Dynamics of Sir3 spreading in budding yeast: secondary recruitment sites and euchromatic localization

Abstract

Chromatin domains are believed to spread via a polymerization-like mechanism in which modification of a given nucleosome recruits a modifying complex, which can then modify the next nucleosome in the polymer. In this study, we carry out genome-wide mapping of the Sir3 component of the Sir silencing complex in budding yeast during a time course of establishment of heterochromatin. Sir3 localization patterns do not support a straightforward model for nucleation and polymerization, instead showing strong but spatially delimited binding to silencers, and weaker and more variable Ume6-dependent binding to novel secondary recruitment sites at the seripauperin (PAU) genes. Genomewide nucleosome mapping revealed that Sir binding to subtelomeric regions was associated with overpackaging of subtelomeric promoters. Sir3 also bound to a surprising number of euchromatic sites, largely at genes expressed at high levels, and was dynamically recruited to GAL genes upon galactose induction. Together, our results indicate that heterochromatin complex localization cannot simply be explained by nucleation and linear polymerization, and show that heterochromatin complexes associate with highly expressed euchromatic genes in many different organisms.

Highly expressed loci are vulnerable to misleading ChIP localization of multiple unrelated proteins

“…For example, Sir2 was reported to be enriched at CDC19 by Li et al, a locus we find to be hyper-ChIPpable, but no biological consequence for this enrichment was found (21). Additionally, Sir3 was reported to associate with the GAL1-10 locus specifically upon induction by galactose, which is consistent with our findings that this nonspecific enrichment occurs only when genes are highly expressed (9). By inference, interpreting the enrichment profiles of proteins that cause high-level expression may be especially problematic because the consequence of their function is to set the stage for this artifact.…”

The Nuts and Bolts of Transcriptionally Silent Chromatin in Saccharomyces cerevisiae

“…In various studies, de novo assembly has been triggered by reintroduction of a Sir protein that was experimentally withheld (usually Sir3). At model telomeres and sites distant from strong silencers, timedependent expansion of silent chromatin domains was detected (Katan-Khaykovich and Struhl 2005;Lynch and Rusche 2009;Radman-Livaja et al 2011). Even in these best-case scenarios, however, true processivity of the spreading reaction (i.e., template commitment) was not demonstrated.…”

The Chromatin and Transcriptional Landscape of NativeSaccharomyces cerevisiaeTelomeres and Subtelomeric Domains

“…Additionally, there were three genes that MACS called as significantly enriched for at least one of the three Sir proteins but whose expression did not change in the sir mutants: IRC7, VBA5, and PAU20. PAU20 was previously implicated as a secondary recruitment site for Sir3 (Radman-Livaja et al 2011). Thus Sir proteins can be recruited to a loci without repressing the adjacent gene.…”

“…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).…”