Characterization of lysine 56 of histone H3 as an acetylation site in Saccharomyces cerevisiae

Post-translational histone modifications abound and regulate multiple nuclear processes. Most modifications are targeted to the amino-terminal domains of histones. Here we report the identification and characterization of acetylation of lysine 56 within the core domain of histone H3. In the crystal structure of the nucleosome, lysine 56 contacts DNA. Phenotypic analysis suggests that lysine 56 is critical for histone function and that it modulates formamide resistance, ultraviolet radiation sensitivity, and sensitivity to hydroxyurea. We show that the acetylated form of histone H3 lysine 56 (H3-K56) is present during interphase, metaphase, and S phase. Finally, reverse genetic analysis indicates that none of the known histone acetyltransferases is solely responsible for H3-K56 acetylation in Saccharomyces cerevisiae.     

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.     

Whole-genome analysis of histone H3 lysine 4 and lysine 27 methylation in human embryonic stem cells

We mapped Polycomb-associated H3K27 trimethylation (H3K27me3) and Trithorax-associated H3K4 trimethylation (H3K4me3) across the whole genome in human embryonic stem (ES) cells. The vast majority of H3K27me3 colocalized on genes modified with H3K4me3. These commodified genes displayed low expression levels and were enriched in developmental function. Another significant set of genes lacked both modifications and was also expressed at low levels in ES cells but was enriched for gene function in physiological responses rather than development. Commodified genes could change expression levels rapidly during differentiation, but so could a substantial number of genes in other modification categories. SOX2, POU5F1, and NANOG, pluripotency-associated genes, shifted from modification by H3K4me3 alone to colocalization of both modifications as they were repressed during differentiation. Our results demonstrate that H3K27me3 modifications change during early differentiation, both relieving existing repressive domains and imparting new ones, and that colocalization with H3K4me3 is not restricted to pluripotent cells.     

Selective interactions between vertebrate polycomb homologs and the SUV39H1 histone lysine methyltransferase suggest that histone H3-K9 methylation contributes to chromosomal targeting of Polycomb group proteins

Polycomb group (PcG) proteins form multimeric chromatin-associated protein complexes that are involved in heritable repression of gene activity. Two distinct human PcG complexes have been characterized. The EED/EZH2 PcG complex utilizes histone deacetylation to repress gene activity. The HPC/HPH PcG complex contains the HPH, RING1, BMI1, and HPC proteins. Here we show that vertebrate Polycomb homologs HPC2 and XPc2, but not M33/MPc1, interact with the histone lysine methyltransferase (HMTase) SUV39H1 both in vitro and in vivo. We further find that overexpression of SUV39H1 induces selective nuclear relocalization of HPC/HPH PcG proteins but not of the EED/EZH2 PcG proteins. This SUV39H1-dependent relocalization concentrates the HPC/HPH PcG proteins to the large pericentromeric heterochromatin domains (1q12) on human chromosome 1. Within these PcG domains we observe increased H3-K9 methylation. Finally, we show that H3-K9 HMTase activity is associated with endogenous HPC2. Our findings suggest a role for the SUV39H1 HMTase and histone H3-K9 methylation in the targeting of human HPC/HPH PcG proteins to modified chromatin structures.     

Regulation of histone H3 lysine 56 acetylation in Schizosaccharomyces pombe

In Saccharomyces cerevisiae, acetylation of lysine 56 (Lys-56) in the globular domain of histone H3 plays an important role in response to genotoxic agents that interfere with DNA replication. However, the regulation and biological function of this modification are poorly defined in other eukaryotes. Here we show that Lys-56 acetylation in Schizosaccharomyces pombe occurs transiently during passage through S-phase and is normally removed in G(2). Genotoxic agents that cause DNA double strand breaks during replication elicit a delay in deacetylation of histone H3 Lys-56. In addition, mutant cells that cannot acetylate Lys-56 are acutely sensitive to genotoxic agents that block DNA replication. Moreover, we show that Spbc342.06cp, a previously uncharacterized open reading frame, encodes the functional homolog of S. cerevisiae Rtt109, and that this protein acetylates H3 Lys-56 both in vitro and in vivo. Altogether, our results indicate that both the regulation of histone H3 Lys-56 acetylation by its histone acetyltransferase and histone deacetylase and its role in the DNA damage response are conserved among two distantly related yeast model organisms.     

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.