuclease activity. Finally, we speculate on the implications of the specificity of the L1 endonuclease for the parasitic relationship between retroelements and the human genome.

Using a genetic screen aimed at identifying cellular factors involved in Ty1 transposition, we have identified a mutation in a host gene that reduces Ty1 transposition frequency. The mutant, dbr1, is also defective in the process of intron turnover. In dbr1 cells, excised introns derived from a variety of pre-mRNAs are remarkably stable and accumulate to levels exceeding that of the corresponding mRNA. The stable excised introns accumulate in the form of a lariat that is missing the linear sequences 3' of the branchpoint. The DBR1 gene has been isolated by complementation of the transposition phenotype. DBR1 is shown to encode debranching enzyme, an RNA processing activity that hydrolyzes the 2'-5' phosphodiester linkage at the branchpoint of excised intron lariats. In Saccharomyces cerevisiae, debranching enzyme plays a requisite role in the rapid turnover of excised introns, yet its function is not essential for viability.

ABSTRACT Transcriptional silencing in Saccharomyces cerevisiae occurs at several genetic loci, including the ribosomal DNA (rDNA). Silencing at telomeres (telomere position effect [TPE]) and the cryptic mating-type loci ( HML and HMR ) depends on the silent information regulator genes, SIR1 , SIR2 , SIR3 , and SIR4 . However, silencing of polymerase II-transcribed reporter genes integrated within the rDNA locus (rDNA silencing) requires only SIR2 . The mechanism of rDNA silencing is therefore distinct from TPE and HM silencing. Few genes other than SIR2 have so far been linked to the rDNA silencing process. To identify additional non-Sir factors that affect rDNA silencing, we performed a genetic screen designed to isolate mutations which alter the expression of reporter genes integrated within the rDNA. We isolated two classes of mutants: those with a loss of rDNA silencing ( lrs ) phenotype and those with an increased rDNA silencing ( irs ) phenotype. Using transposon mutagenesis, lrs mutants were found in 11 different genes, and irs mutants were found in 22 different genes. Surprisingly, we did not isolate any genes involved in rRNA transcription. Instead, multiple genes associated with DNA replication and modulation of chromatin structure were isolated. We describe these two gene classes, and two previously uncharacterized genes, LRS4 and IRS4 . Further characterization of the lrs and irs mutants revealed that many had alterations in rDNA chromatin structure. Several lrs mutants, including those in the cdc17 and rfc1 genes, caused lengthened telomeres, consistent with the hypothesis that telomere length modulates rDNA silencing. Mutations in the HDB (RPD3) histone deacetylase complex paradoxically increased rDNA silencing by a SIR2 -dependent, SIR3 -independent mechanism. Mutations in rpd3 also restored mating competence selectively to sir3Δ MAT α strains, suggesting restoration of silencing at HMR in a sir3 mutant background.

Sir2 proteins are NAD(+)-dependent protein deacetylases that play key roles in transcriptional regulation, DNA repair, and life span regulation. The structure of an archaeal Sir2 enzyme, Sir2-Af2, bound to an acetylated p53 peptide reveals that the substrate binds in a cleft in the enzyme, forming an enzyme-substrate beta sheet with two flanking strands in Sir2-Af2. The acetyl-lysine inserts into a conserved hydrophobic tunnel that contains the active site histidine. Comparison with other structures of Sir2 enzymes suggests that the apoenzyme undergoes a conformational change upon substrate binding. Based on the Sir2-Af2 substrate complex structure, mutations were made in the other A. fulgidus sirtuin, Sir2-Af1, that increased its affinity for the p53 peptide.

The biological significance of recently described modifiable residues in the globular core of the bovine nucleosome remains elusive. We have mapped these modification sites onto the Saccharomyces cerevisiae histones and used a genetic approach to probe their potential roles both in heterochromatic regions of the genome and in the DNA repair response. By mutating these residues to mimic their modified and unmodified states, we have generated a total of 39 alleles affecting 14 residues in histones H3 and H4. Remarkably, despite the apparent evolutionary pressure to conserve these near-invariant histone amino acid sequences, the vast majority of mutant alleles are viable. However, a subset of these variant proteins elicit an effect on transcriptional silencing both at the ribosomal DNA locus and at telomeres, suggesting that posttranslational modification(s) at these sites regulates formation and/or maintenance of heterochromatin. Furthermore, we provide direct mass spectrometry evidence for the existence of histone H3 K56 acetylation in yeast. We also show that substitutions at histone H4 K91, K59, S47, and R92 and histone H3 K56 and K115 lead to hypersensitivity to DNA-damaging agents, linking the significance of the chemical identity of these modifiable residues to DNA metabolism. Finally, we allude to the possible molecular mechanisms underlying the effects of these modifications.

