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.     

Methylation of H3 K4 and K79 is not strictly dependent on H2B K123 ubiquitylation

Covalent modifications of histone proteins have profound consequences on chromatin structure and function. Specific modification patterns constitute a code read by effector proteins. Studies from yeast found that H3 trimethylation at K4 and K79 is dependent on ubiquitylation of H2B K123, which is termed a “trans-tail pathway.” In this study, we show that a strain unable to be ubiquitylated on H2B (K123R) is still proficient for H3 trimethylation at both K4 and K79, indicating that H3 methylation status is not solely dependent on H2B ubiquitylation. However, additional mutations in H2B result in loss of H3 methylation when combined with htb1-K123R. Consistent with this, we find that the original strain used to identify the trans-tail pathway has a genomic mutation that, when combined with H2B K123R, results in defective H3 methylation. Finally, we show that strains lacking the ubiquitin ligase Bre1 are defective for H3 methylation, suggesting that there is an additional Bre1 substrate that in combination with H2B K123 facilitates H3 methylation.     

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.     

Distinctive core histone post-translational modification patterns in Arabidopsis thaliana

Post-translational modifications of histones play crucial roles in the genetic and epigenetic regulation of gene expression from chromatin. Studies in mammals and yeast have found conserved modifications at some residues of histones as well as non-conserved modifications at some other sites. Although plants have been excellent systems to study epigenetic regulation, and histone modifications are known to play critical roles, the histone modification sites and patterns in plants are poorly defined. In the present study we have used mass spectrometry in combination with high performance liquid chromatography (HPLC) separation and phospho-peptide enrichment to identify histone modification sites in the reference plant, Arabidopsis thaliana. We found not only modifications at many sites that are conserved in mammalian and yeast cells, but also modifications at many sites that are unique to plants. These unique modifications include H4 K20 acetylation (in contrast to H4 K20 methylation in non-plant systems), H2B K6, K11, K27 and K32 acetylation, S15 phosphorylation and K143 ubiquitination, and H2A K144 acetylation and S129, S141 and S145 phosphorylation, and H2A.X S138 phosphorylation. In addition, we found that lysine 79 of H3 which is highly conserved and modified by methylation and plays important roles in telomeric silencing in non-plant systems, is not modified in Arabidopsis. These results suggest distinctive histone modification patterns in plants and provide an invaluable foundation for future studies on histone modifications in plants.     

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.     

Histone H2B Ubiquitylation Is Not Required for Histone H3 Methylation at Lysine 4 in Tetrahymena

Ubiquitylation of histone H2B and/or a component of the system that ubiquitylates H2B is required for methylation of histone H3 at lysine 4 (H3K4) in yeasts and probably in humans. In this study, the single ubiquitylation site was mapped to conserved lysine 115 of the C-terminal region of histone H2B in the single-cell model organism Tetrahymena thermophila. In strains lacking H2B ubiquitylation, H3K4 methylation was not detectably affected. As in other organisms, the E2 ubiquitin-conjugating enzyme Ubc2 and the E3 ubiquitin ligase Bre1 were required for H2B ubiquitylation. However, neither enzyme was required for H3K4 methylation. These studies argue that, in T. thermophila, the histone ubiquitylation mechanism is not required for H3K4 methylation, demonstrating that different organisms can speak different languages in the “cross-talk” among post-translational modifications on different histones.     

H3 lysine 9 methylation is maintained on a transcribed inverted repeat by combined action of SUVH6 and SUVH4 methyltransferases

Transcribed inverted repeats are potent triggers for RNA interference and RNA-directed DNA methylation in plants through the production of double-stranded RNA (dsRNA). For example, a transcribed inverted repeat of endogenous genes in Arabidopsis thaliana, PAI1-PAI4, guides methylation of itself as well as two unlinked duplicated PAI genes, PAI2 and PAI3. In previous work, we found that mutations in the SUVH4/KYP histone H3 lysine 9 (H3 K9) methyltransferase cause a loss of DNA methylation on PAI2 and PAI3, but not on the inverted repeat. Here we use chromatin immunoprecipitation analysis to show that the transcribed inverted repeat carries H3 K9 methylation, which is maintained even in an suvh4 mutant. PAI1-PAI4 H3 K9 methylation and DNA methylation are also maintained in an suvh6 mutant, which is defective for a gene closely related to SUVH4. However, both epigenetic modifications are reduced at this locus in an suvh4 suvh6 double mutant. In contrast, SUVH6 does not play a significant role in maintenance of H3 K9 or DNA methylation on PAI2, transposon sequences, or centromere repeat sequences. Thus, SUVH6 is preferentially active at a dsRNA source locus versus targets for RNA-directed chromatin modifications.     

Aging changes the chromatin configuration and histone methylation of mouse oocytes at germinal vesicle stage

Aging decreases the fertility of mammalian females. In old oocytes at metaphase II stage (MII) there are alterations of the chromatin configuration and chromatin modifications such as histone acetylation. Recent data indicate that alterations of histone acetylation at MII initially arise at germinal vesicle stage (GV). Therefore, we hypothesized that the chromatin configuration and histone methylation could also change in old GV oocytes. In agreement with our hypothesis, young GV oocytes had non-surrounded nucleolus (NSN) and surrounded nucleolus (SN) chromatin configurations, while old GV oocytes also had chromatin configurations that could not be classified as NSN or SN. Regarding histone methylation, young GV and MII oocytes showed dimethylation of lysines 4, 9, 36 and 79 in histone 3 (H3K4me2, H3K9me2, H3K36me2, H3K79me2), lysine 20 in histone H4 (H4K20me2) and trimethylation of lysine 9 in histone 3 (H3K9me3) while a significant percentage of old GV and MII oocytes lacked H3K9me3, H3K36me2, H3K79me2 and H4K20me2. The percentage of old oocytes lacking histone methylation was similar at GV and MII suggesting that alterations of histone methylation in old MII oocytes initially arise at GV. Besides, the expression of the histone methylation-related factors Cbx1 and Sirt1 was also found to change in old GV oocytes. In conclusion, our study reports changes of chromatin configuration and histone methylation in old GV oocytes, which could be very useful for further understanding of human infertility caused by aging.     

Lineage-specific transition of histone signatures in the killer cell Ig-like receptor locus from hematopoietic progenitor to NK cells

The clonal distribution and stable expression of killer cell Ig-like receptor (KIR) genes is epigenetically regulated. To assess the epigenetic changes that occur during hemopoietic development we examined DNA methylation and chromatin structure of the KIR locus in early hemopoietic progenitor cells and major lymphocyte lineages. In hemopoietic progenitor cells, KIR genes exhibited the major hallmarks of epigenetic repression, which are dense DNA methylation, inaccessibility of chromatin to Micrococcus nuclease digest, and a repressive histone signature, characterized by strong H3K9 dimethylation and reduced H4K8 acetylation. In contrast, KIR genes of NK cells showed active histone signatures characterized by absence of H3K9 dimethylation and presence of H4K8 acetylation. Histone modifications correlated well with the competence of different lymphocyte lineages to express KIR; whereas H4K8 acetylation was high in NK and CD8+ T cells, it was almost absent in CD4+ T cells and B cells and, in the latter case, replaced by H3K9 dimethylation. In KIR-competent lineages, active histone signatures were also observed in silent KIR genes and in this case found in combination with dense DNA methylation of the promoter and nearby regions. The study suggests a two-step model of epigenetic regulation in which lineage-specific acquisition of euchromatic histone marks is a prerequisite for subsequent gene-specific DNA demethylation and expression of KIR genes.