The DNA damage checkpoint response requires histone H2B ubiquitination by Rad6-Bre1 and H3 methylation by Dot1

The cellular response to DNA lesions entails the recruitment of several checkpoint and repair factors to damaged DNA, and chromatin modifications may play a role in this process. Here we show that in Saccharomyces cerevisiae epigenetic modification of histones is required for checkpoint activity in response to a variety of genotoxic stresses. We demonstrate that ubiquitination of histone H2B on lysine 123 by the Rad6-Bre1 complex, is necessary for activation of Rad53 kinase and cell cycle arrest. We found a similar requirement for Dot1-dependent methylation of histone H3. Loss of H3-Lys(79) methylation does not affect Mec1 activation, whereas it renders cells checkpoint-defective by preventing phosphorylation of Rad9. Such results suggest that histone modifications may have a role in checkpoint function by modulating the interactions of Rad9 with chromatin and active Mec1 kinase.     

Cellular aging is associated with increased ubiquitylation of histone H2B in yeast telomeric heterochromatin

Epigenetic changes in chromatin state are associated with aging. Notably, two histone modifications have recently been implicated in lifespan regulation, namely acetylation at H4 lysine 16 in yeast and methylation at H3 lysine 4 (H3K4) in nematodes. However, less is known about other histone modifications. Here, we report that cellular aging is associated with increased ubiquitylation of histone H2B in yeast telomeric heterochromatin. An increase in ubiquitylation at histone H2B lysine 123 and methylations at both H3K4 and H3 lysine 79 (H3K79) was observed at the telomere-proximal regions of replicatively aged cells, coincident with decreased Sir2 abundance. Moreover, deficiencies in the H2B ubiquitylase complex Rad6/Bre1 as well as the deubiquitylase Ubp10 reduced the lifespan by altering both H3K4 and H3K79 methylation and Sir2 recruitment. Thus, these results show that low levels of H2B ubiquitylation are a prerequisite for a normal lifespan and the trans-tail regulation of histone modifications regulates age-associated Sir2 recruitment through telomeric silencing.     

DOT1—— 一类新的组蛋白赖氨酸甲基转移酶

组蛋白甲基化是一种重要的组蛋白共价修饰, 在染色质结构和基因表达的调控过程中起着重要的、多样化的作用。DOT1催化核心球体部位的组蛋白H3第79位赖氨酸(H3K79)使其发生甲基化, 是首个被发现的无SET结构域的组蛋白赖氨酸甲基转移酶, 代表了一类新的组蛋白赖氨酸甲基转移酶。DOT1及H3K79甲基化的特点决定了其可能具有重要的、特殊的生物学功能。文章重点综述了DOT1蛋白的结构及特点, DOT1及H3K79甲基化的生物学功能以及组蛋白泛素化修饰对H3K79甲基化的反式调控。     

The RAD6/BRE1 histone modification pathway in Saccharomyces confers radiation resistance through a RAD51-dependent process that is independent of RAD18

We examine ionizing radiation (IR) sensitivity and epistasis relationships of several Saccharomyces mutants affecting post-translational modifications of histones H2B and H3. Mutants bre1Delta, lge1Delta, and rtf1Delta, defective in histone H2B lysine 123 ubiquitination, show IR sensitivity equivalent to that of the dot1Delta mutant that we reported on earlier, consistent with published findings that Dot1p requires H2B K123 ubiquitination to fully methylate histone H3 K79. This implicates progressive K79 methylation rather than mono-methylation in IR resistance. The set2Delta mutant, defective in H3 K36 methylation, shows mild IR sensitivity whereas mutants that abolish H3 K4 methylation resemble wild type. The dot1Delta, bre1Delta, and lge1Delta mutants show epistasis for IR sensitivity. The paf1Delta mutant, also reportedly defective in H2B K123 ubiquitination, confers no sensitivity. The rad6Delta, rad51null, rad50Delta, and rad9Delta mutations are epistatic to bre1Delta and dot1Delta, but rad18Delta and rad5Delta show additivity with bre1Delta, dot1Delta, and each other. The bre1Delta rad18Delta double mutant resembles rad6Delta in sensitivity; thus the role of Rad6p in ubiquitinating H2B accounts for its extra sensitivity compared to rad18Delta. We conclude that IR resistance conferred by BRE1 and DOT1 is mediated through homologous recombinational repair, not postreplication repair, and confirm findings of a G1 checkpoint role for the RAD6/BRE1/DOT1 pathway.     

