Abstract
Heterochromatin consists of highly ordered nucleosomes with characteristic histone modifications. There is evidence implicating chromatin remodeling proteins in heterochromatin formation, but their exact roles are not clear. We demonstrate in Saccharomyces cerevisiae that the Fun30p and Isw1p chromatin remodeling factors are similarly required for transcriptional silencing at the HML locus, but they differentially contribute to the structure and stability of HML heterochromatin. In the absence of Fun30p, only a partially silenced structure is established at HML. Such a structure resembles fully silenced heterochromatin in histone modifications but differs markedly from both fully silenced and derepressed chromatin structures regarding nucleosome arrangement. This structure likely represents an intermediate state of heterochromatin that can be converted by Fun30p to the mature state. Moreover, Fun30p removal reduces the rate of de novo establishment of heterochromatin, suggesting that Fun30p assists the silencing machinery in forming heterochromatin. We also find evidence suggesting that Fun30p functions together with, or after, the action of the silencing machinery. On the other hand, Isw1p is dispensable for the formation of heterochromatin structure but is instead critically required for maintaining its stability. Therefore, chromatin remodeling proteins may rearrange nucleosomes during the formation of heterochromatin or serve to stabilize/maintain heterochromatin structure.
Highlights
DNA in eukaryotes is packed into chromatin through the formation of nucleosomes
We demonstrate in Saccharomyces cerevisiae that the Fun30p and Isw1p chromatin remodeling factors are required for transcriptional silencing at the HML locus, but they differentially contribute to the structure and stability of HML heterochromatin
FUN30 and ISW1 Are Required for Efficient Transcriptional Silencing at the HML Locus— heterochromatin at HML and HMR loci is formed via a common mechanism, the extent and regulation of transcriptional silencing at each locus is not identical
Summary
Replacement of a gene with the kanMX or natMX marker was achieved by transforming the parental strain to Geneticin- or nourseothricin-resistant with a PCR-generated fragment composed of kanMX or natMX bracketed by 5Ј- and 3Ј-flanking sequences of the coding region of the gene to be disrupted. Replacing a gene with the HIS3 or URA3 marker was achieved by transforming the parental strain to histidine or uracil prototrophy with a PCR-generated fragment composed of the HIS3 or URA3 gene bracketed by 5Ј- and 3Ј-flanking sequences of the coding region of the gene to be disrupted. An aliquot of permeabilized spheroplasts not treated with MNase was used to isolate genome (naked) DNA that was digested with MNase at 7.5 units/ml. Cross-linked chromatin from 50 ml of mid-log phase cell culture was sonicated with Branson Sonifer 450 to yield DNA fragments of an average size of 500 bp. The means of data from three independent experiments together with corresponding S.D. were presented
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