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The RSC chromatin-remodeling complex completely disassembles a nucleosome in the presence of the histone chaperone Nap1 and ATP. Disassembly occurs in a stepwise manner, with the removal of H2A͞H2B dimers, followed by the rest of the histones and the release of naked DNA. RSC and related chromatin-remodeling complexes may be responsible for the removal of promoter nucleosomes during transcriptional activation in vivo.Asf1 ͉ histones ͉ Nap1 ͉ RSC ͉ yeast T he remodeling of promoter chromatin is a prerequisite for transcription. Remodeling relieves repression by the nucleosome; it exposes promoter DNA for interaction with RNA polymerase and associated proteins. Remodeling has been thought to involve a ''reconfiguration'' rather than removal of the nucleosome (1). This view, until recently widely held, was based on two lines of evidence. First, promoter DNA becomes more accessible to nuclease attack after transcriptional activation. Second, histones remain associated with the DNA but in a highly modified state, as shown with the use of antibodies against acetylated, phosphorylated, methylated, and other forms of the N-terminal ''tails.'' Exposure of DNA was reconciled with the retention of histones by the hypothesis of an altered nucleosome, whose modified structure would be conducive to transcription.This hypothesis has been challenged by a reexamination of promoter chromatin structure and a reinterpretation of the evidence. Quantitative measurements of DNA topology, nuclease digestion rate, and sedimentation profile were performed on the yeast PHO5 promoter. The results of these three very different types of analysis were in close quantitative agreement with one another (2), showing that nucleosomes are present on the activated promoter at levels 18-60% of those at the repressed promoter, and that activated promoter nucleosomes are indistinguishable in structure from repressed promoter nucleosomes (2). On this basis, it was proposed that transcriptional activation is accompanied by the continual removal and reformation of promoter nucleosomes. The modified histones detected on the activated promoter by chromatin immunoprecipitation (3) were interpreted as intermediates in the processes of removal and reformation (4).Evidence has been presented for the removal of a nucleosome from the TATA box of a promoter by sliding of the histone octamer to an adjacent position on the DNA (5). The alternative is that nucleosomes are removed by dissociation of the octamer from the DNA. These possibilities could be distinguished for the PHO5 promoter by transcriptional activation on small chromatin circles (6). Nucleosomes were lost from the circles, demonstrating octamer dissociation. This result was obtained with a TATA box mutant, so it did not depend on replacement of the octamer by TATA-binding protein. Rather it reflects a natural mechanism for denuding promoter DNA.What enzyme system(s) might be responsible for the removal of promoter nucleosomes? Genetic studies have implicated the SWI͞SNF and closely related RSC complexe...

Gene activator proteins comprise distinct DNA-binding and transcriptional activation domains (ADs). Because few ADs have been described, we tested domains tiling all yeast transcription factors for activation in vivo and identified 150 ADs. By mRNA display, we showed that 73% of ADs bound the Med15 subunit of Mediator, and that binding strength was correlated with activation. AD-Mediator interaction in vitro was unaffected by a large excess of free activator protein, pointing to a dynamic mechanism of interaction. Structural modeling showed that ADs interact with Med15 without shape complementarity (‘fuzzy’ binding). ADs shared no sequence motifs, but mutagenesis revealed biochemical and structural constraints. Finally, a neural network trained on AD sequences accurately predicted ADs in human proteins and in other yeast proteins, including chromosomal proteins and chromatin remodeling complexes. These findings solve the longstanding enigma of AD structure and function and provide a rationale for their role in biology.

