Asymmetric nucleosomes flank promoters in the budding yeast genome

Abstract

Nucleosomes in active chromatin are dynamic, but whether they have distinct structural conformations is unknown. To identify nucleosomes with alternative structures genome-wide, we used H4S47C-anchored cleavage mapping, which revealed that 5% of budding yeast (Saccharomyces cerevisiae) nucleosome positions have asymmetric histone-DNA interactions. These asymmetric interactions are enriched at nucleosome positions that flank promoters. Micrococcal nuclease (MNase) sequencebased profiles of asymmetric nucleosome positions revealed a corresponding asymmetry in MNase protection near the dyad axis, suggesting that the loss of DNA contacts around H4S47 is accompanied by protection of the DNA from MNase. Chromatin immunoprecipitation mapping of selected nucleosome remodelers indicated that asymmetric nucleosomes are bound by the RSC chromatin remodeling complex, which is required for maintaining nucleosomes at asymmetric positions. These results imply that the asymmetric nucleosome-RSC complex is a metastable intermediate representing partial unwrapping and protection of nucleosomal DNA on one side of the dyad axis during chromatin remodeling.[Supplemental material is available for this article.]Nucleosomes, the fundamental units of chromatin, are dynamic structures characterized by spontaneous conformational fluctuations that lead to reversible loss of histone-DNA and histone-histone contacts. Every nucleosome in the genome has to disassemble at least once during the cell cycle to allow for passage of the DNA replication machinery (Annunziato 2005), and nucleosomes at active regions might turn over several times during each cell cycle (Dion et al. 2007;Deal et al. 2010). Intrinsic to nucleosome dynamics is the formation of nucleosomal intermediates with alternative structures. However, the nature of such intermediate nucleosome structures formed in vivo is not known.Intermediate nucleosome structures can potentially be identified in vivo using base-pair resolution methods that interrogate histone-DNA contacts genome-wide. The traditional method for high-resolution mapping of nucleosomes is to use micrococcal nuclease (MNase) digestion, which digests away linker regions between nucleosomes (Reeves and Jones 1976). Subjecting MNase-digested DNA fragments to paired-end sequencing (MNase-seq) results in a high-resolution map of nucleosome positions (Hughes and Rando 2014). An alternative method for mapping nucleosomes is H4S47C-anchored cleavage mapping (Brogaard et al. 2012b), which has been used to determine the precise position of nucleosomes in yeast genomes (Brogaard et al. 2012a;Moyle-Heyrman et al. 2013). In this method, histone H4 mutant S47C is derivatized ex vivo with a phenanthroline ligand, converting H4 into a site-specific DNA cleavage agent. Using a modified library preparation and a structural model for H4S47C-anchored cleavage, we extended this method to determine the precise position and orientation of half-nucleosomes (hemisomes) at centromeres (Henikoff et al. 2014), thus showing that alternative nucle...

A nucleosome turnover map reveals that the stability of histone H4 Lys20 methylation depends on histone recycling in transcribed chromatin

“…S1B). We also observed the recently described asymmetry of the nucleosome dyads surrounding the NDR (Rhee et al 2014;Ramachandran et al 2015). The ChIP-exo data were used to identify locus-specific nucleosome turnover at high resolution across the genome (Fig.…”

Chromatin-remodeling and the initiation of transcription

“…Incorporation of a copper-phenanthroline moiety in histone H4 and cleavage of the associated DNA with hydrogen peroxide revealed a pronounced asymmetry of +1 nucleosomes (Ramachandran et al 2015): cleavage was more frequent on one side of the dyad axis of the nucleosome than on the other. By contrast, the vast majority of yeast nucleosomes showed only symmetric cleavage.…”

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

“…To determine the relative contribution of each histone PTM in targeting the NuA3 complex, we reasoned that histone PTMs that promote the interaction of NuA3 with chromatin would colocalize with Sas3. To this end, we mapped NuA3-bound nucleosomes at high resolution in vivo using a MNase-based ChIP-seq approach, which has previously been used to map chromatin remodelers (Koerber et al 2009;Floer et al 2010;Yen et al 2012;Ramachandran et al 2015). We immunoprecipitated HA-tagged Sas3, in parallel with an untagged control, from cross-linked MNase-digested chromatin and performed paired-end sequencing.…”