Maintenance of Heterochromatin Boundary and Nucleosome Composition at Promoters by the Asf1 Histone Chaperone and SWR1-C Chromatin Remodeler in Saccharomyces cerevisiae

Abstract

Chromatin remodeling complexes cooperate to regulate gene promoters and to define chromatin neighborhoods. Here, we identified genetic and functional connections between two silencing-related chromatin factors in the maintenance of native heterochromatic structures and nucleosome composition at promoters. Building on a previously reported link between the histone chaperone Asf1 and the Yaf9 subunit of the SWR1-C chromatin remodeler, we found that ASF1 broadly interacted with genes encoding for SWR1-C subunits. Asf1 and Yaf9 were required for maintaining expression of heterochromatin-proximal genes and they worked cooperatively to prevent repression of telomere-proximal genes by limiting the spread of SIR complexes into nearby regions. Genome-wide Sir2 profiling, however, revealed that the cooperative heterochromatin regulation of Asf1 and SWR1-C occurred only on a subset of yeast telomeres. Extensive analyses demonstrated that formation of aberrant heterochromatin structures in the absence of ASF1 and YAF9 was not causal for the pronounced growth and transcriptional defects in cells lacking both these factors. Instead, genetic and molecular analysis revealed that H3K56 acetylation was required for efficient deposition of H2A.Z at subtelomeric and euchromatic gene promoters, pointing to a role for Asf1-dependent H3K56 acetylation in SWR1-C biology.T HE fundamental building block of chromatin is a nucleosome composed of 146 bp of DNA wrapped around a histone octamer. Protein complexes involved in post-translational modification of histones, nucleosome movement, or replacement alter chromatin dynamics to regulate various chromosomal processes. Often, these chromatin-modifying processes intersect and interact cooperatively to regulate chromatin structure.Transcriptionally silent heterochromatin structures are a prime example of the multilayered activities of chromatin modifying complexes. In budding yeast, Saccharomyces cerevisiae, there are three well-defined regions of silent chromatin: the mating loci (HMR and HML), the rDNA locus, and telomeres. These regions are characterized by a distinct set of histone modifications and associated factors that distinguish them from adjacent transcriptionally active euchromatin (Rusche et al. 2003). Chief among these is the silent information regulator (SIR) complex, which not only constitutes the main structural component during establishment and maintenance of heterochromatin but also harbors an enzymatic function (Rusche et al. 2003). Specifically, the Sir2 subunit is a NAD+ dependent histone deacetylase (HDAC) that deacetylates H4K16ace, a process critical for promoting initial SIR complex formation at heterochromatin loci as well as for formation of heterochromatin boundaries. The latter is biologically important to prevent antagonistic silencing of neighboring euchromatic genes by encroaching heterochromatic structures (Imai et al. 2000;Rusche et al. 2003).Several additional histone modifying enzymes contribute to demarcating the boundary between heterochromatin...

Histone Modifications in Ageing and Lifespan Regulation

“…The acetylation of lysine 56 on histone H3 (H3K56ac) is evolutionarily conserved from yeast to man [46]. In S. cerevisiae, it promotes de novo nucleosome assembly, genomic stability, transcription, and formation of heterochromatin/euchromatin boundaries [47][48][49][50][51][52]. In mammals, the role of H3K56ac remains elusive, but recent evidence indicates that it also promotes genomic stability [46].…”

Cdk8 directly regulates glycolysis via phosphorylation of Gcr2

“…Cells with the indicated mutations were 10-fold serially diluted, spotted on SC-TRP media and grown for 3-5 days with the indicated carbon source or drug concentrations. (B) RT-qPCR analysis of mRNA levels for representative genes encoding glycolysis enzymes ( PGI1 , TPI1 , PGK1 and PYK1 ) normalized to TUB1 mRNA levels (Lu and Kobor, 2014). Error bars represent standard deviation from three independent biological replicates.…”

The RNAPII-CTD Maintains Genome Integrity through Inhibition of Retrotransposon Gene Expression and Transposition

“…cDNA was analyzed using a Rotor-Gene 600 (Corbett Research) and PerfeCTa SYBR Green FastMix (Quanta Biosciences). Samples were analyzed in triplicate from three independent RNA preparations and the protein-coding gene TUB1 was used as a control given that its expression is not altered upon truncation of the RNAPII-CTD [ 62 ]. For measuring Ty1 mRNA levels 6 pg/μl of cDNA were used in a 15 μl PCR reaction.…”