Murine Chd1 (chromodomain helicase DNA-binding protein 1), a chromodomain-containing chromatin remodeling protein, is necessary for embryonic stem (ES) cell pluripotency. Chd1 binds to nucleosomes trimethylated at histone 3 Lys 4 (H3K4me3) near the beginning of active genes but not to bivalent domains also containing H3K27me3. To address the mechanism of this specificity, we reproduced H3K4me3-and CHD1-stimulated gene activation in HeLa extracts. Multidimensional protein identification technology (MuDPIT) and immunoblot analyses of purified preinitiation complexes (PICs) revealed the recruitment of CHD1 to naive chromatin but enhancement on H3K4me3 chromatin. Studies in depleted extracts showed that the Mediator coactivator complex, which controls PIC assembly, is also necessary for CHD1 recruitment. MuDPIT analyses of CHD1-associated proteins support the recruitment data and reveal numerous components of the PIC, including Mediator. In vivo, CHD1 and Mediator are recruited to an inducible gene, and genome-wide binding of the two proteins correlates well with active gene transcription in mouse ES cells. Finally, coimmunoprecipitation of CHD1 and Mediator from cell extracts can be ablated by shRNA knockdown of a specific Mediator subunit. Our data support a model in which the Mediator coordinates PIC assembly along with the recruitment of CHD1. The combined action of the PIC and H3K4me3 provides specificity in targeting CHD1 to active genes.

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.

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. N ucleosomes pose a strong barrier to RNA polymerase II (pol II) elongation (1-3). An understanding of how this barrier is overcome will reveal principles central to all eukaryotic organisms. Genome-wide chromatin immunoprecipitation analyses reveal that histone density is inversely proportional to transcriptional activity (4). However, with the exception of the heat shock loci (5), most genes maintain limited nucleosome density in the coding region during transcription. Maintenance of nucleosome density prevents cryptic transcription, which can have potentially deleterious effects on gene expression and genomic integrity (6).Transcription through chromatin in vivo requires ATP-dependent remodeling machines, histone modification enzymes, and histone chaperones (7). ATP-dependent remodeling enzymes such as SWI/SNF and remodels structure of chromatin (RSC) can mobilize and/or evict nucleosomes to create an unimpeded DNA template for transcription (8). These enzymes are found at the promoters of genes and within ORFs (9-11). Furthermore, SWI/SNF has been shown to travel with pol II in vivo, evicting histones on active genes (12), and RSC has been shown to directly interact with the RNA pol subunit Rpb5 (13). In mammalian cells, pol II pauses at an artificially introduced 601 positioning sequence when SWI/ SNF is knocked down by RNAi (14). Histone chaperones such as ASF1, SPT6, and FACT (SPT16/POB3) also travel with pol II throughout transcription and probably assist in the removal of histones in front of pol II and redeposition behind (15-17). Indeed, one study suggests that FACT redeposits the original histones behind pol II (18).Studies of pol II elongation on chromatin in vitro have revealed insights into the mechanism. Experiments by Studitsky and coworkers showed that pol II frequently stalls and backtracks from the nucleosome (19). Higher ionic strength, which weakens DNA-histone contacts, abrogates the barrier allowing pol II to pass (20). The reaction is stimulated by TFIIS (19). Although nucleosomal passage by pol II occurs at physiological salt concentration (i.e., 150 mM), the efficiency increases with increasing ionic strength (20). Further, one orientation of the 601 positioned nucleosome is more permissive to tran...

Pervasive transcription initiates from cryptic promoters and is observed in eukaryotes ranging from yeast to mammals. The Set2-Rpd3 regulatory system prevents cryptic promoter function within expressed genes. However, conserved systems that control pervasive transcription within intergenic regions have not been well established. Here we show that Mot1, Ino80 chromatin remodeling complex (Ino80C), and NC2 colocalize on chromatin and coordinately suppress pervasive transcription in S. cerevisiae and murine embryonic stem cells (mESCs). In yeast, all three proteins bind subtelomeric heterochromatin through a Sir3-stimulated mechanism and to euchromatin via a TBP-stimulated mechanism. In mESCs, the proteins bind to active and poised TBP-bound promoters along with promoters of polycomb-silenced genes apparently lacking TBP. Depletion of Mot1, Ino80C or NC2 by anchor away in yeast or RNAi in mESCs leads to near-identical transcriptome phenotypes, with new subtelomeric transcription in yeast, and greatly increased pervasive transcription in both yeast and mESCs.

Summary The S. cerevisiae Rpd3 large (Rpd3L) and small (Rpd3S) histone deacetylase (HDAC) complexes are prototypes for understanding transcriptional repression in eukaryotes [1]. The current view is that they function by deacetylating chromatin, thereby limiting accessibility of the transcriptional machinery to the underlying DNA. However, one study showed that an Rpd3 catalytic mutant retains substantial repression capability when targeted to a promoter as a LexA fusion protein [2]. We investigated the HDAC-independent properties of the Rpd3 complexes biochemically and discovered a chaperone function, which promotes histone deposition onto DNA in vitro, and a novel activity, which prevents nucleosome eviction but not remodeling mediated by the ATP-dependent RSC complex. These HDAC-independent activities inhibit Pol II transcription on a nucleosomal template in vitro. Importantly, the functions of the endogenous Rpd3 complexes can be recapitulated with recombinant Rpd3 core complex comprising Sin3, Rpd3 and Ume1, but not with the individual subunits. To test the hypothesis that Rpd3 contributes to chromatin stabilization in vivo, we measured histone H3 density genomewide and found it was reduced at promoters in an Rpd3 deletion mutant but partially restored in a catalytic mutant. Importantly, the effects on H3 density are most apparent on RSC-enriched genes [3]. Our data suggest that the Rpd3 core complex could contribute to repression via a novel nucleosome stabilization function.