Retroviruses and their relatives, the LTR-containing retrotransposons, integrate newly replicated cDNA copies of their genomes into the genomes of their hosts using element-encoded integrases. Although target site selection is not well understood for this general class of elements, it is becoming clear that some elements target their integration events to very specific regions of their host genomes. Evidence is accumulating that the yeast retrotransposon Ty1 behaves in this manner. Ty1 is found frequently adjacent to tRNA genes in the yeast genome and experimental evidence implicates these regions as preferred integration sites. To determine the basis for Ty1 targeting, we developed an in vivo integration assay using a Ty1 donor plasmid and a second target plasmid that could be used to measure the relative frequency of Ty1 integration into sequences cloned from various regions of the yeast genome. Targets containing genes transcribed by RNA polymerase III (Pol III) were up to several hundredfold more active as integration targets than "cold" sequences lacking such genes. High-frequency targeting was dependent on Pol III transcription, and integration was "region specific," occurring exclusively upstream of the transcription start sites of these genes. Thus, Ty1 has evolved a powerful targeting mechanism, requiring Pol III transcription to integrate its DNA at very specific locations within the yeast genome.

<h2>Summary</h2> Nucleosome structural integrity underlies the regulation of DNA metabolism and transcription. Using a synthetic approach, a versatile library of 486 systematic histone H3 and H4 substitution and deletion mutants that probes the contribution of each residue to nucleosome function was generated in <i>Saccharomyces cerevisiae</i>. We probed fitness contributions of each residue to perturbations of chromosome integrity and transcription, mapping global patterns of chemical sensitivities and requirements for transcriptional silencing onto the nucleosome surface. Each histone mutant was tagged with unique molecular barcodes, facilitating identification of histone mutant pools through barcode amplification, labeling, and TAG microarray hybridization. Barcodes were used to score complex phenotypes such as competitive fitness in a chemostat, DNA repair proficiency, and synthetic genetic interactions, revealing new functions for distinct histone residues and new interdependencies among nucleosome components and their modifiers.

Model organisms have played an important role in the elucidation of multiple genes and cellular processes that regulate aging. In this study we utilized the budding yeast, Saccharomyces cerevisiae, in a large-scale screen for genes that function in the regulation of chronological lifespan, which is defined by the number of days that non-dividing cells remain viable. A pooled collection of viable haploid gene deletion mutants, each tagged with unique identifying DNA "bar-code" sequences was chronologically aged in liquid culture. Viable mutants in the aging population were selected at several time points and then detected using a microarray DNA hybridization technique that quantifies abundance of the barcode tags. Multiple short- and long-lived mutants were identified using this approach. Among the confirmed short-lived mutants were those defective for autophagy, indicating a key requirement for the recycling of cellular organelles in longevity. Defects in autophagy also prevented lifespan extension induced by limitation of amino acids in the growth media. Among the confirmed long-lived mutants were those defective in the highly conserved de novo purine biosynthesis pathway (the ADE genes), which ultimately produces IMP and AMP. Blocking this pathway extended lifespan to the same degree as calorie (glucose) restriction. A recently discovered cell-extrinsic mechanism of chronological aging involving acetic acid secretion and toxicity was suppressed in a long-lived ade4Delta mutant and exacerbated by a short-lived atg16Delta autophagy mutant. The identification of multiple novel effectors of yeast chronological lifespan will greatly aid in the elucidation of mechanisms that cells and organisms utilize in slowing down the aging process.

