Distinguishing between recruitment and spread of silent chromatin structures in <i>Saccharomyces cerevisiae</i>.

Abstract

The formation of heterochromatin at <i>HML</i>, <i>HMR</i>, and telomeres in <i>Saccharomyces cerevisiae</i> involves two main steps: the recruitment of Sir proteins to silencers and their spread throughout the silenced domain. We developed a method to study these two processes at single basepair resolution. Using a fusion protein between the heterochromatin protein Sir3 and the nonsite-specific bacterial adenine methyltransferase M.EcoGII, we mapped sites of Sir3-chromatin interactions genome-wide using long-read Nanopore sequencing to detect adenines methylated by the fusion protein and by ChIP-seq to map the distribution of Sir3-M.EcoGII. A silencing-deficient mutant of Sir3 lacking its Bromo-Adjacent Homology (BAH) domain, <i>sir3-bah∆</i>, was still recruited to <i>HML</i>, <i>HMR</i>, and telomeres. However, in the absence of the BAH domain, it was unable to spread away from those recruitment sites. Overexpression of Sir3 did not lead to further spreading at <i>HML</i>, <i>HMR</i>, and most telomeres. A few exceptional telomeres, like 6R, exhibited a small amount of Sir3 spreading, suggesting that boundaries at telomeres responded variably to Sir3-M.EcoGII overexpression. Finally, by using a temperature-sensitive allele of <i>SIR3</i> fused to <i>M.ECOGII</i>, we tracked the positions first methylated after induction and found that repression of genes at <i>HML</i> and <i>HMR</i> began before Sir3 occupied the entire locus.