The Ino80 complex prevents invasion of euchromatin into silent chromatin.

Here we show that the Ino80 chromatin remodeling complex (Ino80C) directly prevents euchromatin from invading transcriptionally silent chromatin within intergenic regions and at the border of euchromatin and heterochromatin. Deletion of Ino80C subunits leads to increased H3K79 methylation and noncoding RNA polymerase II (Pol II) transcription centered at the Ino80C-binding sites. The effect of Ino80C is direct, as it blocks H3K79 methylation by Dot1 in vitro. Heterochromatin stimulates the binding of Ino80C in vitro and in vivo. Our data reveal that Ino80C serves as a general silencing complex that restricts transcription to gene units in euchromatin.

Mechanism for epigenetic variegation of gene expression at yeast telomeric heterochromatin.

Yeast contains heterochromatin at telomeres and the silent mating-type loci (HML/HMR). Genes positioned within the telomeric heterochromatin of Saccharomyces cerevisiae switch stochastically between epigenetically bistable ON and OFF expression states. Important aspects of the mechanism of variegated gene expression, including the chromatin structure of the natural ON state and the mechanism by which it is maintained, are unknown. To address this issue, we developed approaches to select cells in the ON and OFF states. We found by chromatin immunoprecipitation (ChIP) that natural ON telomeres are associated with Rap1 binding and, surprisingly, also contain known characteristics of OFF telomeres, including significant amounts of Sir3 and H4K16 deacetylated nucleosomes. Moreover, we found that H3K79 methylation (H3K79me), H3K4me, and H3K36me, which are depleted from OFF telomeres, are enriched at ON telomeres. We demonstrate in vitro that H3K79me, but not H3K4me or H3K36me, disrupts transcriptional silencing. Importantly, H3K79me does not significantly reduce Sir complex binding in vivo or in vitro. Finally, we show that maintenance of H3K79me at ON telomeres is dependent on transcription. Therefore, although Sir proteins are required for silencing, we propose that epigenetic variegation of telomeric gene expression is due to the bistable enrichment/depletion of H3K79me and not the fluctuation in the amount of Sir protein binding to nucleosomes.

Histone density is maintained during transcription mediated by the chromatin remodeler RSC and histone chaperone NAP1 in vitro.

ATPases and histone chaperones facilitate RNA polymerase II (pol II) elongation on chromatin. In vivo, the coordinated action of these enzymes is necessary to permit pol II passage through a nucleosome while restoring histone density afterward. We have developed a biochemical system recapitulating this basic process. Transcription through a nucleosome in vitro requires the ATPase remodels structure of chromatin (RSC) and the histone chaperone nucleosome assembly protein 1 (NAP1). In the presence of NAP1, RSC generates a hexasome. Despite the propensity of RSC to evict histones, NAP1 reprograms the reaction such that the hexasome is retained on the template during multiple rounds of transcription. This work has implications toward understanding the mechanism of pol II elongation on chromatin.