Combined Action of Histone Reader Modules Regulates NuA4 Local Acetyltransferase Function but Not Its Recruitment on the Genome

Abstract

e Recognition of histone marks by reader modules is thought to be at the heart of epigenetic mechanisms. These protein domains are considered to function by targeting regulators to chromosomal loci carrying specific histone modifications. This is important for proper gene regulation as well as propagation of epigenetic information. The NuA4 acetyltransferase complex contains two of these reader modules, an H3K4me3-specific plant homeodomain (PHD) within the Yng2 subunit and an H3K36me2/3-specific chromodomain in the Eaf3 subunit. While each domain showed a close functional interaction with the respective histone mark that it recognizes, at the biochemical level, genetic level (as assessed with epistatic miniarray profile screens), and phenotypic level, cells with the combined loss of both readers showed greatly enhanced phenotypes. Chromatin immunoprecipitation coupled with next-generation sequencing experiments demonstrated that the Yng2 PHD specifically directs H4 acetylation near the transcription start site of highly expressed genes, while Eaf3 is important downstream on the body of the genes. Strikingly, the recruitment of the NuA4 complex to these loci was not significantly affected. Furthermore, RNA polymerase II occupancy was decreased only under conditions where both PHD and chromodomains were lost, generally in the second half of the gene coding regions. Altogether, these results argue that methylated histone reader modules in NuA4 are not responsible for its recruitment to the promoter or coding regions but, rather, are required to orient its acetyltransferase catalytic site to the methylated histone 3-bearing nucleosomes in the surrounding chromatin, cooperating to allow proper transition from transcription initiation to elongation. Chromatin is a dynamic nucleoprotein structure that compacts the long DNA molecule into the small nuclear space, simultaneously creating a mechanism to regulate DNA access to machineries implicated in essential nuclear processes, namely, transcription, DNA replication, DNA repair, and meiotic recombination. Modulation of the chromatin structure is highly regulated by four broad classes of nuclear factors: chromatin remodelers, histone chaperones, histone variants, and histone modifiers. These players essentially target nucleosomes, basic units of chromatin consisting of 146 bp of DNA wrapped around an octamer of histone proteins. Histone modifiers catalyze posttranslational modifications (PTM), including acetylation, phosphorylation, and methylation, that can directly affect DNA-histone contacts (e.g., acetylation) and/or create a binding platform for PTM readers (1, 2). These readers correspond to proteins/complexes containing distinct domains/modules that can bind specific modified histone residues. Such motifs include bromodomains that recognize acetylated residues; chromodomains (CHD); PWWP, Tudor, and MBT domains for methylated residues; BRCT, BIR, and 14-3-3 domains for phosphorylated histones; and plant homeodomain (PHD) fingers that can bind different modification...

The majority of histone acetylation is a consequence of transcription

“…We note however that this observation could not be generalized to other chromatin-modifying enzymes, as the occupancy of the H3K4 methyltransferase, Set1, positively correlated with H3K4me3 ( Figure 3B and S4B). Thus, specifically for HATs, we observe poor correspondence between HAT localization and histone acetylation, which is consistent with previous suggestions that regulation of HAT activity, following chromatin binding, is a major determinant of histone acetylation genome-wide 21,26,37,38 .…”

“…To confirm that histone acetylation loss is a direct consequence of transcription inhibition, we treated S. cerevisiae cells with 1,10-pt for 15 minutes and performed ChIPseq for H3K23ac, H4K8ac, and H4K12ac. Consistent with previous studies 25,26 , we used non-transcribed regions to account for global changes in ChIP-seq experiments (see methods). While the mechanism of action for transcription inhibition by 1,10-pt has not been extensively characterized, Figure 1B and our previous work 27 indicate inhibition of both transcription initiation and elongation.…”

“…Similar to other groups 25,26 , ChIP-seq datasets comparing across RNAPII perturbations were normalized to silent regions. The genome was divided into 250 bp bins, bins outside the interquartile range for coverage in the input were discarded, the 100 regions with the lowest Rpb3 signal were defined as silent regions, and these silent regions were used to normalized ChIP-seq datasets for cross-condition comparisons (Table S2).…”

Cryo-electron microscopy of chromatin biology

“…A semi-flexible Tudor domain within Esa1 was docked into density proximal to the catalytically engaged Esa HAT domain, in close proximity to nucleosomal DNA. Interestingly, the complete NuA4 complex contains chromatin reader domains that are not present in this study but are required to direct histone acetylation (Steunou et al, 2016). How these extra domains interact within an NCP remains an unanswered question.…”

Tri-methylation of Histone H3 Lysine 4 Facilitates Gene Expression in Ageing Cells

“…Multiple H3K4me3-binding proteins are present in yeast, which in turn are members of histone modifying and repositioning complexes. Binding of Yng1, Yng2 or Sgf29 would recruit NuA3, NuA4 or SAGA histone acetyl transferase complexes respectively, promoting local histone acetylation that should enhance transcriptional activation (Agalioti et al, 2002;Bian et al, 2011;Pokholok et al, 2005;Steunou et al, 2016;Taverna et al, 2006). Loss of recruitment of NuA3 and NuA4 to H3K4me3 has remarkably little effect on gene expression under normal conditions (Choy et al, 2001;Taverna et al, 2006), but may well impact a specific subset of genes when induced or during dramatic chromatin remodelling of the sort that occurs during ageing; indeed delays in induction of individual genes have been reported in mutants lacking H3K4me3-linked acetyltransferase recruitment (Bian et al, 2011;Morillon et al, 2005).…”