Recent studies of histone methylation have yielded fundamental new insights pertaining to the role of this modification in gene activation as well as in gene silencing. While a number of methylation sites are known to occur on histones, only limited information exists regarding the relevant enzymes that mediate these methylation events. We thus sought to identify native histone methyltransferase (HMT) activities from Saccharomyces cerevisiae. Here, we describe the biochemical purification and characterization of Set2, a novel HMT that is site-specific for lysine 36 (Lys36) of the H3 tail. Using an antiserum directed against Lys36 methylation in H3, we show that Set2, via its SET domain, is responsible for methylation at this site in vivo. Tethering of Set2 to a heterologous promoter reveals that Set2 represses transcription, and part of this repression is mediated through the HMT activity of the SET domain. These results suggest that Set2 and methylation at H3 Lys36 play a role in the repression of gene transcription.Eukaryotic DNA is complexed in cells by histone proteins to form the fundamental repeating unit of chromatin, the nucleosome. Stretches of nucleosomes are further folded upon themselves to create higher-order chromatin structures that are currently not well defined. Compaction of DNA in this manner imposes a severe impediment to proteins that require access to the DNA template. Clear examples of this impediment have been shown to exist for the machinery that drives DNA transcription (28,38,41). However, this same impediment faces all aspects of DNA metabolism, including replication, repair and recombination (18,40).Posttranslational modifications of histone amino termini are recognized to play a central role in the control of chromatin structure and function. A diverse array of covalent histone modifications have been documented that take place on the tail domains of histones which protrude away from the nucleosome (9, 39). We and others have proposed that these modifications form a histone code which directly regulates chromatin function either by altering the specific structure of the chromatin polymer itself and/or by recruiting proteins or protein complexes that uniquely recognize a single or combinatorial set of modifications on one or more histone tails (14,35,37). For example, recent evidence showing that the bromodomains of various histone acetyltransferases, including PCAF, GCN5 and TAF II 250, bind to acetylated lysines in the histone tails suggests that specific recruitment of the transcriptional apparatus to promoters is one likely mechanism to explain how histone modifications influence transcription (8,22). It appears that other histone modifications, including methylation, function in the same manner (see below).Histone methylation is a posttranslational modification that occurs on lysine and arginine residues in the H3 and H4 tail domains (reviewed in reference 42). In histone H3, lysines 4, 9, 27, and 36 are well-documented sites of methylation, while in histone H4, lysine methylati...

The Gcn5p histone acetyltransferase exhibits a limited substrate specificity in vitro. However, neither the specificity of this enzyme in vivo nor the importance of particular acetylated residues to transcription or cell growth are well defined. To probe these questions, we mutated specific lysines in the N-termini of histones H3 and H4 and examined the effects of these mutations in yeast strains with and without functional GCN5. We found that in vivo, GCN5 is required either directly or indirectly for the acetylation of several sites in H3 and H4 in addition to those recognized by the recombinant enzyme in vitro. Moreover, in the absence of GCN5, cells accumulate in G 2 /M indicating that Gcn5p functions are important for normal cell-cycle progression. Mutation of K14 in H3, which serves as the major target of recombinant Gcn5p acetylation in vitro, confers a strong, synthetic growth defect in gcn5 cells. Synergistic growth defects were also observed in gcn5 cells carrying mutations in lysine pairs (K8/K16 or K5/K12) in histone H4. Strikingly, simultaneous mutation of K14 in H3 and K8 and K16 in H4 to arginine, or deletion of either the H3 or the H4 N-terminal tail, results in the death of gcn5 cells. Mutation of these same three sites to glutamine is not lethal. Indeed, this combination of mutations largely bypasses the need for GCN5 for transcriptional activation by Gal4-VP16, supporting an important role for histone acetylation in Gcn5p-mediated regulation of transcription. Our data indicate that acetylation of particular lysines in histones H3 and H4 serves both unique and overlapping functions important for normal cell growth, and that a critical overall level of histone acetylation is essential for cell viability.

Dosage compensation in Drosophila occurs by an increase in transcription of genes on the X chromosome in males. This elevated expression requires the function of at least four loci, known collectively as the male-specific lethal (ms/) genes. The proteins encoded by two of these genes, maleless (m/e) and male-specific lethal-1 (ms/-/), are found associated with the X chromosome in males, suggesting that they act as positive regulators of dosage compensation. A specific acetylated isoform of histone H4, H4Acl6, is also detected predominantly on the male X chromosome. We have found that MLE and MSL-1 bind to the X chromosome in an identical pattern and that the pattern of H4Acl6 on the X is largely coincident with that of MLE/MSL-1. We fail to detect H4AcI6 on the X chromosome in homozygous msl males, correlating with the lack of dosage compensation in these mutants. Conversely, in Sxl mutants, we detect H4Acl6 on the female X chromosomes, coincident with an inappropriate increase in X chromosome transcription. These data suggest that synthesis or localization of H4Acl6 is controlled by the dosage compensation regulatory hierarchy. Dosage compensation may involve H4Acl6 function, potentially through interaction with the products of the msl genes.

Ssn6-Tup1 regulates multiple genes in yeast, providing a paradigm for corepressor functions. Tup1 interacts directly with histones H3 and H4, and mutation of these histones synergistically compromises Ssn6-Tup1-mediated repression. In vitro, Tup1 interacts preferentially with underacetylated isoforms of H3 and H4, suggesting that histone acetylation may modulate Tup1 functions in vivo. Here we report that histone hyperacetylation caused by combined mutations in genes encoding the histone deacetylases (HDACs) Rpd3, Hos1, and Hos2 abolishes Ssn6-Tup1 repression. Unlike HDAC mutations that do not affect repression, this combination of mutations causes concomitant hyperacetylation of both H3 and H4. Strikingly, two of these class I HDACs interact physically with Ssn6-Tup1. These findings suggest that Ssn6-Tup1 actively recruits deacetylase activities to deacetylate adjacent nucleosomes and promote Tup1-histone interactions.

MSL complexes bind hundreds of sites along the single male X chromosome to achieve dosage compensation in Drosophila. Previously, we proposed that approximately 35 "high-affinity" or "chromatin entry" sites (CES) might nucleate spreading of MSL complexes in cis to paint the X chromosome. This was based on analysis of the first characterized sites roX1 and roX2. roX transgenes attract MSL complex to autosomal locations where it can spread long distances into flanking chromatin. roX1 and roX2 also produce noncoding RNA components of the complex. Here we identify a third site from the 18D10 region of the X chromosome. Like roX genes, 18D binds full and partial MSL complexes in vivo and encompasses a male-specific DNase I hypersensitive site (DHS). Unlike roX genes, the 510 bp 18D site is apparently not transcribed and shows high affinity for MSL complex and spreading only as a multimer. While mapping 18D, we discovered MSL binding to X cosmids that do not carry one of the approximately 35 high-affinity sites. Based on additional analyses of chromosomal transpositions, we conclude that spreading in cis from the roX genes or the approximately 35 originally proposed "entry sites" cannot be the sole mechanism for MSL targeting to the X chromosome.