Cargando…

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

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 fus...

Descripción completa

Detalles Bibliográficos
Autores principales: Brothers, Molly, Rine, Jasper
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8830885/
https://www.ncbi.nlm.nih.gov/pubmed/35073254
http://dx.doi.org/10.7554/eLife.75653
_version_ 1784648378243612672
author Brothers, Molly
Rine, Jasper
author_facet Brothers, Molly
Rine, Jasper
author_sort Brothers, Molly
collection PubMed
description 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.
format Online
Article
Text
id pubmed-8830885
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher eLife Sciences Publications, Ltd
record_format MEDLINE/PubMed
spelling pubmed-88308852022-02-14 Distinguishing between recruitment and spread of silent chromatin structures in Saccharomyces cerevisiae Brothers, Molly Rine, Jasper eLife Chromosomes and Gene Expression 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. eLife Sciences Publications, Ltd 2022-01-24 /pmc/articles/PMC8830885/ /pubmed/35073254 http://dx.doi.org/10.7554/eLife.75653 Text en © 2022, Brothers and Rine https://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited.
spellingShingle Chromosomes and Gene Expression
Brothers, Molly
Rine, Jasper
Distinguishing between recruitment and spread of silent chromatin structures in Saccharomyces cerevisiae
title Distinguishing between recruitment and spread of silent chromatin structures in Saccharomyces cerevisiae
title_full Distinguishing between recruitment and spread of silent chromatin structures in Saccharomyces cerevisiae
title_fullStr Distinguishing between recruitment and spread of silent chromatin structures in Saccharomyces cerevisiae
title_full_unstemmed Distinguishing between recruitment and spread of silent chromatin structures in Saccharomyces cerevisiae
title_short Distinguishing between recruitment and spread of silent chromatin structures in Saccharomyces cerevisiae
title_sort distinguishing between recruitment and spread of silent chromatin structures in saccharomyces cerevisiae
topic Chromosomes and Gene Expression
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8830885/
https://www.ncbi.nlm.nih.gov/pubmed/35073254
http://dx.doi.org/10.7554/eLife.75653
work_keys_str_mv AT brothersmolly distinguishingbetweenrecruitmentandspreadofsilentchromatinstructuresinsaccharomycescerevisiae
AT rinejasper distinguishingbetweenrecruitmentandspreadofsilentchromatinstructuresinsaccharomycescerevisiae