Distinguishing between recruitment and spread of silent chromatin structures in Saccharomyces cerevisiae.

Molly Brothers,Jasper Rine

doi:10.7554/elife.75653

Abstract

The formation of heterochromatin at HML, HMR, and telomeres in Saccharomyces cerevisiae 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, sir3-bah∆, was still recruited to HML, HMR, 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 HML, HMR, 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 SIR3 fused to M.ECOGII, we tracked the positions first methylated after induction and found that repression of genes at HML and HMR began before Sir3 occupied the entire locus.

Highlights

Cells have an interest in coordinating the expression of genes: It allows them to turn sets of genes on and off in response to various stimuli or ensure certain genes are always expressed or always repressed to create and maintain cell identity
We developed a new method to study the process of recruitment and spread of the S. cerevisiae heterochromatin protein Sir3 in living cells with a resolution approximating the frequency of single A-T base pairs
We harnessed the power of single base-pair resolution afforded by Nanopore sequencing to distinguish between recruitment and spread of Sir3 by studying a mutant of Sir3 whose distribution and binding profile had not yet been characterized, sir3-bah∆

Summary

INTRODUCTION

Cells have an interest in coordinating the expression of genes: It allows them to turn sets of genes on and off in response to various stimuli or ensure certain genes are always expressed or always repressed to create and maintain cell identity. Almost any gene placed within the defined domain can be transcriptionally silenced, establishing the locusspecific, gene non-specific nature of heterochromatic silencing (Dodson and Rine, 2015; Gottschling et al, 1990; Saxton and Rine, 2019; Schnell and Rine, 1986; Sussel et al, 1993) This difference in sequence dependence between recruitment and spread implies they are separable processes that rely on different factors and interactions. Using long-read sequencing, we used this new method to resolve the distinction between the recruitment and spread of Sir and to track the establishment of gene silencing in heterochromatin over time These data pinpointed when the process of transcriptional silencing begins

RESULTS

DISCUSSION

Limitations