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.     

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.     

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.     

Trimethylation of histone H3 lysine 4 impairs methylation of histone H3 lysine 9

Chromatin is broadly compartmentalized in two defined states: euchromatin and heterochromatin. Generally, euchromatin is trimethylated on histone H3 lysine 4 (H3K4^me3) while heterochromatin contains the H3K9^me3 marks. The H3K9^me3 modification is added by lysine methyltransferases (KMTs) such as SETDB1. Herein, we show that SETDB1 interacts with its substrate H3, but only in the absence of the euchromatic mark H3K4^me3. In addition, we show that SETDB1 fails to methylate substrates containing the H3K4^me3 mark. Likewise, the functionally related H3K9 KMTs G9A, GLP, and SUV39H1 also fail to bind and to methylate H3K4^me3 substrates. Accordingly, we provide in vivo evidence that H3K9^me2-enriched histones are devoid of H3K4^me2/3 and that histones depleted of H3K4^me2/3 have elevated H3K9^me2/3. The correlation between the loss of interaction of these KMTs with H3K4^me3 and concomitant methylation impairment leads to the postulate that, at least these four KMTs, require stable interaction with their respective substrates for optimal activity. Thus, novel substrates could be discovered via the identification of KMT interacting proteins. Indeed, we find that SETDB1 binds to and methylates a novel substrate, the inhibitor of growth protein ING2, while SUV39H1 binds to and methylates the heterochromatin protein HP1α. Thus, our observations suggest a mechanism of post-translational regulation of lysine methylation and propose a potential mechanism for the segregation of the biologically opposing marks, H3K4^me3 and H3K9^me3. Furthermore, the correlation between H3-KMTs interaction and substrate methylation highlights that the identification of novel KMT substrates may be facilitated by the identification of interaction partners.     

Cross-talk between histone modifications in response to histone deacetylase inhibitors: MLL4 links histone H3 acetylation and histone H3K4 methylation

Histones are subject to a wide variety of post-translational modifications that play a central role in gene activation and silencing. We have used histone modification-specific antibodies to demonstrate that two histone modifications involved in gene activation, histone H3 acetylation and H3 lysine 4 methylation, are functionally linked. This interaction, in which the extent of histone H3 acetylation determines both the abundance and the "degree" of H3K4 methylation, plays a major role in the epigenetic response to histone deacetylase inhibitors. A combination of in vivo knockdown experiments and in vitro methyltransferase assays shows that the abundance of H3K4 methylation is regulated by the activities of two opposing enzyme activities, the methyltransferase MLL4, which is stimulated by acetylated substrates, and a novel and as yet unidentified H3K4me3 demethylase.     

DNA methylation, histone H3 methylation, and histone H4 acetylation in the genome of a crustacean.

In this work, we used antibodies against histone H3 trimethylated at lysine 9 (H3K9m3); against histone H4 acetylated at lysines 5, 8, 12, and 16 (H4ac); and against DNA methylated at 5C cytosine (m5C) to study the presence and distribution of these markers in the genome of the isopod crustacean Asellus aquaticus. The use of these 3 antibodies to immunolabel spermatogonial metaphases yields reproducible patterns on the chromosomes of this crustacean. The X and Y chromosomes present an identical banding pattern with each of the antibodies. The heterochromatic telomeric regions and the centromeric regions are rich in H3K9m3, but depleted in m5C and H4ac. Thus, m5C does not seem to be required to stabilize the silence of these regions in this organism.     

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.