The Saccharomyces cerevisiae gene deletion project revealed that approximately 20% of yeast genes are required for viability. The analysis of essential genes traditionally relies on conditional mutants, typically temperature-sensitive (ts) alleles. We developed a systematic approach (termed "diploid shuffle") useful for generating a ts allele for each essential gene in S. cerevisiae and for improved genetic manipulation of mutant alleles and gene constructs in general. Importantly, each ts allele resides at its normal genomic locus, flanked by specific cognate UPTAG and DNTAG bar codes. A subset of 250 ts mutants, including ts alleles for all uncharacterized essential genes and prioritized for genes with human counterparts, is now ready for distribution. The importance of this collection is demonstrated by biochemical and genetic screens that reveal essential genes involved in RNA processing and maintenance of chromosomal stability.

Yeast Ty1 elements are retrotransposons that transpose via an RNA intermediate found in a virus-like particle (Ty-VLP). A Ty-encoded reverse transcriptase activity found inside the particles is capable of giving rise to full-length reverse transcripts. The predominant form of these reverse transcripts is a full-length linear duplex DNA. We have developed a cell-free system for transposition of Ty1 DNA molecules into a bacteriophage lambda target. Purified Ty-VLPs and target DNA are the only macromolecular components required for the transposition reaction. A TYB-encoded protein, p90-TYB, contains amino acid sequences that are similar to those of retroviral integrase proteins. Mutations in the integrase coding region abolish transposition both in vivo and in vitro.

Integrated HIV-1 genomes are found within actively transcribed host genes in latently infected CD4+ T cells. Readthrough transcription of the host gene might therefore suppress HIV-1 gene expression and promote the latent infection that allows viral persistence in patients on therapy. To address the effect of host gene readthrough, we used homologous recombination to insert HIV-1 genomes in either orientation into an identical position within an intron of an actively transcribed host gene, hypoxanthine-guanine phosphoribosyltransferase (HPRT). Constructs were engineered to permit or block readthrough transcription of HPRT. Readthrough transcription inhibited HIV-1 gene expression for convergently orientated provirus but enhanced HIV-1 gene expression when HIV-1 was in the same orientation as the host gene. Orientation had a >10-fold effect on HIV-1 gene expression. Due to the nature of HIV-1 integration sites in vivo, this orientation-dependent regulation can influence the vast majority of infected cells and adds complexity to the maintenance of latency.

Genes with small open reading frames (sORFs; &lt;100 amino acids) represent an untapped source of important biology. sORFs largely escaped analysis because they were difficult to predict computationally and less likely to be targeted by genetic screens. Thus, the substantial number of sORFs and their potential importance have only recently become clear. To investigate sORF function, we undertook the first functional studies of sORFs in any system, using the model eukaryote Saccharomyces cerevisiae . Based on independent experimental approaches and computational analyses, evidence exists for 299 sORFs in the S. cerevisiae genome, representing ∼5% of the annotated ORFs. We determined that a similar percentage of sORFs are annotated in other eukaryotes, including humans, and 184 of the S. cerevisiae sORFs exhibit similarity with ORFs in other organisms. To investigate sORF function, we constructed a collection of gene-deletion mutants of 140 newly identified sORFs, each of which contains a strain-specific “molecular barcode,” bringing the total number of sORF deletion strains to 247. Phenotypic analyses of the new gene-deletion strains identified 22 sORFs required for haploid growth, growth at high temperature, growth in the presence of a nonfermentable carbon source, or growth in the presence of DNA damage and replication-arrest agents. We provide a collection of sORF deletion strains that can be integrated into the existing deletion collection as a resource for the yeast community for elucidating gene function. Moreover, our analyses of the S. cerevisiae sORFs establish that sORFs are conserved across eukaryotes and have important biological functions.

LINE-1 (L1) retrotransposons are replicating repetitive elements that, by mass, are the most-abundant sequences in the human genome. Over one-third of mammalian genomes are the result, directly or indirectly, of L1 retrotransposition. L1 encodes two proteins: ORF1, an RNA-binding protein, and ORF2, an endonuclease/reverse transcriptase. Both proteins are required for L1 mobilization. Apart from the obvious function of self-replication, it is not clear what other roles, if any, L1 plays within its host. The sheer magnitude of L1 sequences in our genome has fueled speculation that over evolutionary time L1 insertions may structurally modify endogenous genes and regulate gene expression. Here we provide a review of L1 replication and its potential functional consequences. BioEssays 27:775–784, 2005. © 2005 Wiley Periodicals, Inc.

