Persistence of an alternate chromatin structure at silenced loci in vitro

Athar Ansari, Marc R. Gartenberg

Research output: Contribution to journalArticlepeer-review

26 Scopus citations

Abstract

In Saccharomyces cerevisiae, transcriptional repression at the HM mating-type loci and telomeres results from the formation of a heterochromatin-like structure. Silencing requires at least three Sir proteins (Sir2p-4p), which are recruited to chromatin by silencers at the HM loci and TG(1-3) tracts at telomeres. Sir proteins and telomeres colocalize at the nuclear periphery, suggesting that this subnuclear position may also contribute to transcriptional repression. To evaluate the contribution of nuclear context to silencing, we developed methodology to isolate silent chromatin for analysis in vitro. Site-specific recombination was used in vivo to produce DNA rings from the silent HMR locus, and differential centrifugation was used to isolate the rings from whole-cell lysate. The partially purified rings retained many of the intracellular hallmarks of transcriptionally repressed domains. Specifically, rings from repressed strains were resistant to restriction endonuclease digestion, bore an altered DNA topology, and were associated with Sir3p. The recombination approach also was used to form rings from HMR that lacked silencers. Despite the uncoupling of these cis-acting regulatory elements, similar but nonidentical results were obtained. We conclude that an alternate chromatin structure at silent loci can persist in vitro in the absence of silencers and nuclear compartmentalization.

Original languageEnglish (US)
Pages (from-to)343-348
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume96
Issue number2
DOIs
StatePublished - Jan 19 1999

All Science Journal Classification (ASJC) codes

  • General

Keywords

  • Heterochromatin
  • Mating-type locus
  • Recombination
  • Saccharomyces cerevisiae
  • Transcriptional repression and silencers

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