Dot1-dependent histone H3K79 methylation promotes the formation of meiotic double-strand breaks in the absence of histone H3K4 methylation in budding yeast.
ABSTRACT: Epigenetic marks such as histone modifications play roles in various chromosome dynamics in mitosis and meiosis. Methylation of histones H3 at positions K4 and K79 is involved in the initiation of recombination and the recombination checkpoint, respectively, during meiosis in the budding yeast. Set1 promotes H3K4 methylation while Dot1 promotes H3K79 methylation. In this study, we carried out detailed analyses of meiosis in mutants of the SET1 and DOT1 genes as well as methylation-defective mutants of histone H3. We confirmed the role of Set1-dependent H3K4 methylation in the formation of double-strand breaks (DSBs) in meiosis for the initiation of meiotic recombination, and we showed the involvement of Dot1 (H3K79 methylation) in DSB formation in the absence of Set1-dependent H3K4 methylation. In addition, we showed that the histone H3K4 methylation-defective mutants are defective in SC elongation, although they seem to have moderate reduction of DSBs. This suggests that high levels of DSBs mediated by histone H3K4 methylation promote SC elongation.
Project description:Histone modification is a critical determinant of the frequency and location of meiotic double-strand breaks (DSBs), and thus recombination. Set1-dependent histone H3K4 methylation and Dot1-dependent H3K79 methylation play important roles in this process in budding yeast. Given that the RNA polymerase II associated factor 1 complex, Paf1C, promotes both types of methylation, we addressed the role of the Paf1C component, Rtf1, in the regulation of meiotic DSB formation. Similar to a set1 mutation, disruption of RTF1 decreased the occurrence of DSBs in the genome. However, the rtf1 set1 double mutant exhibited a larger reduction in the levels of DSBs than either of the single mutants, indicating independent contributions of Rtf1 and Set1 to DSB formation. Importantly, the distribution of DSBs along chromosomes in the rtf1 mutant changed in a manner that was different from the distributions observed in both set1 and set1 dot1 mutants, including enhanced DSB formation at some DSB-cold regions that are occupied by nucleosomes in wild-type cells. These observations suggest that Rtf1, and by extension the Paf1C, modulate the genomic DSB landscape independently of H3K4 methylation.
Project description:Regulation of Set1-COMPASS-mediated H3K4 methylation and Dot1-mediated H3K79 methylation by H2BK123 ubiquitination (H2Bub1) is an evolutionarily conserved trans-histone crosstalk mechanism. How H2Bub1 impacts chromatin structure and affects Set1-COMPASS/Dot1 functions has not been fully defined. Ubiquitin was proposed to bind proteins to physically bridge H2Bub1 with Set1-COMPASS/Dot1. Alternatively, the bulky ubiquitin was thought to be a "wedge" that loosens the nucleosome for factor access. Contrary to the latter possibility, recent discoveries provide evidence for nucleosome stabilization by H2Bub1 via preventing the constant H2A-H2B eviction. Recent data has also uncovered a "docking-site" on H2B for Set1-COMPASS. Collectively, these findings invoke a model, where ubiquitin acts as a "glue" to bind the nucleosome together for supporting Set1-COMPASS/Dot1 functions. This review provides an overview of these novel findings. Additionally, how H2Bub1 and its deubiquitination might alter the chromatin dynamics during transcription is discussed. Possible models for nucleosome stabilization by ubiquitin are also provided.
Project description:During meiosis, accurate chromosome segregation relies on the proper interaction between homologous chromosomes, including synapsis and recombination. The meiotic recombination checkpoint is a quality control mechanism that monitors those crucial events. In response to defects in synapsis and/or recombination, this checkpoint blocks or delays progression of meiosis, preventing the formation of aberrant gametes. Meiotic recombination occurs in the context of chromatin and histone modifications, which play crucial roles in the maintenance of genomic integrity. Here, we unveil the role of Dot1-dependent histone H3 methylation at lysine 79 (H3K79me) in this meiotic surveillance mechanism. We demonstrate that the meiotic checkpoint function of Dot1 relies on H3K79me because, like the dot1 deletion, H3-K79A or H3-K79R mutations suppress the checkpoint-imposed meiotic delay of a synapsis-defective zip1 mutant. Moreover, by genetically manipulating Dot1 catalytic activity, we find that the status of H3K79me modulates the meiotic checkpoint response. We also define the phosphorylation events involving activation of the meiotic checkpoint effector Mek1 kinase. Dot1 is required for Mek1 autophosphorylation, but not for its Mec1/Tel1-dependent phosphorylation. Dot1-dependent H3K79me also promotes Hop1 activation and its proper distribution along zip1 meiotic chromosomes, at least in part, by regulating Pch2 localization. Furthermore, HOP1 overexpression bypasses the Dot1 requirement for checkpoint activation. We propose that chromatin remodeling resulting from unrepaired meiotic DSBs and/or faulty interhomolog interactions allows Dot1-mediated H3K79-me to exclude Pch2 from the chromosomes, thus driving localization of Hop1 along chromosome axes and enabling Mek1 full activation to trigger downstream responses, such as meiotic arrest.