Derivatives of filamentous phage, f1, fd, and M 13, useful as cloning vectors are listed, and procedures for their use are reviewed. Methods for growing phage, preparing single- and double-stranded DNA, and cloning are given in the “cook-book” form. These procedures minimize the practical problem often associated with filamentous-phage cloning, i.e., deletion of inserts.

As the rough draft of the human genome sequence nears a finished product and other genome-sequencing projects accumulate sequence data exponentially, bioinformatics is emerging as an important tool for studies of transposon biology. In particular, L1 elements exhibit a variety of sequence structures after insertion into the human genome that are amenable to computational analysis. We carried out a detailed analysis of the anatomy and distribution of L1 elements in the human genome using a new computer program, TSDfinder, designed to identify transposon boundaries precisely.Structural variants of L1 elements shared similar trends in the length and quality of their target site duplications (TSDs) and poly(A) tails. Furthermore, we found no correlation between the composition and genomic location of the pre-insertion locus and the resulting anatomy of the L1 insertion. We verified that L1 insertions with TSDs have the 5'-TTAAAA-3' cleavage site associated with L1 endonuclease activity. In addition, the second target DNA cut required for L1 insertion weakly matches the consensus pattern TTAAAA. On the other hand, the L1-internal breakpoints of deleted and inverted L1 elements do not resemble L1 endonuclease cleavage sites. Finally, the genome sequence data indicate that whereas singly inverted elements are common, doubly inverted elements are almost never found.The sequence data give no indication that the creation of L1 structural variants depends on characteristics of the insertion locus. In addition, the formation of 5' truncated and 5' inverted L1s are probably not due to the action of the L1 endonuclease.

Loss or duplication of chromosome segments can lead to further genomic changes associated with cancer. However, it is not known whether only a select subset of genes is responsible for driving further changes. To determine whether perturbation of any given gene in a genome suffices to drive subsequent genetic changes, we analyzed the yeast knockout collection for secondary mutations of functional consequence. Unlike wild-type, most gene knockout strains were found to have one additional mutant gene affecting nutrient responses and/or heat-stress-induced cell death. Moreover, independent knockouts of the same gene often evolved mutations in the same secondary gene. Genome sequencing identified acquired mutations in several human tumor suppressor homologs. Thus, mutation of any single gene may cause a genomic imbalance, with consequences sufficient to drive adaptive genetic changes. This complicates genetic analyses but is a logical consequence of losing a functional unit originally acquired under pressure during evolution.

Many biological processes rely upon protein-protein interactions. Hence, detailed analysis of these interactions is critical for their understanding. Due to the complexities involved, genetic approaches are often needed. In yeast and phage, genetic characterizations of protein complexes are possible. However, in multicellular organisms, such characterizations are limited by the lack of powerful selection systems. Herein we describe genetic selections that allow single amino acid changes that disrupt protein-protein interactions to be selected from large libraries of randomly generated mutant alleles. The strategy, based on a yeast reverse two-hybrid system, involves a first-step negative selection for mutations that affect interaction, followed by a second-step positive selection for a subset of these mutations that maintain expression of full-length protein (two-step selection). We have selected such mutations in the transcription factor E2F1 that affect its ability to heterodimerize with DP1. The mutations obtained identified a putative helix in the marked box, a region conserved among E2F family members, as an important determinant for interaction. This two-step selection procedure can be used to characterize any interaction domain that can be tested in the two-hybrid system.

We describe a detailed deletion analysis of the anchoring domain of a model membrane protein. Removal of the 23 contiguous uncharged amino acids from the carboxy terminus of the bacteriophage fl gene III protein (pIII) converts it from an integral membrane protein to a secreted periplasmic form. Deletions that remove six or fewer residues of the hydrophobic core result in no diminution of the protein's capacity to anchor in the membrane. Longer deletions into this hydrophobic domain gradually destablize the protein-membrane association. pIII derivatives with over half of the hydrophobic core deleted retain substantial residual anchor function. The basic residues, arginine and lysine, which provide a carboxy-terminal boundary for this domain, can be deleted without loss of anchoring capacity.