AT-rich DNA is concentrated in the nucleosome-free regions (NFRs) associated with transcription start sites of most genes. We tested the hypothesis that AT-rich DNA engenders NFR formation by virtue of its rigidity and consequent exclusion of nucleosomes. We found that the AT-rich sequences present in many NFRs have little effect on the stability of nucleosomes. Rather, these sequences facilitate the removal of nucleosomes by the RSC chromatin remodeling complex. RSC activity is stimulated by AT-rich sequences in nucleosomes and inhibited by competition with AT-rich DNA. RSC may remove NFR nucleosomes without effect on adjacent ORF nucleosomes. Our findings suggest that many NFRs are formed and maintained by an active mechanism involving the ATPdependent removal of nucleosomes rather than a passive mechanism due to the intrinsic instability of nucleosomes on AT-rich DNA sequences.[Keywords: RSC; poly(dA:dT) tracts; yeast] Supplemental material is available for this article.Received August 9, 2014; revised version accepted October 17, 2014.The assembly of promoters in nucleosomes prevents the initiation of transcription in vitro (Lorch et al. 1987), and depletion of nucleosomes leads to gene activation in yeast in vivo (Han and Grunstein 1988). Nucleosomes may be regarded at a fundamental level as general gene repressors. Relief from repression is achieved by the removal of nucleosomes by either the occurrence of nucleosome-free regions (NFRs) at the transcription start sites (TSSs) of TATA-less promoters (;80% of promoters in yeast) (Yuan et al. 2005;Zhang et al. 2011) or chromatin remodeling of genes with TATA-containing promoters (which may have distinctive nucleosomal configurations, but not NFRs, as defined by size and proximity to TSSs) (Svaren and Horz 1997;Boeger et al. 2003).The uniform coverage of eukaryote genomes by nucleosomes is punctuated at NFRs by apparently naked DNA regions and by well-defined locations of the nucleosomes nearby. The formation of NFRs is generally believed to be an intrinsic property of the DNA sequence; it is attributed to their high content of AT base pairs and in particular of poly(dA:dT) tracts (Yuan et al. 2005) with consequent destabilization of nucleosomes. Poly(dA:dT) is comparatively rigid and resists bending around the histone core of the nucleosome. The evidence for this passive mechanism of NFR formation comes from nucleosome positioning analysis, chromatin reconstitution, and effects on transcription in vivo. Poly(dA:dT) tracts greater than or equal to seven residues in length are confined to the first two turns of the double helix at the ends of the DNA in chicken nucleosomes (Satchwell et al. 1986). Pure poly(dA:dT) DNA is refractory to nucleosome formation (Kunkel and Martinson 1981;Prunell 1982). Poly(dA:dT) tracts of 17-42 residues stimulate transcription from the HIS3 promoter in yeast (Iyer and Struhl 1995). Poly(dG:dC) tracts have the same effect, leading to the conclusion that a structural property of the sequence element rather than interaction with a pro...

ATP-dependent chromatin-remodeling complexes, such as RSC, can reposition, evict or restructure nucleosomes. A structure of a RSC-nucleosome complex with a nucleosome determined by cryo-EM shows the nucleosome bound in a central RSC cavity. Extensive interaction of RSC with histones and DNA seems to destabilize the nucleosome and lead to an overall ATP-independent rearrangement of its structure. Nucleosomal DNA appears disordered and largely free to bulge out into solution as required for remodeling, but the structure of the RSC-nucleosome complex indicates that RSC is unlikely to displace the octamer from the nucleosome to which it is bound. Consideration of the RSC-nucleosome structure and published biochemical information suggests that ATP-dependent DNA translocation by RSC may result in the eviction of histone octamers from adjacent nucleosomes.The mobilization of nucleosomes is a prerequisite for DNA transactions. Nucleosomes are repeatedly removed and reassembled in promoter regions, resulting in transient exposure of the DNA for interaction with the transcription machinery 1,2 . Chromatin modification is also essential in the process of double-strand break repair 3 . The prime candidates for nucleosome removal are the SWI/SNF family of chromatin-remodeling complexes, which relieve repression by nucleosomes in vivo and perturb nucleosome structure in an ATP-dependent manner in vitro.Whereas SWI and SNF genes are nonessential in yeast, and their protein products are present at low levels, homologs of these genes encode the components of the essential, abundant RSC complex 4 . Biochemical and structural studies of RSC have illuminated the chromatinremodeling process. RSC binds a nucleosome core particle with nanomolar affinity 5 and reduces digestion of nucleosomal DNA by nucleases 6 . A three-dimensional reconstruction of RSC calculated from EM images of single particles preserved in stain revealed a cavity able to accommodate a nucleosome and likely to afford the observed nuclease protection 6 . Addition of ATP to a RSC-nucleosome complex leads to sliding of the histone octamer along the DNA 7 or to transfer of the octamer, either to a histone chaperone 8 or to another DNA molecule 9 . The underlying principle of these activities 10 is the coupling of ATP hydrolysis to