Eaf5/7/3 form a functionally independent NuA4 submodule linked to RNA polymerase II ‐coupled nucleosome recycling

Abstract

The NuA4 histone acetyltransferase complex is required for gene regulation, cell cycle progression, and DNA repair. Dissection of the 13-subunit complex reveals that the Eaf7 subunit bridges Eaf5 with Eaf3, a H3K36me3-binding chromodomain protein, and this Eaf5/7/3 trimer is anchored to NuA4 through Eaf5. This trimeric subcomplex represents a functional module, and a large portion exists in a native form outside the NuA4 complex. Gene-specific and genome-wide location analyses indicate that Eaf5/7/3 correlates with transcription activity and is enriched over the coding region. In agreement with a role in transcription elongation, the Eaf5/7/3 trimer interacts with phosphorylated RNA polymerase II and helps its progression. Loss of Eaf5/7/3 partially suppresses intragenic cryptic transcription arising in set2 mutants, supporting a role in nucleosome destabilization. On the other hand, loss of the trimer leads to an increase of replicationindependent histone exchange over the coding region of transcribed genes. Taken together, these results lead to a model where Eaf5/7/3 associates with elongating polymerase to promote the disruption of nucleosomes in its path, but also their refolding in its wake.

Histone H3K4 and H3K36 Methylation Independently Recruit the NuA3 Histone Acetyltransferase in Saccharomyces cerevisiae

“…This is unsurprising, however, as other studies have shown that HAT occupancy is a poor predictor of histone acetylation (Xue-Franzén et al 2010;Rossetto et al 2014). Instead, these results suggest that there is a level of regulation of histone acetylation that is independent of HAT recruitment.…”

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

“…Tandem affinity purification of complexes was performed as described previously (39) with a few modifications reported elsewhere (12,20). Cultures of 250 ml of exponentially growing cells were harvested at an OD 600 of 1 to 2 and washed in 10 mM Tris, pH 8, 350 mM NaCl, and cell lysis was achieved with glass beads at 4°C in lysis buffer (10 mM Tris-HCl, pH 8, 350 mM NaCl, 10% glycerol, 0.1% NP-40, 1 mM PMSF, 0.5 mM dithiothreitol [DTT], 2 g/ml pepstatin, 2 g/ml leupeptin, 5 g/ml aprotinin, 5 mM ␤-glycerophosphate, 5 mM Na butyrate).…”

“…Chromatin immunoprecipitations were performed essentially as previously described (20). Chromatin samples were prepared from 200 ml of exponentially growing cells using the standard protocol.…”

“…NuA4 acetylates lysines on the N-terminal tails of H4, H2A, and H2AZ (Htz1) histones (7,(9)(10)(11)(12), and its histone acetyltransferase (HAT) activity is important for the regulation of gene transcription (13)(14)(15)(16)(17)(18)(19)(20)(21) and also for the cellular response to DNA dam-age, recombination, and DNA double-strand break repair (22)(23)(24)(25)(26)(27). More recently, NuA4 was also shown to regulate life span and autophagy through its ability to acetylate nonhistone proteins (28)(29)(30).…”

“…More recently, NuA4 was also shown to regulate life span and autophagy through its ability to acetylate nonhistone proteins (28)(29)(30). While the NuA4 complex was first demonstrated to be recruited to specific gene promoters, where it locally acetylates nucleosomal histones (9,14,15,18), more recent studies have shown that NuA4 is also present on actively transcribed coding sequences (open reading frames [ORFs]) (19,20). Its enrichment at promoters correlates with an increase of histone acetylation and chromatin relaxation, allowing access to DNA for the transcription machinery, and its targeting to promoters is thought to be mediated by transcription activators (13,15,21,31).…”