The Saccharomyces genome-deletion project created >5900 'molecularly barcoded' yeast knockout mutants (YKO mutants). The YKO mutant collections have facilitated large-scale analyses of a multitude of mutant phenotypes. For example, both synthetic genetic array (SGA) and synthetic-lethality analysis by microarray (SLAM) methods have been used for synthetic-lethality screens. Global analysis of synthetic lethality promises to identify cellular pathways that 'buffer' each other biologically. The combination of global synthetic-lethality analysis, together with global protein-protein interaction analyses, mRNA expression profiling and functional profiling will, in principle, enable construction of a cellular 'wiring diagram' that will help frame a deeper understanding of human biology and disease.

Histone acetylation and deacetylation are among the principal mechanisms by which chromatin is regulated during transcription, DNA silencing, and DNA repair. We analyzed patterns of genetic interactions uncovered during comprehensive genome-wide analyses in yeast to probe how histone acetyltransferase (HAT) and histone deacetylase (HDAC) protein complexes interact. The genetic interaction data unveil an underappreciated role of HDACs in maintaining cellular viability, and led us to show that deacetylation of the histone variant Htz1p at Lys 14 is mediated by Hda1p. Studies of the essential nucleosome acetyltransferase of H4 (NuA4) revealed acetylation-dependent protein stabilization of Yng2p, a potential nonhistone substrate of NuA4 and Rpd3C, and led to a new functional organization model for this critical complex. We also found that DNA double-stranded breaks (DSBs) result in local recruitment of the NuA4 complex, followed by an elaborate NuA4 remodeling process concomitant with Rpd3p recruitment and histone deacetylation. These new characterizations of the HDA and NuA4 complexes demonstrate how systematic analyses of genetic interactions may help illuminate the mechanisms of intricate cellular processes.

To broaden the range of tools available for proteomic research, we generated a library of 16,368 unique full-length human ORFs that are expressible as N-terminal GST-His(6) fusion proteins. Following expression in yeast, these proteins were then individually purified and used to construct a human proteome microarray. To demonstrate the usefulness of this reagent, we developed a streamlined strategy for the production of monospecific monoclonal antibodies that used immunization with live human cells and microarray-based analysis of antibody specificity as its central components. We showed that microarray-based analysis of antibody specificity can be performed efficiently using a two-dimensional pooling strategy. We also demonstrated that our immunization and selection strategies result in a large fraction of monospecific monoclonal antibodies that are both immunoblot and immunoprecipitation grade. Our data indicate that the pipeline provides a robust platform for the generation of monoclonal antibodies of exceptional specificity.

Modern molecular biology has brought many new tools to the geneticist as well as an exponentially expanding database of genomes and new genes for study. Of particular use in the analysis of these genes is the synthetic gene, a nucleotide sequence designed to the specifications of the investigator. Typically, synthetic genes encode the same product as the gene of interest, but the synthetic nucleotide sequence for that protein may contain modifications affecting expression or base composition. Other desirable changes typically involve the revision of restriction sites. Designing synthetic genes by hand is a time-consuming and error-prone process that may involve several computer programs. We have developed a tools environment that combines many modules to provide a platform for rapid synthetic gene design for multikilobase sequences. We have used GeneDesign to successfully design a synthetic Ty1 element and a large variety of other synthetic sequences. GeneDesign has been implemented as a publicly accessible Web-based resource and can be found at http://slam.bs.jhmi.edu/gd.

Acetylation of histone and nonhistone proteins is an important posttranslational modification affecting many cellular processes. Here, we report that NuA4 acetylation of Sip2, a regulatory β subunit of the Snf1 complex (yeast AMP-activated protein kinase), decreases as cells age. Sip2 acetylation, controlled by antagonizing NuA4 acetyltransferase and Rpd3 deacetylase, enhances interaction with Snf1, the catalytic subunit of Snf1 complex. Sip2-Snf1 interaction inhibits Snf1 activity, thus decreasing phosphorylation of a downstream target, Sch9 (homolog of Akt/S6K), and ultimately leading to slower growth but extended replicative life span. Sip2 acetylation mimetics are more resistant to oxidative stress. We further demonstrate that the anti-aging effect of Sip2 acetylation is independent of extrinsic nutrient availability and TORC1 activity. We propose a protein acetylation-phosphorylation cascade that regulates Sch9 activity, controls intrinsic aging, and extends replicative life span in yeast.

Synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) generates combinatorial genomic diversity through rearrangements at designed recombinase sites. We applied SCRaMbLE to yeast synthetic chromosome arm synIXR (43 recombinase sites) and then used a computational pipeline to infer or unscramble the sequence of recombinations that created the observed genomes. Deep sequencing of 64 synIXR SCRaMbLE strains revealed 156 deletions, 89 inversions, 94 duplications, and 55 additional complex rearrangements; several duplications are consistent with a double rolling circle mechanism. Every SCRaMbLE strain was unique, validating the capability of SCRaMbLE to explore a diverse space of genomes. Rearrangements occurred exclusively at designed loxPsym sites, with no significant evidence for ectopic rearrangements or mutations involving synthetic regions, the 99% nonsynthetic nuclear genome, or the mitochondrial genome. Deletion frequencies identified genes required for viability or fast growth. Replacement of 3' UTR by non-UTR sequence had surprisingly little effect on fitness. SCRaMbLE generates genome diversity in designated regions, reveals fitness constraints, and should scale to simultaneous evolution of multiple synthetic chromosomes.

Extant species have wildly different numbers of chromosomes, even among taxa with relatively similar genome sizes (for example, insects)1,2. This is likely to reflect accidents of genome history, such as telomere–telomere fusions and genome duplication events3–5. Humans have 23 pairs of chromosomes, whereas other apes have 24. One human chromosome is a fusion product of the ancestral state6. This raises the question: how well can species tolerate a change in chromosome numbers without substantial changes to genome content? Many tools are used in chromosome engineering in Saccharomyces cerevisiae7–10, but CRISPR–Cas9-mediated genome editing facilitates the most aggressive engineering strategies. Here we successfully fused yeast chromosomes using CRISPR–Cas9, generating a near-isogenic series of strains with progressively fewer chromosomes ranging from sixteen to two. A strain carrying only two chromosomes of about six megabases each exhibited modest transcriptomic changes and grew without major defects. When we crossed a sixteen-chromosome strain with strains with fewer chromosomes, we noted two trends. As the number of chromosomes dropped below sixteen, spore viability decreased markedly, reaching less than 10% for twelve chromosomes. As the number of chromosomes decreased further, yeast sporulation was arrested: a cross between a sixteen-chromosome strain and an eight-chromosome strain showed greatly reduced full tetrad formation and less than 1% sporulation, from which no viable spores could be recovered. However, homotypic crosses between pairs of strains with eight, four or two chromosomes produced excellent sporulation and spore viability. These results indicate that eight chromosome–chromosome fusion events suffice to isolate strains reproductively. Overall, budding yeast tolerates a reduction in chromosome number unexpectedly well, providing a striking example of the robustness of genomes to change. Yeast chromosomes have been fused to produce viable strains with only two chromosomes that are reproductively isolated from the sixteen-chromosome wild type, but otherwise show high fitness in mitosis and meiosis.

Pancreatic ductal adenocarcinoma (PDAC) is typically diagnosed after the disease has metastasized; it is among the most lethal forms of cancer. We recently described aberrant expression of an open reading frame 1 protein, ORF1p, encoded by long interspersed element-1 (LINE-1; L1) retrotransposon, in PDAC. To test whether LINE-1 expression leads to somatic insertions of this mobile DNA, we used a targeted method to sequence LINE-1 insertion sites in matched PDAC and normal samples. We found evidence of 465 somatic LINE-1 insertions in 20 PDAC genomes, which were absent from corresponding normal samples. In cases in which matched normal tissue, primary PDAC and metastatic disease sites were available, insertions were found in primary and metastatic tissues in differing proportions. Two adenocarcinomas secondarily involving the pancreas, but originating in the stomach and duodenum, acquired insertions with a similar discordance between primary and metastatic sites. Together, our findings show that LINE-1 contributes to the genetic evolution of PDAC and suggest that somatic insertions are acquired discontinuously in gastrointestinal neoplasms.