Histone crosstalk between H2B monoubiquitination and H3 methylation mediated by COMPASS

COMPASS, the yeast homolog of the mammalian MLL complex, is a histone H3 lysine 4 (H3K4) methylase consisting of Set1 (KMT2) and seven other polypeptides, including Cps35, the only essential subunit. Histone H2B monoubiquitination by Rad6/Bre1 is required for both H3K4 methylation by COMPASS, and H3K79 methylation by Dot1. However, the molecular mechanism for such histone crosstalk is poorly understood. Here, we demonstrate that histone H2B monoubiquitination controls the binding of Cps35 with COMPASS complex. Cps 35 is required for COMPASS' catalytic activity in vivo, and the addition of exogenous purified Cps35 to COMPASS purified from a Deltarad6 background results in the generation of a methylation competent COMPASS. Cps35 associates with the chromatin of COMPASS-regulated genes in a H2BK123 monoubiquitination-dependent but Set1-independent manner. Cps35 is also required for proper H3K79 trimethylation. These findings offer insight into the molecular role of Cps35 in translating the H2B monoubiquitination signal into H3 methylation.     

Role of Dot1-dependent histone H3 methylation in G1 and S phase DNA damage checkpoint functions of Rad9

We screened radiation-sensitive yeast mutants for DNA damage checkpoint defects and identified Dot1, the conserved histone H3 Lys 79 methyltransferase. DOT1 deletion mutants (dot1Delta) are G1 and intra-S phase checkpoint defective after ionizing radiation but remain competent for G2/M arrest. Mutations that affect Dot1 function such as Rad6-Bre1/Paf1 pathway gene deletions or mutation of H2B Lys 123 or H3 Lys 79 share dot1Delta checkpoint defects. Whereas dot1Delta alone confers minimal DNA damage sensitivity, combining dot1Delta with histone methyltransferase mutations set1Delta and set2Delta markedly enhances lethality. Interestingly, set1Delta and set2Delta mutants remain G1 checkpoint competent, but set1Delta displays a mild S phase checkpoint defect. In human cells, H3 Lys 79 methylation by hDOT1L likely mediates recruitment of the signaling protein 53BP1 via its paired tudor domains to double-strand breaks (DSBs). Consistent with this paradigm, loss of Dot1 prevents activation of the yeast 53BP1 ortholog Rad9 or Chk2 homolog Rad53 and decreases binding of Rad9 to DSBs after DNA damage. Mutation of Rad9 to alter tudor domain binding to methylated Lys 79 phenocopies the dot1Delta checkpoint defect and blocks Rad53 phosphorylation. These results indicate a key role for chromatin and methylation of histone H3 Lys 79 in yeast DNA damage signaling.     

Methylation of histone H3 by COMPASS requires ubiquitination of histone H2B by Rad6

The DNA of eukaryotes is wrapped around nucleosomes and packaged into chromatin. Covalent modifications of the histone proteins that comprise the nucleosome alter chromatin structure and have major effects on gene expression. Methylation of lysine 4 of histone H3 by COMPASS is required for silencing of genes located near chromosome telomeres and within the rDNA (Krogan, N. J, Dover, J., Khorrami, S., Greenblatt, J. F., Schneider, J., Johnston, M., and Shilatifard, A. (2002) J. Biol. Chem. 277, 10753-10755; Briggs, S. D., Bryk, M., Strahl, B. D., Cheung, W. L., Davie, J. K., Dent, S. Y., Winston, F., and Allis, C. D. (2001) Genes. Dev. 15, 3286-3295). To learn about the mechanism of histone methylation, we surveyed the genome of the yeast Saccharomyces cerevisiae for genes necessary for this process. By analyzing approximately 4800 mutant strains, each deleted for a different non-essential gene, we discovered that the ubiquitin-conjugating enzyme Rad6 is required for methylation of lysine 4 of histone H3. Ubiquitination of histone H2B on lysine 123 is the signal for the methylation of histone H3, which leads to silencing of genes located near telomeres.     

Loss of Histone H3 Methylation at Lysine 4 Triggers Apoptosis in Saccharomyces cerevisiae

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.     

A conserved RING finger protein required for histone H2B monoubiquitination and cell size control

Monoubiquitination of histone H2B is required for methylation of histone H3 on lysine 4 (K4), a modification associated with active chromatin. The identity of the cognate ubiquitin ligase is unknown. We identify Bre1 as an evolutionarily conserved RING finger protein required in vivo for both H2B ubiquitination and H3 K4 methylation. The RING domain of Bre1 is essential for both of these modifications as is Lge1 (Large 1), a protein required for cell size control that copurifies with Bre1. In cells lacking the euchromatin-associated histone variant H2A.Z, BRE1, RAD6, and LGE1 are each essential for cell viability, supporting redundant functions for H2B ubiquitination and H2A substitution in the formation of active chromatin. Notably, analysis of mutants demonstrates a function for Bre1/Lge1-dependent H2B monoubiquitination in the control of cell size.