Barrier Proteins Remodel and Modify Chromatin To Restrict Silenced Domains

Abstract

Transcriptionally active and inactive domains are frequently found adjacent to one another in the eukaryotic nucleus. To better understand the underlying mechanisms by which domains maintain opposing transcription patterns, we performed a systematic genomewide screen for proteins that may block the spread of silencing in yeast. This analysis identified numerous proteins with efficient silencing blocking activities, and some of these have previously been shown to be involved in chromatin dynamics. We isolated subunits of Swi/Snf, mediator, and TFIID, as well as subunits of the Sas-I, SAGA, NuA3, NuA4, Spt10p, Rad6p, and Dot1p complexes, as barrier proteins. We demonstrate that histone acetylation and chromatin remodeling occurred at the barrier and correlated with a block to the spread of silencing. Our data suggest that multiple overlapping mechanisms were involved in delimiting silenced and active domains in vivo.The eukaryotic nucleus is organized into active and inactive domains, and the modulation of genes within these domains is mediated by different regulatory elements. Enhancers, promoters, and locus control regions activate genes, whereas silencers repress the transcription of genes. Junctions between the active and inactive gene domains commonly occur along chromosomes (reviewed in reference 54), and specific elements and activities that maintain opposing transcription states in adjacent domains have been previously identified.The budding yeast Saccharomyces cerevisiae also possesses active and inactive chromatin domains, and one of the bestcharacterized chromatin domains is the silenced HMR locus. The genes present at HMR are flanked by silencer elements, and proteins that bind these elements recruit the Sir proteins that mediate silencing. Following recruitment, the Sir proteins are thought to spread along the DNA to form a specialized chromatin state that is inaccessible to various enzymatic probes. The silent domains extend beyond the silencers and up to barrier elements that block the spread (3,13,15,21,40,57).While the left boundary of the silenced chromatin domain at HMR is not well defined, the right boundary has been studied in great detail. The deletion of this barrier element at the native HMR locus leads to an increased spread of silenced chromatin and to concomitant repression of neighboring euchromatic genes (13, 15), while the ectopic insertion of this barrier between a silencer and a promoter blocks the repressive effects of the silencer. A specific tRNA gene acts as a barrier at HMR, and mutations in the promoter of this gene or in the RNA polymerase III transcription factors weaken the barrier activity mediated by this tRNA gene. In addition, mutations in the acetyltransferases Sas2p and Gcn5p advertently affect barrier activity mediated by the tRNA gene.The left boundary of the Sir proteins at HML maps to the upstream activation sequence of YCL069w (3, 37), while the right boundary maps to the promoter of the CHA1 gene (37)-though silencing is believed to terminate at HML-I (4). Howev...

Long-Range Communication between the Silencers of HMR

“…These results with the dual reporter systems are concordant with our earlier studies of single tethered barrier proteins (47). Silencing adjacent to HMR-I requires the HMR-I silencer if a barrier blocks the action of HMR-E.…”

“…Plasmids RO590 and RO635 contained the full-length SAS2 or NUP2 coding regions fused in frame to the Gal4 DNA binding domain (GBD), with transcription driven by the ADH1 promoter in the pGBK-RC-TRP1 base plasmid (pGBD) (47).…”

Histone H3 Lysine 36 Methylation Antagonizes Silencing in Saccharomyces cerevisiae Independently of the Rpd3S Histone Deacetylase Complex

“…Therefore, Set2-mediated anti-silencing acts at least in part in a pathway that does not require the recruitment of Rpd3S (Figure 5b). It is possible that the eaf3D mutation confers an anti-silencing defect due to the association of Eaf3 with the NuA4 complex, which also antagonizes silencing (Oki et al 2004), and not Eaf3's association with Rpd3S, since absence of the Rpd3S-specific subunit Rco1 conferred no detectable anti-silencing defect. Taken together, our data imply that methylation of H3-K36 functions through at least two different effector mechanisms.…”

Regulation of Histone Gene Expression in Budding Yeast

“…Unfortunately, Spt21 has no identifiable domains to offer clues as to its mode of action. Genetic evidence implicates both Spt21 and Spt10 in transcriptional silencing (Oki et al 2004;Chang and Winston 2011), but this is probably an indirect effect, since neither protein could be detected at telomeres by ChIP. Intriguingly, Spt21 inhibits the formation of "T-bodies," cytoplasmic granules containing Ty1 RNA (Malagon and Jensen 2008).…”