Although the design of the synthetic yeast genome Sc2.0 is highly conservative with respect to gene content, the deletion of several classes of repeated sequences and the introduction of thousands of designer changes may affect genome organization and potentially alter cellular functions. We report here the Hi-C-determined three-dimensional (3D) conformations of Sc2.0 chromosomes. The absence of repeats leads to a smoother contact pattern and more precisely tractable chromosome conformations, and the large-scale genomic organization is globally unaffected by the presence of synthetic chromosome(s). Two exceptions are synIII, which lacks the silent mating-type cassettes, and synXII, specifically when the ribosomal DNA is moved to another chromosome. We also exploit the contact maps to detect rearrangements induced in SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution) strains.

Interspersed repeat sequences comprise much of our DNA, although their functional effects are poorly understood. The most commonly occurring repeat is the Alu short interspersed element. New Alu insertions occur in human populations, and have been responsible for several instances of genetic disease. In this study, we sought to determine if there are instances of polymorphic Alu insertion variants that function in a common variant, common disease paradigm. We cataloged 809 polymorphic Alu elements mapping to 1,159 loci implicated in disease risk by genome-wide association study (GWAS) (P < 10-8). We found that Alu insertion variants occur disproportionately at GWAS loci (P = 0.013). Moreover, we identified 44 of these Alu elements in linkage disequilibrium (r2 > 0.7) with the trait-associated SNP. This figure represents a >20-fold increase in the number of polymorphic Alu elements associated with human phenotypes. This work provides a broader perspective on how structural variants in repetitive DNAs may contribute to human disease.

Compatibility between host cells and heterologous pathways is a challenge for constructing organisms with high productivity or gain of function. Designer yeast cells incorporating the Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) system provide a platform for generating genotype diversity. Here we construct a genetic AND gate to enable precise control of the SCRaMbLE method to generate synthetic haploid and diploid yeast with desired phenotypes. The yield of carotenoids is increased to 1.5-fold by SCRaMbLEing haploid strains and we determine that the deletion of YEL013W is responsible for the increase. Based on the SCRaMbLEing in diploid strains, we develop a strategy called Multiplex SCRaMbLE Iterative Cycling (MuSIC) to increase the production of carotenoids up to 38.8-fold through 5 iterative cycles of SCRaMbLE. This strategy is potentially a powerful tool for increasing the production of bio-based chemicals and for mining deep knowledge.

LINE-1/L1 retrotransposon sequences comprise 17% of the human genome. Among the many classes of mobile genetic elements, L1 is the only autonomous retrotransposon that still drives human genomic plasticity today. Through its co-evolution with the human genome, L1 has intertwined itself with host cell biology. However, a clear understanding of L1’s lifecycle and the processes involved in restricting its insertion and intragenomic spread remains elusive. Here we identify modes of L1 proteins’ entrance into the nucleus, a necessary step for L1 proliferation. Using functional, biochemical, and imaging approaches, we also show a clear cell cycle bias for L1 retrotransposition that peaks during the S phase. Our observations provide a basis for novel interpretations about the nature of nuclear and cytoplasmic L1 ribonucleoproteins (RNPs) and the potential role of DNA replication in L1 retrotransposition.

Sensor proteins that exploit principles of linkage-specific avidity reveal topology-related functions of polyubiquitin in diverse cell types and pathways. Polyubiquitin chain topology is thought to direct modified substrates to specific fates, but this function-topology relationship is poorly understood, as are the dynamics and subcellular locations of specific polyubiquitin signals. Experimental access to these questions has been limited because linkage-specific inhibitors and in vivo sensors have been unavailable. Here we present a general strategy to track linkage-specific polyubiquitin signals in yeast and mammalian cells, and to probe their functions. We designed several high-affinity Lys63 polyubiquitin–binding proteins and demonstrate their specificity in vitro and in cells. We apply these tools as competitive inhibitors to dissect the polyubiquitin-linkage dependence of NF-κB activation in several cell types, inferring the essential role of Lys63 polyubiquitin for signaling via the IL-1β and TNF-related weak inducer of apoptosis (TWEAK) but not TNF-α receptors. We anticipate live-cell imaging, proteomic and biochemical applications for these tools and extension of the design strategy to other polymeric ubiquitin-like protein modifications.