Project description:Genomic features of DSB re-landscaping in rtf1 mutants. Histone modification is a critical determinant of frequency and location of double-strand breaks (DSBs), which induce recombination during meiosis. The Set1-dependent histone H3K4 and Dot1-dependent H3K79 methylations play an important role in DSB formations in budding yeast. Both methylations are promoted by the RNA polymerase II associated factor 1 (Paf1) complex, Paf1C. This study addressed a role of the Paf1C component Rtf1, which is critical for H3K4 and H3K79 methylations, for the regulation of meiotic DSB formation. Similar to set1 mutation, rtf1 mutation decreased the occurrence of DSBs in the genome. The rtf1 set1 double mutant exhibited a larger reduction in the levels of DSBs than the frequency of DSBs detected in either of the single mutants; this indicates independent roles of Rtf1 and Set1 in DSB formation. Importantly, the distribution of DSBs along chromosomes in the rtf1 mutant changed in a different manner than the pattern observed in the set1 and set1 dot1 mutants; this was characterized by enhanced DSB formation at some DSB-cold regions. These observations suggest that Rtf1, and, possibly, the Paf1C, determine DSB landscape in the genome, independent of H3K4 methylation.
Project description:The trans-histone regulatory cross talk between H2BK123 ubiquitination (H2Bub1) and H3K4 and H3K79 methylation is not fully understood. In this study, we report that the residues arginine 119 and threonine 122 in the H2B C-terminal helix are important for transcription and cell growth and play a direct role in controlling H2Bub1 and H3K4 methylation. These residues modulate H2Bub1 levels by controlling the chromatin binding and activities of the deubiquitinases. Furthermore, we find an uncoupling of the H2Bub1-mediated coregulation of both H3K4 and -K79 methylation, as these H2B C-terminal helix residues are part of a distinct surface that affects only Set1-COMPASS (complex proteins associated with Set1)-mediated H3K4 methylation without affecting the functions of Dot1. Importantly, we also find that these residues interact with Spp1 and control the chromatin association, integrity, and overall stability of Set1-COMPASS independent of H2Bub1. Therefore, we have uncovered a novel role for the H2B C-terminal helix in the trans-histone cross talk as a binding surface for Set1-COMPASS. We provide further insight into the trans-histone cross talk and propose that H2Bub1 stabilizes the nucleosome by preventing H2A-H2B eviction and, thereby, retains the "docking site" for Set1-COMPASS on chromatin to maintain its stable chromatin association, complex stability, and processive methylation.
Project description:Monoubiquitination of histone H2B lysine 123 regulates methylation of histone H3 lysine 4 (H3K4) and 79 (H3K79) and the lack of H2B ubiquitination in Saccharomyces cerevisiae coincides with metacaspase-dependent apoptosis. Here, we discovered that loss of H3K4 methylation due to depletion of the methyltransferase Set1p (or the two COMPASS subunits Spp1p and Bre2p, respectively) leads to enhanced cell death during chronological aging and increased sensitivity to apoptosis induction. In contrast, loss of H3K79 methylation due to DOT1 disruption only slightly affects yeast survival. SET1 depleted cells accumulate DNA damage and co-disruption of Dot1p, the DNA damage adaptor protein Rad9p, the endonuclease Nuc1p, and the metacaspase Yca1p, respectively, impedes their early death. Furthermore, aged and dying wild-type cells lose H3K4 methylation, whereas depletion of the H3K4 demethylase Jhd2p improves survival, indicating that loss of H3K4 methylation is an important trigger for cell death in S. cerevisiae. Given the evolutionary conservation of H3K4 methylation this likely plays a role in apoptosis regulation in a wide range of organisms.
