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.     

Ubiquitination of histone H2B by Rad6 is required for efficient Dot1-mediated methylation of histone H3 lysine 79

Dot1 is a non-SET domain protein that methylates histone H3 at lysine 79, a surface-exposed residue that lies within the globular domain. In the context of a nucleosome, H3 lysine 79 is located in close proximity with lysine 123 of histone H2B, a major site for ubiquitination by Rad6. Here we show that Rad6-mediated ubiquitination of H2B lysine 123 is important for efficient methylation of lysine 79, but not lysine 36, of histone H3. In contrast, lysine 79 methylation of H3 is not required for ubiquitination of H2B. Our study provides a new example of trans-histone regulation between modifications on different histones. In addition, it suggests that Rad6 affects telomeric silencing, at least in part, by influencing methylation of histone H3.     

Coupling of glucose deprivation with impaired histone H2B monoubiquitination in tumors

Metabolic reprogramming is associated with tumorigenesis. However, glucose metabolism in tumors is poorly understood. Here, we report that glucose levels are significantly lower in bulk tumor specimens than those in normal tissues of the same tissue origins. We show that mono-ubiquitinated histone H2B (uH2B) is a semi-quantitative histone marker for glucose. We further show that loss of uH2B occurs specifically in cancer cells from a wide array of tumor specimens of breast, colon, lung and additional 23 anatomic sites. In contrast, uH2B levels remain high in stromal tissues or non-cancerous cells in the tumor specimens. Taken together, our data suggest that glucose deficiency and loss of uH2B are novel properties of cancer cells in vivo, which may represent important regulatory mechanisms of tumorigenesis.     

The structure of ubiquitinated histone H2B.

Ubiquitinated histone H2B (uH2B) has been purified from both calf and pig thymus by exclusion chromatography in 7 M urea. Digestion of uH2B with Staphylococcus aureus V8 protease yielded the peptide 114-125 containing the ubiquitin moiety. Further digestion of this peptide with trypsin removed the ubiquitin and three H2B residues from the N-terminus. Edman degradations of both peptides established that ubiquitin is attached to the epsilon-amino group of lysine 120 in both calf and pig uH2B by an iso-peptide bond to the C-terminal glycine 76 of ubiquitin.     

Structure of polyubiquitinated histone H2A

We have recently demonstrated that trout liver histones H2A, H2B, and H2A.Z can be polyubiquitinated [Davie, J.R., Delcuve, G.P., Nickel, B.E., Moyer, R., & Bailey, G. (1987) Cancer Res. 47, 5407-5410]. In the present study we determined the arrangement of the ubiquitin molecules in polyubiquitinated histone H2A. Trout liver chromatin fragments. which had histone H1 removed, were digested with Staphylococcus aureus (V8 strain) protease which cleaves specifically on the carboxyl side of glutamic acid residues under the conditions used. The V8 protease readily degraded histone H2A and ubiquitinated (u) H2A at equivalent rates. One site in H2A and uH2A, the peptide bond between Glu 121 and Lys 122, was cleaved, yielding protein species cH2A and cuH2A, respectively. None of the other nucleosomal histones (H2B, H2A.Z, H3, and H4) including uH2B and uH2A.Z were sensitive to digestion. Trout liver histones cleaved with either V8 protease, histone H2A specific protease, or cyanogen bromide were resolved by two-dimensional gel electrophoresis and ubiquitinated peptides detected with anti-ubiquitin IgG. The results suggest that the major arrangement of ubiquitin in polyubiquitinated H2A is a chain of ubiquitin molecules joined to each other by isopeptide bonds to a ubiquitin molecule that is attached to the epsilon-amino group of lysine 119 of histone H2A.     

The C-terminus of histone H2B is involved in chromatin compaction specifically at telomeres, independently of its monoubiquitylation at lysine 123

Telomeric heterochromatin assembly in budding yeast propagates through the association of Silent Information Regulator (SIR) proteins with nucleosomes, and the nucleosome array has been assumed to fold into a compacted structure. It is believed that the level of compaction and gene repression within heterochromatic regions can be modulated by histone modifications, such as acetylation of H3 lysine 56 and H4 lysine 16, and monoubiquitylation of H2B lysine 123. However, it remains unclear as to whether or not gene silencing is a direct consequence of the compaction of chromatin. Here, by investigating the role of the carboxy-terminus of histone H2B in heterochromatin formation, we identify that the disorderly compaction of chromatin induced by a mutation at H2B T122 specifically hinders telomeric heterochromatin formation. H2B T122 is positioned within the highly conserved AVTKY motif of the αC helix of H2B. Heterochromatin containing the T122E substitution in H2B remains inaccessible to ectopic dam methylase with dramatically increased mobility in sucrose gradients, indicating a compacted chromatin structure. Genetic studies indicate that this unique phenotype is independent of H2B K123 ubiquitylation and Sir4. In addition, using ChIP analysis, we demonstrate that telomere structure in the mutant is further disrupted by a defect in Sir2/Sir3 binding and the resulting invasion of euchromatic histone marks. Thus, we have revealed that the compaction of chromatin per se is not sufficient for heterochromatin formation. Instead, these results suggest that an appropriately arrayed chromatin mediated by H2B C-terminus is required for SIR binding and the subsequent formation of telomeric chromatin in yeast, thereby identifying an intrinsic property of the nucleosome that is required for the establishment of telomeric heterochromatin. This requirement is also likely to exist in higher eukaryotes, as the AVTKY motif of H2B is evolutionarily conserved.     

