Identification of Histone H3 Lysine 36 Acetylation as a Highly Conserved Histone Modification

The Rockefeller University, New York, New York, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 04/2007; 282(10):7632-40. DOI: 10.1074/jbc.M607909200
Source: PubMed


Histone lysine acetylation is a major mechanism by which cells regulate the structure and function of chromatin, and new sites of acetylation continue to be discovered. Here we identify and characterize histone H3K36 acetylation (H3K36ac). By mass spectrometric analyses of H3 purified from Tetrahymena thermophila and Saccharomyces cerevisiae (yeast), we find that H3K36 can be acetylated or methylated. Using an antibody specific to H3K36ac, we show that this modification is conserved in mammals. In yeast, genome-wide ChIP-chip experiments show that H3K36ac is localized predominantly to the promoters of RNA polymerase II-transcribed genes, a pattern inversely related to that of H3K36 methylation. The pattern of H3K36ac localization is similar to that of other sites of H3 acetylation, including H3K9ac and H3K14ac. Using histone acetyltransferase complexes purified from yeast, we show that the Gcn5-containing SAGA complex that regulates transcription specifically acetylates H3K36 in vitro. Deletion of GCN5 completely abolishes H3K36ac in vivo. These data expand our knowledge of the genomic targets of Gcn5, show H3K36ac is highly conserved, and raise the intriguing possibility that the transition between H3K36ac and H3K36me acts as an "acetyl/methyl switch" governing chromatin function along transcription units.

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    • "We hypothesized that while H3K14ac may be critical for transcription, secondary acetylation sites may also play a critical role in biology by improving the binding of bromodomains or competing with methylation (e.g. H3K9) which is critical for gene silencing [35]. This hypothesis is supported by the fact that deletions of gcn5 in vivo result in a loss of acetylation in residues acetylated after K14 [17], . "
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    ABSTRACT: Lysine acetyltransferases (KATs) play a unique role in regulating gene transcription as well as maintaining the epigenetic state of the cell. KATs such as Gcn5 and p300/CBP can modify multiple residues on a single histone; however, order and specificity of acetylation can be altered by factors such as histone chaperones, subunit proteins or external stimulus. While the importance of acetylation is well documented, it has been difficult to quantitatively measure the specificity and selectivity of acetylation at different residues within a histone. In this paper, we demonstrate a label-free quantitative high throughput mass spectrometry-based assay capable of quantitatively monitoring all known acetylation sites of H3 simultaneously. Using this assay, we are able to analyze the steady-state enzyme kinetics of Gcn5, an evolutionarily conserved KAT. In doing so, we measured Gcn5-mediated acetylation at six residues (K14>K9 ≈ K23> K18> K27 ≈ K36) and the catalytic efficiency (k(cat)/K(m)) for K9, K14, K18, and K23 as well as the nonenzymatic acetylation rate. We observed selectivity differences of up to -4 kcal/mol between K14 and K18, the highest and lowest measurable k(cat)/K(m). These data provide a first look at quantitating the specificity and selectivity of multiple lysines on a single substrate (H3) by Gcn5.
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    • "Toward this end, we performed chromatin immunoprecipitation coupled with high-resolution tiling arrays (ChIP-chip) and global gene expression studies to identify and characterize BES1 direct target genes in the Arabidopsis genome. ChIP-chip has been used to generate high-resolution maps of genome-wide distributions of histone modifications (Morris et al., 2007; Opel et al., 2007; Zhang et al., 2007; Minsky et al., 2008) and transcription factor binding sites in several species. The latter can be exemplified by studies on transcription factors such as Twist, Biniou and Ladybird in Drosophila (Jakobsen et al., 2007; Sandmann et al., 2007), estrogen receptor and homeobox C6 in human cancer cells (Carroll et al., 2006; McCabe et al., 2008), as well as HY5, AGL15, PIL5 and FLP in Arabidopsis (Lee et al., 2007; Oh et al., 2009a; Zheng et al., 2009; Xie et al., 2010). "
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    • "Recent studies demonstrated that H3K36 can be modified by Set2-mediated methylation and Gcn5-dependent acetylation (59). Acetylation at H3K36 is localized predominantly at the promoters of RNA polymerase II-transcribed genes and functions as a prelude to transcriptional initiation (59,60). The set2Δ eliminated only H3K36 methylation, and H3K36A wiped out both methylation and acetylation at the PHR1 promoter. "
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