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

Abstract
Data availability
Article and author information
Metrics

Abstract

The formation of heterochromatin at HML, HMR, and telomeres in Saccharomyces cerevisiae involves two main steps: Recruitment of Sir proteins to silencers and their spread throughout the silenced domain. We developed a method to study these two processes at single base-pair resolution. Using a fusion protein between the heterochromatin protein Sir3 and the non-site-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 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.

Data availability

All strains (Supplementary File 1) and plasmids (Supplementary File 2) are available upon request. Sequencing data is available in GEO under the SuperSeries GSE190137. ChIP-seq and DIP-seq data are under accession code GSE189038 in the SuperSeries. Nanopore data are under accession code GSE190136 in the SuperSeries.

The following data sets were generated

1. Brothers M
2. Rine J
(2021) Distinguishing between recruitment and spread of silent chromatin structures in Saccharomyces cerevisiae
NCBI Gene Expression Omnibus, GSE190137.

https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE190137
1. Brothers M
2. Rine J
(2021) Distinguishing between recruitment and spread of silent chromatin structures in Saccharomyces cerevisiae [I]
NCBI Gene Expression Omnibus, GSE189038.

https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE189038
1. Brothers M
2. Rine J
(2022) Distinguishing between recruitment and spread of silent chromatin structures in Saccharomyces cerevisiae [II]
NCBI Gene Expression Omnibus, GSE190136.

https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE190136

The following previously published data sets were used

(2017) Precise genome-wide mapping of single nucleosomes and linkers in vivo
NCBI Gene Expression Omnibus, GSE97290.

https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE97290

Article and author information

Author details

Molly Brothers

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-6114-3393
Jasper Rine

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

For correspondence
jrine@berkeley.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0003-2297-9814

Funding

National Science Foundation (1752814)

Molly Brothers

National Institutes of Health (R35 GM139488)

Jasper Rine

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.