The mystery of the histones also derived from their bizarre biochemical behavior. Purified histones formed all manner of aggregates, both individually and in mixtures with one another (Edwards and Shooter, 1969; D'Anna and Isenberg, 1974), discouraging attempts at
A novel 15-subunit complex with the capacity to remodel the structure of chromatin, termed RSC, has been isolated from S. cerevisiae on the basis of homology to the SWI/SNF complex. At least three RSC subunits are related to SWI/SNF polypeptides: Sth1p, Rsc6p, and Rsc8p are significantly similar to Swi2/Snf2p, Swp73p, and Swi3p, respectively, and were identified by mass spectrometric and sequence analysis of peptide fragments. Like SWI/SNF, RSC exhibits a DNA-dependent ATPase activity stimulated by both free and nucleosomal DNA and a capacity to perturb nucleosome structure. RSC is, however, at least 10-fold more abundant than SWI/SNF complex and is essential for mitotic growth. Contrary to a report for SWII/SNF complex, no association of RSC (nor of SWI/SNF complex) with RNA polymerase II holoenzyme was detected.
GRF2, an abundant yeast protein of Mr -127,000, binds to the GAL upstream activating sequence (UASc) and creates a nucleosome-free region of -230 bp. Purified GRF2 binds to sequences found in many other UASs, in the 35S rRNA enhancer, at centromeres, and at telomeres. Although GRF2 stimulates transcription only slightly on its own, it combines with a neighboring weak activator to give as much as a 170-fold enhancement. This effect of GRF2 is strongly distance-dependent, declining by 85% when 22 bp is interposed between the GRF2 and neighboring activator sites.
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...
RSC, an abundant, essential chromatin-remodeling complex related to SWI/SNF complex, catalyzes the transfer of a histone octamer from a nucleosome core particle to naked DNA. The newly formed octamer-DNA complex is identical with a nucleosome in all respects. The reaction requires ATP and involves an activated RSC-nucleosome intermediate. The mechanism may entail formation of a duplex displacement loop on the nucleosome, facilitating the entry of exogeneous DNA and the release of the endogenous molecule.
RSC, an abundant, essential chromatin-remodeling complex, related to SWI/SNF complex, binds nucleosomes and naked DNA with comparable affinities, as shown by gel shift analysis. The RSC-nucleosome complex is converted in the presence of ATP to a slower migrating form. This activated complex exhibits greatly increased susceptibility to endo- and exonucleases but retains a full complement of histones. Activation persists in the absence of ATP, and on removal of RSC, the nucleosome is released in an altered form, with a diminished electrophoretic mobility, greater sedimentation rate, and marked instability at elevated ionic strength. The reaction is reversible in the presence of RSC and ATP, with conversion of the altered form back to the nucleosome.
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...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.