Systematic genetic and proteomic screens during gametogenesis identify H2BK34 methylation as an evolutionary conserved meiotic mark.

BACKGROUND: Gametes are highly differentiated cells specialized to carry and protect the parental genetic information. During male germ cell maturation, histone proteins undergo distinct changes that result in a highly compacted chromatin organization. Technical difficulties exclude comprehensive analysis of precise histone mutations during mammalian spermatogenesis. The model organism Saccharomyces cerevisiae possesses a differentiation pathway termed sporulation which exhibits striking similarities to mammalian spermatogenesis. This study took advantage of this yeast pathway to first perform systematic mutational and proteomics screens on histones, revealing amino acid residues which are essential for the formation of spores. METHODS: A systematic mutational screen has been performed on the histones H2A and H2B, generating ~ 250 mutants using two genetic backgrounds and assessing their ability to form spores. In addition, histones were purified at key stages of sporulation and post-translational modifications analyzed by mass spectrometry. RESULTS: The mutation of 75 H2A H2B residues affected sporulation, many of which were localized to the nucleosome lateral surface. The use of different genetic backgrounds confirmed the importance of many of the residues, as 48% of yeast histone mutants exhibited impaired formation of spores in both genetic backgrounds. Extensive proteomic analysis identified 67 unique post-translational modifications during sporulation, 27 of which were previously unreported in yeast. Furthermore, 33 modifications are located on residues that were found to be essential for efficient sporulation in our genetic mutation screens. The quantitative analysis of these modifications revealed a massive deacetylation of all core histones during the pre-meiotic phase and a close interplay between H4 acetylation and methylation during yeast sporulation. Methylation of H2BK37 was also identified as a new histone marker of meiosis and the mouse paralog, H2BK34, was also enriched for methylation during meiosis in the testes, establishing conservation during mammalian spermatogenesis. CONCLUSION: Our results demonstrate that a combination of genetic and proteomic approaches applied to yeast sporulation can reveal new aspects of chromatin signaling pathways during mammalian spermatogenesis.

MLL1 is essential for the senescence-associated secretory phenotype.

Oncogene-induced senescence (OIS) and therapy-induced senescence (TIS), while tumor-suppressive, also promote procarcinogenic effects by activating the DNA damage response (DDR), which in turn induces inflammation. This inflammatory response prominently includes an array of cytokines known as the senescence-associated secretory phenotype (SASP). Previous observations link the transcription-associated methyltransferase and oncoprotein MLL1 to the DDR, leading us to investigate the role of MLL1 in SASP expression. Our findings reveal direct MLL1 epigenetic control over proproliferative cell cycle genes: MLL1 inhibition represses expression of proproliferative cell cycle regulators required for DNA replication and DDR activation, thus disabling SASP expression. Strikingly, however, these effects of MLL1 inhibition on SASP gene expression do not impair OIS and, furthermore, abolish the ability of the SASP to enhance cancer cell proliferation. More broadly, MLL1 inhibition also reduces "SASP-like" inflammatory gene expression from cancer cells in vitro and in vivo independently of senescence. Taken together, these data demonstrate that MLL1 inhibition may be a powerful and effective strategy for inducing cancerous growth arrest through the direct epigenetic regulation of proliferation-promoting genes and the avoidance of deleterious OIS- or TIS-related tumor secretomes, which can promote both drug resistance and tumor progression.

H4K44 Acetylation Facilitates Chromatin Accessibility during Meiosis.

Meiotic recombination hotspots are associated with histone post-translational modifications and open chromatin. However, it remains unclear how histone modifications and chromatin structure regulate meiotic recombination. Here, we identify acetylation of histone H4 at Lys44 (H4K44ac) occurring on the nucleosomal lateral surface. We show that H4K44 is acetylated at pre-meiosis and meiosis and displays genome-wide enrichment at recombination hotspots in meiosis. Acetylation at H4K44 is required for normal meiotic recombination, normal levels of double-strand breaks (DSBs) during meiosis, and optimal sporulation. Non-modifiable H4K44R results in increased nucleosomal occupancy around DSB hotspots. Our results indicate that H4K44ac functions to facilitate chromatin accessibility favorable for normal DSB formation and meiotic recombination.

H3K36 methylation promotes longevity by enhancing transcriptional fidelity.

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.

Inactivation of yeast Isw2 chromatin remodeling enzyme mimics longevity effect of calorie restriction via induction of genotoxic stress response.

