The molecular topography of silenced chromatin in Saccharomyces cerevisiae

Abstract

Heterochromatin imparts regional, promoter-independent repression of genes and is epigenetically heritable. Understanding how silencing achieves this regional repression is a fundamental problem in genetics and development. Current models of yeast silencing posit that Sir proteins, recruited by transcription factors bound to the silencers, spread throughout the silenced region. To test this model directly at high resolution, we probed the silenced chromatin architecture by chromatin immunoprecipitation (ChIP) followed by next-generation sequencing (ChIP-seq) of Sir proteins, histones, and a key histone modification, H4K16-acetyl. These analyses revealed that Sir proteins are strikingly concentrated at and immediately adjacent to the silencers, with lower levels of enrichment over the promoters at HML and HMR, the critical targets for transcriptional repression. The telomeres also showed discrete peaks of Sir enrichment yet a continuous domain of hypoacetylated histone H4K16. Surprisingly, ChIP-seq of cross-linked chromatin revealed a distribution of nucleosomes at silenced loci that was similar to Sir proteins, whereas native nucleosome maps showed a regular distribution throughout silenced loci, indicating that cross-linking captured a specialized chromatin organization imposed by Sir proteins. This specialized chromatin architecture observed in yeast informs the importance of a steric contribution to regional repression in other organisms.

The Chromatin and Transcriptional Landscape of NativeSaccharomyces cerevisiaeTelomeres and Subtelomeric Domains

“…There was no evidence of a gradient of Sir proteins, as envisioned by early models of telomere position effect (Hecht et al 1996). The discontinuous distribution of Sir proteins has been reported previously for specific telomeres (Zill et al 2010;Thurtle and Rine 2014). Overall, this analysis clearly established the generality of the discrete nature of Sir protein association at all 32 telomeres.…”

“…Sir proteins associated at discrete positions at natural telomeres To investigate Sir protein association at the 32 natural telomeres of S. cerevisiae, we analyzed ChIP-Seq data sets in the 20-kbp subtelomeric region of Myc-tagged Sir2, Sir3, and Sir4 from our previous Sir ChIP-Seq studies (Thurtle and Rine 2014) (Figure 1). Additionally, we analyzed ChIP-Seq data sets for green fluorescent protein endowed with a nuclear localization signal (GFP-NLS) and a no-tag sample immunoprecipitated with the Myc antibody as controls for artifacts of ChIP-Seq analyses and nonspecific enrichment, respectively (Teytelman et al 2013) (Figure S1 and Figure S2).…”

“…For each Sir protein chromatin sample, MACS was run on two biological replicates of ChIP-Seq data from chromatin sheared by sonication and on a third sample for each Sir protein in which the chromatin sample was prepared by enzymatic digestion with MNase (Thurtle and Rine 2014). For each chromatin sample analyzed with MACS, the IP sample was the "treatment," and the input sample was the "control."…”

“…ChIP-Seq read mapping: ChIP-Seq reads analyzed were from previous Sir protein ChIP studies (Teytelman et al 2013;Thurtle and Rine 2014), deposited in the NCBI Sequence Read Archive under accession nos. SRP030670 and SRP034921, respectively.…”

“…For the few natural telomeres at which URA3 appears repressed (TEL13R, TEL11L, and TEL02R), silencing is discontinuous across the length of the telomere and largely restricted to positions close to the X element. Similarly, Sir proteins also associate discretely at select natural telomeres, with the highest levels of enrichment proximal to the X element (Zill et al 2010;Radman-Livaja et al 2011;Thurtle and Rine 2014). The natural telomeres that repress the URA3 transgene exhibit a characteristic array of phased nucleosomes specific to those telomeres (Loney et al 2009).…”