Polycomb group proteins Ring1A/B link ubiquitylation of histone H2A to heritable gene silencing and X inactivation

In many higher organisms, 5%-15% of histone H2A is ubiquitylated at lysine 119 (uH2A). The function of this modification and the factors involved in its establishment, however, are unknown. Here we demonstrate that uH2A occurs on the inactive X chromosome in female mammals and that this correlates with recruitment of Polycomb group (PcG) proteins belonging to Polycomb repressor complex 1 (PRC1). Based on our observations, we tested the role of the PRC1 protein Ring1B and its closely related homolog Ring1A in H2A ubiquitylation. Analysis of Ring1B null embryonic stem (ES) cells revealed extensive depletion of global uH2A levels. On the inactive X chromosome, uH2A was maintained in Ring1A or Ring1B null cells, but not in double knockout cells, demonstrating an overlapping function for these proteins in development. These observations link H2A ubiquitylation, X inactivation, and PRC1 PcG function, suggesting an unanticipated and novel mechanism for chromatin-mediated heritable gene silencing.     

组蛋白H2B单泛素化参与DNA损伤修复的调控机制

作为一种重要的组蛋白修饰形式,H2B的单泛素化(uH2B)广泛地参与DNA复制、基因的表达与转录、DNA损伤修复及异染色质维持等生物学事件.在裂殖酵母中,H2B的单泛素化发生在其羧基端的119位赖氨酸(K119),并依赖于Rhp6/Bre1泛素连接酶复合体.研究表明,uH2B通过破坏H2A/H2B二聚体的结构促进mRNA在转录过程中的延伸,同时促进H3K4的三甲基化激活基因的表达及参与DNA损伤修复.本研究发现,Rhp6能够对核糖核苷酸还原酶抑制基因(Spd1)位点进行活跃的染色质修饰,促进H2B的单泛素化并抑制基因表达,从而促进dNTP的合成并调控DNA复制及损伤修复.重要的是,本研究发现,该过程不依赖于H3K4而决定于H3K9的三甲基化.同时uH2B直接在DNA双链断裂位点富集,通过改变染色质的结构参与DNA损伤修复,该过程中可能存在其他更为复杂的分子机制.     

A haploid affair: core histone transitions during spermatogenesis.

The process of meiosis reduces a diploid cell to four haploid gametes and is accompanied by extensive recombination. Thus, the dynamics of chromatin during meiosis are significantly different than in mitotic cells. As spermatogenesis progresses, there is a widespread reorganization of the haploid genome followed by extensive DNA compaction. It has become increasingly clear that the dynamic composition of chromatin plays a critical role in the activities of enzymes and processes that act upon it. Therefore, an analysis of the role of histone variants and modifications in these processes may shed light upon the mechanisms involved and the control of chromatin structure in general. Histone variants such as histone H3.3, H2AX, and macroH2A appear to play key roles in the various stages of spermiogenesis, in addition to the specifically modulated acetylation of histone H4 (acH4), ubiquitination of histones H2A and H2B (uH2A, uH2B), and phosphorylation of histone H3 (H3p). This review will examine recent discoveries concerning the role of histone modifications and variants during meiosis and spermatogenesis.     

Histone H2B deacetylation at lysine 11 is required for yeast apoptosis induced by phosphorylation of H2B at serine 10

Chromatin alterations, induced by covalent histone modifications, mediate a wide range of DNA-templated processes, including apoptosis. Apoptotic chromatin condensation has been causally linked to the phosphorylation of histone H2B (serine 14 in human; serine 10 in yeast, H2BS10ph) in human and yeast cells. Here, we extend these studies by demonstrating a unidirectional, crosstalk pathway between H2BS10 phosphorylation and lysine 11 acetylation (H2BK11ac) in yeast. We demonstrate that the H2BK11 acetyl mark, which exists in growing yeast, is removed upon H(2)O(2) treatment but before H2BS10ph occurs, in a unidirectional fashion. H2B K11Q mutants are resistant to cell death elicited by H(2)O(2), while H2B K11R mutants that mimic deacetylation promote cell death. Our results suggest that Hos3 HDAC deacetylates H2BK11ac, which in turn mediates H2BS10ph by Ste20 kinase. Together, these studies underscore a concerted series of enzyme reactions governing histone modifications that promote a switch from cell proliferation to cell death.     

Decoding the trans-histone crosstalk: methods to analyze H2B ubiquitination, H3 methylation and their regulatory factors

Regulation of histone H3 lysine 4 and 79 methylation by histone H2B lysine 123 monoubiquitination is an evolutionarily conserved trans-histone crosstalk mechanism, which demonstrates a functional role for histone ubiquitination within the cell. The regulatory enzymes, factors and processes involved in the establishment and dynamic modulation of these modifications and their genome-wide distribution patterns have been determined in many model systems. Rapid progress in understanding this trans-histone crosstalk has been made using the standard experimental tools of chromatin biology in budding yeast (Saccharomyces cerevisiae), a highly tractable model organism. Here, we provide a set of modified and refined experimental procedures that can be used to gain further insights into the underlying mechanisms that govern this crosstalk in budding yeast. Importantly, the improved procedures and their underlying principles can also be applied to other model organisms. Methods presented here provide a rapid and efficient means to prepare enriched protein extracts to better preserve and assess the steady state levels of histones, non-histone proteins and their modifications. Improved chromatin immunoprecipitation and double immunoprecipitation protocols are provided to measure the occupancy and distribution of proteins and their modified forms at specific chromatin regions or loci. A quick and easy method to measure overall protein abundance and changes in protein-protein and protein-DNA interactions on native chromatin is also described.