ATP-dependent chromatin remodeling is involved in all DNA transactions and is linked to numerous human diseases. We explored functions of chromatin remodelers during cellular aging. Deletion of ISW2, or mutations inactivating the Isw2 enzyme complex, extends yeast replicative lifespan. This extension by ISW2 deletion is epistatic to the longevity effect of calorie restriction (CR), and this mechanism is distinct from suppression of TOR signaling by CR. Transcriptome analysis indicates that isw2Δ partially mimics an upregulated stress response in CR cells. In particular, isw2Δ cells show an increased response to genotoxic stresses, and the DNA repair enzyme Rad51 is important for isw2Δ-mediated longevity. We show that lifespan is also extended in C. elegans by reducing levels of athp-2, a putative ortholog of Itc1/ACF1, a critical subunit of the enzyme complex. Our findings demonstrate that the ISWI class of ATP-dependent chromatin remodeling complexes plays a conserved role during aging and in CR.

Systematic screen reveals new functional dynamics of histones H3 and H4 during gametogenesis.

Profound epigenetic differences exist between genomes derived from male and female gametes; however, the nature of these changes remains largely unknown. We undertook a systematic investigation of chromatin reorganization during gametogenesis, using the model eukaryote Saccharomyces cerevisiae to examine sporulation, which has strong similarities with higher eukaryotic spermatogenesis. We established a mutational screen of histones H3 and H4 to uncover substitutions that reduce sporulation efficiency. We discovered two patches of residues-one on H3 and a second on H4-that are crucial for sporulation but not critical for mitotic growth, and likely comprise interactive nucleosomal surfaces. Furthermore, we identified novel histone post-translational modifications that mark the chromatin reorganization process during sporulation. First, phosphorylation of H3T11 appears to be a key modification during meiosis, and requires the meiotic-specific kinase Mek1. Second, H4 undergoes amino tail acetylation at Lys 5, Lys 8, and Lys 12, and these are synergistically important for post-meiotic chromatin compaction, occurring subsequent to the post-meiotic transient peak in phosphorylation at H4S1, and crucial for recruitment of Bdf1, a bromodomain protein, to chromatin in mature spores. Strikingly, the presence and temporal succession of the new H3 and H4 modifications are detected during mouse spermatogenesis, indicating that they are conserved through evolution. Thus, our results show that investigation of gametogenesis in yeast provides novel insights into chromatin dynamics, which are potentially relevant to epigenetic modulation of the mammalian process.

Genome-wide mapping of histone H4 serine-1 phosphorylation during sporulation in Saccharomyces cerevisiae.

We previously showed that histone H4 serine-1 phosphorylation (H4S1ph) is evolutionarily conserved during gametogenesis, and contributes to post-meiotic nuclear compaction and to full completion of sporulation in the yeast Saccharomyces cerevisiae. Previous studies showed that H4S1ph and another modification of the same histone, H4 acetylation (H4ac), do not occur together and have opposing roles during DNA double-strand break (DSB) repair. In this study, we investigated the relationship between these marks during yeast sporulation. H4S1ph and H4ac co-exist globally during later stages of sporulation, in contrast to DSB repair. Genome-wide mapping during sporulation reveals accumulation of both marks over promoters of genes. Prevention of H4S1ph deposition delays the decline in transcription that normally occurs during spore maturation. Taken together, our results indicate that H4S1ph deposition reinforces reduced transcription that coincides with full spore compaction, without disrupting the local acetylation signature. These studies indicate distinctive features of a histone H4 modification marking system during sporulation compared with DSB repair.

G9a and Glp methylate lysine 373 in the tumor suppressor p53.

The tumor suppressor p53 is regulated by numerous post-translational modifications. Lysine methylation has recently emerged as a key post-translational modification that alters the activity of p53. Here, we describe a novel lysine methylation site in p53 that is carried out by two homologous histone methyltransferases, G9a and Glp. G9a and Glp specifically methylate p53 at Lys(373), resulting mainly in dimethylation. During DNA damage, the overall level of p53 modified at Lys(373)me2 does not increase, despite the dramatic increase in total p53, indicating that Lys(373)me2 correlates with inactive p53. Further, reduction of G9a and/or Glp levels leads to a larger population of apoptotic cells. Examination of the Oncomine data base shows that G9a and Glp are overexpressed in various cancers compared with corresponding normal tissues, suggesting that they are putative oncogenes. These data reveal a new methylation site within p53 mediated by the methylases G9a and Glp and indicate that G9a is a potential inhibitory target for cancer treatment.