Project description:Homologous recombination is essential for chromosome segregation during meiosis I. Meiotic recombination is initiated by the introduction of double-strand breaks (DSBs) at specific genomic locations called hotspots, which are catalyzed by Spo11 and its partners. DSB hotspots during meiosis are marked with Set1-mediated histone H3K4 methylation. The Spo11 partner complex, Rec114-Mer2-Mei4, essential for the DSB formation, localizes to the chromosome axes. For efficient DSB formation, a hotspot with histone H3K4 methylation on the chromatin loops is tethered to the chromosome axis through the H3K4 methylation reader protein, Spp1, on the axes, which interacts with Mer2. In this study, we found genetic interaction of mutants in a histone modification protein complex called PAF1C with the REC114 and MER2 in the DSB formation in budding yeast Saccharomyces cerevisiae. Namely, the paf1c mutations rtf1 and cdc73 showed synthetic defects in meiotic DSB formation only when combined with a wild-type-like tagged allele of either the REC114 or MER2. The synthetic defect of the tagged REC114 allele in the DSB formation was seen also with the set1, but not with spp1 deletion. These results suggest a novel role of histone modification machinery in DSB formation during meiosis, which is independent of Spp1-mediated loop-axis tethering.
Project description:Histone H3K4 methylation is a feature of meiotic recombination hotspots shared by many organisms including plants and mammals. Meiotic recombination is initiated by programmed double-strand break (DSB) formation that in budding yeast takes place in gene promoters and is promoted by histone H3K4 di/trimethylation. This histone modification is recognized by Spp1, a PHD finger containing protein that belongs to the conserved histone H3K4 methyltransferase Set1 complex. During meiosis, Spp1 binds H3K4me3 and interacts with a DSB protein, Mer2, to promote DSB formation close to gene promoters. How Set1 complex- and Mer2- related functions of Spp1 are connected is not clear. Here, combining genome-wide localization analyses, biochemical approaches and the use of separation of function mutants, we show that Spp1 is present within two distinct complexes in meiotic cells, the Set1 and the Mer2 complexes. Disrupting the Spp1-Set1 interaction mildly decreases H3K4me3 levels and does not affect meiotic recombination initiation. Conversely, the Spp1-Mer2 interaction is required for normal meiotic recombination initiation, but dispensable for Set1 complex-mediated histone H3K4 methylation. Finally, we provide evidence that Spp1 preserves normal H3K4me3 levels independently of the Set1 complex. We propose a model where Spp1 works in three ways to promote recombination initiation: first by depositing histone H3K4 methylation (Set1 complex), next by "reading" and protecting histone H3K4 methylation, and finally by making the link with the chromosome axis (Mer2-Spp1 complex). This work deciphers the precise roles of Spp1 in meiotic recombination and opens perspectives to study its functions in other organisms where H3K4me3 is also present at recombination hotspots.
Project description:Monoubiquitination of histone H2B on Lys 123 (H2BK123ub) is a transient histone modification considered to be essential for establishing H3K4 and H3K79 trimethylation by Set1/COMPASS and Dot1, respectively. Here, we identified Chd1 as a factor that is required for the maintenance of high levels of H2B monoubiquitination, but not for H3K4 and H3K79 trimethylation. Loss of Chd1 results in a substantial loss of H2BK123ub levels with little to no effect on the genome-wide pattern of H3K4 and H3K79 trimethylation. Our data show that nucleosomal occupancy is reduced in gene bodies in both chd1? and, as has been shown, K123A mutant backgrounds. We also demonstrated that Chd1's function in maintaining H2BK123ub levels is conserved from yeast to humans. Our study provides evidence that only small levels of H2BK123ub are necessary for full levels of H3K4 and H3K79 trimethylation in vivo and points to a possible role for Chd1 in positively regulating gene expression through promoting nucleosome reassembly coupled with H2B monoubiquitination.
Project description:Exogenous double-strand breaks (DSBs) induce a DNA damage response during mitosis as well as meiosis. The DNA damage response is mediated by a cascade involving Mec1/Tel1 (ATR/ATM) and Rad53 (Chk2) kinases. Meiotic cells are programmed to form DSBs for the initiation of meiotic recombination. In budding yeast, Spo11-mediated meiotic DSBs activate Mec1/Tel1, but not Rad53; however, the mechanism underlying the insensitivity of Rad53 to meiotic DSBs remains largely unknown. In this study, we found that meiotic cells activate Rad53 in response to exogenous DSBs and that this activation is dependent on an epigenetic marker, Dot1-dependent histone H3K79 methylation, which becomes a scaffold of an Rad53 mediator, Rad9, an ortholog of 53BP1. In contrast, Rad9 is insensitive to meiotic programmed DSBs. This insensitiveness of Rad9 derives from its inability to bind to the DSBs. Indeed, artificial tethering of Rad9 to the meiotic DSBs activated Rad53. The artificial activation of Rad53 kinase in meiosis decreases the repair of meiotic DSBs. These results suggest that the suppression of Rad53 activation is a key event in initiating a meiotic program that repairs programmed DSBs.