ABSTRACT
The exceptionally high positive charge of the histones, concentrated in the N- and C-terminal tails, is believed to contribute to the stability of the nucleosome by neutralizing the negative charge of the nucleosomal DNA. We find, on the contrary, that the high positive charge contributes to instability, performing an essential function in chromatin remodeling. We show that the tails are required for removal of the histone octamer by the RSC chromatin remodeling complex, and this function is not due to direct RSC-tail interaction. We also show that the tails are required for histone octamer transfer from nucleosomes to DNA, and this activity of the tails is a consequence of their positive charge. Thus, the histone tails, intrinsically disordered protein regions, perform a critical role in chromatin structure and transcription, unrelated to their well-known role in regulation through posttranscriptional modification.
Subject(s)
Histones , Nucleosomes , DNA/chemistry , Histones/chemistry , Histones/metabolism , Nucleosomes/metabolism , Transcription Factors/metabolismABSTRACT
Small molecules that bind in the minor groove of DNA are in clinical use as antibiotics and antitumor drugs. Two members of this class of molecules, netropsin and chromomycin, are shown here to displace DNA from the nucleosome and promote transfer of the histone octamer to an acceptor protein. The effects of these groove-binding molecules are exploited to address an outstanding problem in the mechanism of the RSC chromatin remodeling complex. RSC and other remodeling complexes are DNA translocases, acting near the center of the nucleosomal DNA, but translocation is apparently impossible because DNA cannot slide across the histone surface in the nucleosome. Netropsin and chromomycin promote the release of DNA from the histone surface, enhance the formation of a RSC-nucleosome complex, and synergize with RSC in chromatin remodeling. These findings are in keeping with an involvement of bulge translocation in chromatin remodeling.
Subject(s)
Nucleosomes , Saccharomyces cerevisiae Proteins , Histones/metabolism , DNA-Binding Proteins/metabolism , Chromatin Assembly and Disassembly , Clinical Relevance , Netropsin/metabolism , DNA/metabolism , Saccharomyces cerevisiae Proteins/metabolism , ChromatinABSTRACT
The +1 nucleosome of yeast genes, within which reside transcription start sites, is characterized by histone acetylation, by the displacement of an H2A-H2B dimer, and by a persistent association with the RSC chromatin-remodeling complex. Here we demonstrate the interrelationship of these characteristics and the conversion of a nucleosome to the +1 state in vitro. Contrary to expectation, acetylation performs an inhibitory role, preventing the removal of a nucleosome by RSC. Inhibition is due to both enhanced RSC-histone interaction and diminished histone-chaperone interaction. Acetylation does not prevent all RSC activity, because stably bound RSC removes an H2A-H2B dimer on a timescale of seconds in an irreversible manner.
Subject(s)
Chromatin Assembly and Disassembly/physiology , DNA-Binding Proteins/physiology , Histones/physiology , Saccharomyces cerevisiae Proteins/physiology , Transcription Factors/physiology , Acetyl Coenzyme A/metabolism , Acetylation , Animals , DNA-Binding Proteins/metabolism , Histones/metabolism , Nucleosome Assembly Protein 1 , Nucleosomes/physiology , Protein Conformation , Protein Processing, Post-Translational , Rats , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Trans-Activators/metabolism , Transcription Factors/metabolismABSTRACT
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 ATP-dependent removal of nucleosomes rather than a passive mechanism due to the intrinsic instability of nucleosomes on AT-rich DNA sequences.
Subject(s)
Chromatin Assembly and Disassembly/physiology , Nucleosomes/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Adenosine Triphosphate/metabolism , Base Sequence , Chromatin Assembly and Disassembly/genetics , Poly dA-dT/metabolismABSTRACT
Purified chromatin rings, excised from the PHO5 locus of Saccharomyces cerevisiae in transcriptionally repressed and activated states, were remodeled with RSC and ATP. Nucleosomes were translocated, and those originating on the promoter of repressed rings were removed, whereas those originating on the open reading frame (ORF) were retained. Treatment of the repressed rings with histone deacetylase diminished the removal of promoter nucleosomes. These findings point to a principle of promoter chromatin remodeling for transcription, namely that promoter specificity resides primarily in the nucleosomes rather than in the remodeling complex that acts upon them.
Subject(s)
Acid Phosphatase/genetics , Chromatin Assembly and Disassembly/physiology , DNA-Binding Proteins/physiology , Nucleosomes/metabolism , Promoter Regions, Genetic , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/genetics , Transcription Factors/physiology , Acid Phosphatase/chemistry , Chromatin Assembly and Disassembly/drug effects , Histone Deacetylases/pharmacology , Nucleic Acid Conformation , Nucleosomes/chemistry , Nucleosomes/genetics , Open Reading Frames , Saccharomyces cerevisiae Proteins/chemistry , Transcriptional ActivationABSTRACT
Results from biochemical and structural studies of the RSC chromatin-remodeling complex prompt a proposal for the remodeling mechanism: RSC binding to the nucleosome releases the DNA from the histone surface and initiates DNA translocation (through one or a small number of DNA base pairs); ATP binding completes translocation, and ATP hydrolysis resets the system. Binding energy thus plays a central role in the remodeling process. RSC may disrupt histone-DNA contacts by affecting histone octamer conformation and through extensive interaction with the DNA. Bulging of the DNA from the octamer surface is possible, and twisting is unavoidable, but neither is the basis of remodeling.
Subject(s)
Chromatin Assembly and Disassembly , DNA-Binding Proteins/metabolism , Histones/metabolism , Nucleosomes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Transcription Factors/metabolism , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Animals , DNA/metabolism , DNA-Binding Proteins/chemistry , Deoxyribonuclease I/chemistry , Exodeoxyribonucleases/chemistry , Hydrolysis , Rats , Saccharomyces cerevisiae Proteins/chemistry , Transcription Factors/chemistryABSTRACT
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.
Subject(s)
Chromatin Assembly and Disassembly , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/ultrastructure , Nucleosomes/chemistry , Nucleosomes/ultrastructure , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/ultrastructure , Transcription Factors/chemistry , Transcription Factors/ultrastructure , Cryoelectron Microscopy , Image Processing, Computer-Assisted , Models, Molecular , Protein Structure, QuaternaryABSTRACT
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 H2A/H2B 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.