Histone H4 lysine 16 acetylation regulates cellular lifespan.

Cells undergoing developmental processes are characterized by persistent non-genetic alterations in chromatin, termed epigenetic changes, represented by distinct patterns of DNA methylation and histone post-translational modifications. Sirtuins, a group of conserved NAD(+)-dependent deacetylases or ADP-ribosyltransferases, promote longevity in diverse organisms; however, their molecular mechanisms in ageing regulation remain poorly understood. Yeast Sir2, the first member of the family to be found, establishes and maintains chromatin silencing by removing histone H4 lysine 16 acetylation and bringing in other silencing proteins. Here we report an age-associated decrease in Sir2 protein abundance accompanied by an increase in H4 lysine 16 acetylation and loss of histones at specific subtelomeric regions in replicatively old yeast cells, which results in compromised transcriptional silencing at these loci. Antagonizing activities of Sir2 and Sas2, a histone acetyltransferase, regulate the replicative lifespan through histone H4 lysine 16 at subtelomeric regions. This pathway, distinct from existing ageing models for yeast, may represent an evolutionarily conserved function of sirtuins in regulation of replicative ageing by maintenance of intact telomeric chromatin.

The histone variant H3.3 regulates gene expression during lytic infection with herpes simplex virus type 1.

It has been proposed that incorporation of the histone variant H3.3 within actively transcribed regions of a genome helps to facilitate transcription. In this report we use lytic infection by herpes simplex virus type 1 (HSV-1) as a model to examine the temporal profile of histone H3 incorporation and to determine whether the variant histone H3.3 has a direct effect on transcription. We find that canonical H3.1 and variant H3.3 exhibit distinct temporal associations with the genome in cell lines expressing equal amounts of epitope-tagged H3 variants. At the earliest times examined after infection, the HSV-1 genome is incorporated into chromatin that predominantly contains the variant H3.3, whereas incorporation of canonical H3.1 occurs later in infection and is dependent on replication of the HSV-1 genome. Further, inhibition of H3.3 association, via reduced expression of the H3.3 chaperone HIRA, significantly reduces the levels of HSV-1 mRNA. These findings show that incorporation of H3.3 facilitates transcription, and they provide new evidence for a regulatory role of chromatin composition during HSV-1 acute infection.

p53 is regulated by the lysine demethylase LSD1.

p53, the tumour suppressor and transcriptional activator, is regulated by numerous post-translational modifications, including lysine methylation. Histone lysine methylation has recently been shown to be reversible; however, it is not known whether non-histone proteins are substrates for demethylation. Here we show that, in human cells, the histone lysine-specific demethylase LSD1 (refs 3, 4) interacts with p53 to repress p53-mediated transcriptional activation and to inhibit the role of p53 in promoting apoptosis. We find that, in vitro, LSD1 removes both monomethylation (K370me1) and dimethylation (K370me2) at K370, a previously identified Smyd2-dependent monomethylation site. However, in vivo, LSD1 shows a strong preference to reverse K370me2, which is performed by a distinct, but unknown, methyltransferase. Our results indicate that K370me2 has a different role in regulating p53 from that of K370me1: K370me1 represses p53 function, whereas K370me2 promotes association with the coactivator 53BP1 (p53-binding protein 1) through tandem Tudor domains in 53BP1. Further, LSD1 represses p53 function through the inhibition of interaction of p53 with 53BP1. These observations show that p53 is dynamically regulated by lysine methylation and demethylation and that the methylation status at a single lysine residue confers distinct regulatory output. Lysine methylation therefore provides similar regulatory complexity for non-histone proteins and for histones.

Repression of p53 activity by Smyd2-mediated methylation.

