In this report, we identify and characterize a novel site of acetylation on histone H3 at lysine 36. This site was previously determined to be mono-, di-, and trimethylated in a broad range of eukaryotic organisms, and we find that acetylation at this residue is also highly conserved. Furthermore, we have determined that H3K36ac is mediated by the Gcn5-containing SAGA complex in yeast, and is preferentially enriched in the promoter regions of RNAPII-transcribed genes genome-wide. Although the exact function of this modification remains to be elucidated, our data suggests that it is involved in the transcription process.
We and others have found several clues as to how Gcn5 can target H3K36 to acetylate this site. First, previous studies have shown that Gcn5 in isolation can only target H3K14 (43
). However, in its native SAGA complex, this enzyme has an expanded substrate range on H3 that includes H3K9, K18 and K23 (43
). H3K36ac was not detected in this previous study as only H3 synthetic peptides containing amino acid residues from 5 to 28 were investigated. Our data, therefore, reveal an expanded site utilization pattern by SAGA. Second, the amino acid sequence immediately surrounding H3K36 is very similar to that of H3K14, which is a preferred site of Gcn5 acetylation (compare STGGK14
; underlined sequences show identity while bold lysines are the acetyl accepting residues). Structural studies of Gcn5 in complex with an H3 peptide containing H3K14 have identified several critical residues immediately surrounding H3K14 (glycine 13 and proline 16) that are important for substrate recognition and high affinity binding (56
). Importantly, these key residues (G-K14
-X-P) are conserved in the sequence surrounding H3K36. Thus, the high similarity between residues surrounding H3K14 and H3K36 may explain how Gcn5 is capable of acetylating H3K36 in vitro
and in vivo
. Given H3K9/14 acetylation patterns overlap with H3K36ac, it is likely that SAGA mediates a broad H3 acetylation pattern when recruited to gene promoters.
Interestingly, while we found that H3K36ac was present in Tetrahymena
, yeast, and mammalian cells, the general abundance of this modification varied greatly among the organisms we analyzed (). While many possible reasons could account for these differences, one plausible explanation may be due to the fact that budding yeast contains only a single H3 isoform (H3.3), whereas Tetrahymena
and mammalian cells contain multiple H3 variants (H3.1, H3.2 and H3.3; see ). Since H3.1 and H3.2 in flies and mammals are generally associated with silenced chromatin (which make up a large proportion of their genomes), these histone variants are not likely to be H3K36 acetylated and/or associated with active transcription. Thus, much of the bulk-isolated histones from these more complex organisms contain H3 isoforms that harbor “OFF” marks. This idea, along with our H3K36ac observations, is consistent with earlier studies that show that these same multicellular organisms have much lower levels of H3K4 methylation (a modification associated with transcriptionally competent chromatin) compared to those observed in yeast (59
). Given the sequence surrounding H3K36 is highly conserved between these organisms (), the differences observed between species is not likely due to the inability of our antibody to efficiently recognize the site of H3K36.
That H3K36ac is mediated by Gcn5 and is found in the promoters of RNAPII-regulated genes suggests that acetylation at H3K36 may play a role in gene transcription. Previous studies of SAGA and Gcn5 indicate that this enzyme complex is activator recruited and targets acetylation at specific promoters during transcriptional activation (14
). While the exact function of H3K36ac is unknown, we speculate that H3K36ac acts in concert with other Gcn5-mediated sites of acetylation to properly regulate transcriptional induction. Such cooperativity would be in agreement with prior studies that have shown the importance of Gcn5-mediated acetylation of multiple sites on H3 for normal cell growth and transcriptional activation (63
). Additionally, the acetylation of H3K36 by SAGA, as part of this complex’s expanded targeting, is consistent with studies on H4 that show a cumulative effect of acetylation is associated with the promoters of active genes (64
In contrast to H3K36ac, methylation at H3K36 is found in the coding region of genes and is involved in transcriptional elongation. Recent reports have identified a function for this modification in maintaining an environment within coding regions that is repressive to the activation of intergenic transcription by recruiting the deacetylase complex Rpd3(S) (65
). An important question, therefore, is whether acetylation and methylation activities compete for the same target sites, such as H3K36. Well documented is the finding that H3K9 is subject to either methylation or acetylation (see below), and a report co-submitted with this one reveals that a significant number of lysines targeted for acetylation are also targeted for methylation, and vise versa (54
). These results raise the intriguing possibility that functional interplay between two or more posttranslational modifications at a single lysine residue is a general phenomenon that drives distinct biological effects within chromatin. To date, the best example of functional interplay between methylation and acetylation is with H3K9, at which acetylation is removed prior to methylation by Su(var)3–9
in the promoters of genes (68
). This activity is required for the recruitment of HP1/Swi6 that leads to the formation of heterochromatin in both fission yeast and multicellular eukaryotes. Although our analyses do not reveal if functional interplay occurs at this site, future investigations will aim to determine whether SAGA and Set2 regulate transcription initiation events through competition for H3K36.