Methylation of Histone H3 Mediates the Association of the NuA3 Histone Acetyltransferase with Chromatin

Abstract

The SAS3-dependent NuA3 histone acetyltransferase complex was originally identified on the basis of its ability to acetylate histone H3 in vitro. Whether NuA3 is capable of acetylating histones in vivo, or how the complex is targeted to the nucleosomes that it modifies, was unknown. To address this question, we asked whether NuA3 is associated with chromatin in vivo and how this association is regulated. With a chromatin pulldown assay, we found that NuA3 interacts with the histone H3 amino-terminal tail, and loss of the H3 tail recapitulates phenotypes associated with loss of SAS3. Moreover, mutation of histone H3 lysine 14, the preferred site of acetylation by NuA3 in vitro, phenocopies a unique sas3⌬ phenotype, suggesting that modification of this residue is important for NuA3 function. The interaction of NuA3 with chromatin is dependent on the Set1p and Set2p histone methyltransferases, as well as their substrates, histone H3 lysines 4 and 36, respectively. These results confirm that NuA3 is functioning as a histone acetyltransferase in vivo and that histone H3 methylation provides a mark for the recruitment of NuA3 to nucleosomes.In eukaryotes, DNA is packaged into chromatin, a nucleoprotein structure consisting of DNA, histones, and nonhistone proteins. The assembly of DNA into chromatin modulates the access of cellular machinery to DNA and thus regulates transcription, replication, repair, and recombination. Chromatin structure can be regulated by the addition of posttranslational modifications to histones, including acetylation, methylation, phosphorylation, and ubiquitination. Consistent with this, several histone posttranslational modifications have been linked to the regulation of gene expression.Numerous multiprotein complexes are involved in the posttranslational modification of histones. These complexes vary in both their protein components and the modifications they effect. The most well-studied group of modifying complexes is the histone acetyltransferases (HATs), which use acetyl coenzyme A as a substrate for the acetylation of lysine residues within both the tail and globular domains of histones (39,43,45,73). In the budding yeast Saccharomyces cerevisiae, there are at least eight proteins that have been identified as having HAT activity in vitro, including Gcn5p, Hat1p, Esa1p, Elp3p, Nut1p, Hpa2p, Sas2p, and Sas3p (45,73), although for many of these proteins, whether histones represent their true substrates in vivo is not known. Three of these proteins are found in complexes that specifically acetylate the lysines within the tail of histone H3, including the GCN5-dependent SAGA, SLIK/ SALSA, ADA, and HAT-A2 complexes (12,16,49,58,60), the ELP3-dependent elongator complex (70), and the SAS3-dependent NuA3 complex (25). The acetylation of histone tails is associated with regions of transcriptional activity, and histone acetylation is thought to modulate chromatin structure through two different mechanisms. First, the neutralization of charge associated with histone acetylation is believed to re...

MOZ and MORF, two large MYSTic HATs in normal and cancer stem cells

“…One PHD finger of NURF recognizes trimethyl lysine 4 of histone H3 to couple ATP-dependent chromatin remodeling to histone methylation Wysocka et al, 2006), and the PHD finger of ING2 binds to the same mark to stimulate methylation-dependent deacetylation (Pena et al, 2006;Shi et al, 2006). Moreover, methylation of histone H3 at lysine 4 promotes association with yeast Yng1, a subunit of the NuA3 acetyltransferase complex (Martin et al, 2006;Taverna et al, 2006). Like ING2, ING5 binds to trimethyl lysine 4 of histone H3 through its PHD domain , raising the interesting possibility that ING5 facilitates methylation-dependent histone acetylation by MOZ or MORF.…”

The Yng1p Plant Homeodomain Finger Is a Methyl-Histone Binding Module That Recognizes Lysine 4-Methylated Histone H3

“…1B). Deletion of SAS3 did not rescue the Yng1p toxicity, indicating that this phenotype is due to an overabundance of Yng1p We have previously shown that the interaction of NuA3 with chromatin is dependent on the amino-terminal tail of histone H3 (40). If the Yng1p toxicity is due to the interaction of overabundant Yng1p with the H3 tail, then deletion of this tail should rescue the toxicity.…”

“…We attempted to address this issue using chromatin immunoprecipitation analysis, but to date, we have been unable to reproducibly chromatin immunoprecipitate any component of NuA3 to a specific genomic locus. However, using a chromatin pull-down assay, we were able to show that Sas3p, the catalytic subunit of NuA3, interacts with chromatin in vivo, and that this interaction is dependent on the amino-terminal tail of histone H3 (40). To determine whether this interaction is also dependent on Yng1p, we fused a TAP tag to the carboxyl terminus of H2B (Htb1p) to enable us to purify native chromatin and associated proteins using a modified TAP protocol.…”

“…Additionally, other protein complexes which lack these domains have been shown to bind specifically to methylated histones, but the subunits which mediate these interactions are not yet known. These include the Isw1p chromatin remodeling complex and the NuA3 histone acetyltransferase complex (40,55). Thus, it is highly likely that there are unidentified motifs which can mediate the interaction of chromatin modifying factors with posttranslationally modified histones.…”

Systematic Characterization and Prediction of Post-Translational Modification Cross-Talk *

“…phosphate, acetyl, or methyl) to specific amino acid residues (1), which play important roles in regulating gene expression, modifying protein functions, and modulating protein-protein interactions (2)(3)(4)(5)(6). With the development of mass spectrometry (MS) techniques, increasing number of PTM sites have been identified (7,8).…”