Specific sites of lysine methylation on histones correlate with either activation or repression of transcription. The tumour suppressor p53 (refs 4-7) is one of only a few non-histone proteins known to be regulated by lysine methylation. Here we report a lysine methyltransferase, Smyd2, that methylates a previously unidentified site, Lys 370, in p53. This methylation site, in contrast to the known site Lys 372, is repressing to p53-mediated transcriptional regulation. Smyd2 helps to maintain low concentrations of promoter-associated p53. We show that reducing Smyd2 concentrations by short interfering RNA enhances p53-mediated apoptosis. We find that Set9-mediated methylation of Lys 372 inhibits Smyd2-mediated methylation of Lys 370, providing regulatory cross-talk between post-translational modifications. In addition, we show that the inhibitory effect of Lys 372 methylation on Lys 370 methylation is caused, in part, by blocking the interaction between p53 and Smyd2. Thus, similar to histones, p53 is subject to both activating and repressing lysine methylation. Our results also predict that Smyd2 may function as a putative oncogene by methylating p53 and repressing its tumour suppressive function.

Phosphorylation of histone H4 Ser1 regulates sporulation in yeast and is conserved in fly and mouse spermatogenesis.

Sporulation in Saccharomyces cerevisiae is a highly regulated process wherein a diploid cell gives rise to four haploid gametes. In this study we show that histone H4 Ser1 is phosphorylated (H4 S1ph) during sporulation, starting from mid-sporulation and persisting to germination, and is temporally distinct from earlier meiosis-linked H3 S10ph involved in chromosome condensation. A histone H4 S1A substitution mutant forms aberrant spores and has reduced sporulation efficiency. Deletion of sporulation-specific yeast Sps1, a member of the Ste20 family of kinases, nearly abolishes the sporulation-associated H4 S1ph modification. H4 S1ph may promote chromatin compaction, since deletion of SPS1 increases accessibility to antibody immunoprecipitation; furthermore, either deletion of Sps1 or an H4 S1A substitution results in increased DNA volume in nuclei within spores. We find H4 S1ph present during Drosophila melanogaster and mouse spermatogenesis, and similar to yeast, this modification extends late into sperm differentiation relative to H3 S10ph. Thus, H4 S1ph may be an evolutionarily ancient histone modification to mark the genome for gamete-associated packaging.

Histone sumoylation is a negative regulator in Saccharomyces cerevisiae and shows dynamic interplay with positive-acting histone modifications.

Covalent histone post-translational modifications such as acetylation, methylation, phosphorylation, and ubiquitylation play pivotal roles in regulating many cellular processes, including transcription, response to DNA damage, and epigenetic control. Although positive-acting post-translational modifications have been studied in Saccharomyces cerevisiae, histone modifications that are associated with transcriptional repression have not been shown to occur in this yeast. Here, we provide evidence that histone sumoylation negatively regulates transcription in S. cerevisiae. We show that all four core histones are sumoylated and identify specific sites of sumoylation in histones H2A, H2B, and H4. We demonstrate that histone sumoylation sites are involved directly in transcriptional repression. Further, while histone sumoylation occurs at all loci tested throughout the genome, slightly higher levels occur proximal to telomeres. We observe a dynamic interplay between histone sumoylation and either acetylation or ubiquitylation, where sumoylation serves as a potential block to these activating modifications. These results indicate that sumoylation is the first negative histone modification to be identified in S. cerevisiae and further suggest that sumoylation may serve as a general dynamic mark to oppose transcription.

Phosphorylation of histone H4 serine 1 during DNA damage requires casein kinase II in S. cerevisiae.

Distinct patterns of posttranslational histone modifications can regulate DNA-templated events such as mitosis, transcription, replication, apoptosis, and DNA damage, suggesting the presence of a "histone code" in these nuclear processes. Phosphorylation of histone H2A S129 at sites of DNA double-strand breaks (DSBs) has been implicated in damage repair in yeast. Here, we describe another phosphorylation event on serine 1 (S1) of histone H4; this event is also associated with MMS- or phleomycin-induced DSBs but not with UV-induced DNA damage. Chromatin-immunoprecipitation (ChIP) studies of an HO-endonuclease-inducible strain show that S1 phosphorylation is specifically enhanced 20- to 25-fold in nucleosomes proximal to the DSB. In addition, we show that casein kinase II (CK2) can phosphorylate H4 S1 in vitro and that null or temperature-sensitive CK2 yeast mutants are defective for induction of H4 S1 phosphorylation upon DNA damage in vivo. Furthermore, H4 S1 phosphorylation and CK2 play a role in DSB re-joining as indicated by a nonhomologous end-joining (NHEJ) plasmid assay. CK2 has been implicated in regulating a DNA-damage response; our data suggest that histone H4 S1 is one of its physiological substrates. These data suggest that this modification is a part of the DNA-repair histone code.