Abstract
The packaging of the eukaryotic genome into chromatin is likely to be mediated by chromatin assembly factors, including histone chaperones. We investigated the function of the histone H3/H4 chaperones anti-silencing function 1 (Asf1p) and chromatin assembly factor 1 (CAF-1) in vivo. Analysis of chromatin structure by accessibility to micrococcal nuclease and DNase I digestion demonstrated that the chromatin from CAF-1 mutant yeast has increased accessibility to these enzymes. In agreement, the supercoiling of the endogenous 2mu plasmid is reduced in yeast lacking CAF-1. These results indicate that CAF-1 mutant yeast globally under-assemble their genome into chromatin, consistent with a role for CAF-1 in chromatin assembly in vivo. By contrast, asf1 mutants globally over-assemble their genome into chromatin, as suggested by decreased accessibility of their chromatin to micrococcal nuclease and DNase I digestion and increased supercoiling of the endogenous 2mu plasmid. Deletion of ASF1 causes a striking loss of acetylation on histone H3 lysine 9, but this is not responsible for the altered chromatin structure in asf1 mutants. These data indicate that Asf1p may have a global role in chromatin disassembly and an unexpected role in histone acetylation in vivo.
Highlights
The eukaryotic genome is assembled into an ordered nucleoprotein structure known as chromatin
Loss of chromatin assembly factor 1 (CAF-1) Leads to a Subtle Increase in Accessibility to Micrococcal Nuclease, whereas Loss of Asf1p Leads to Decreased Accessibility to Micrococcal Nuclease—To determine whether Asf1p and CAF-1 assemble chromatin in vivo, we examined the structure of chromatin in Saccharomyces cerevisiae containing deletions of the anti-silencing function 1 (ASF1) or CAC1 genes
Loss of CAF-1 Leads to Increased Accessibility to DNase I, whereas Loss of Asf1p Leads to Decreased Accessibility to DNase I Digestion—we examined the accessibility of the chromatin to digestion with DNase I, a more sensitive measure of chromatin structure than MNase digestion [15]
Summary
Yeast Strains and Media—Yeast cultures and genetic manipulations were performed following standard methods [25]. Yeast strains were grown to a density of ϳ1 ϫ 107 cells/ml in yeast extract-peptone-dextrose for all analyses. Supercoiling Analysis—30 ml of the indicated yeast strains were grown to a density of 2 ϫ 107 cells/ml, sedimented, resuspended in 400 l of resuspension buffer
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