ABSTRACT
Nucleosomes contain â¼146 bp of DNA wrapped around a histone protein octamer that controls DNA accessibility to transcription and repair complexes. Posttranslational modification (PTM) of histone proteins regulates nucleosome function. To date, only modest changes in nucleosome structure have been directly attributed to histone PTMs. Histone residue H3(T118) is located near the nucleosome dyad and can be phosphorylated. This PTM destabilizes nucleosomes and is implicated in the regulation of transcription and repair. Here, we report gel electrophoretic mobility, sucrose gradient sedimentation, thermal disassembly, micrococcal nuclease digestion and atomic force microscopy measurements of two DNA-histone complexes that are structurally distinct from nucleosomes. We find that H3(T118ph) facilitates the formation of a nucleosome duplex with two DNA molecules wrapped around two histone octamers, and an altosome complex that contains one DNA molecule wrapped around two histone octamers. The nucleosome duplex complex forms within short â¼150 bp DNA molecules, whereas altosomes require at least â¼250 bp of DNA and form repeatedly along 3000 bp DNA molecules. These results are the first report of a histone PTM significantly altering the nucleosome structure.
Subject(s)
Histones/metabolism , Nucleosomes/chemistry , Nucleosomes/metabolism , Base Sequence , Chromatin/chemistry , DNA/analysis , DNA/chemistry , Histone Chaperones/metabolism , Histones/analysis , Histones/chemistry , Nucleosomes/ultrastructure , Phosphorylation , Threonine/metabolismABSTRACT
Eukaryotic genomes are repetitively wrapped into nucleosomes that then regulate access of transcription and DNA repair complexes to DNA. The mechanisms that regulate extrinsic protein interactions within nucleosomes are unresolved. We demonstrate that modulation of the nucleosome unwrapping rate regulates protein binding within nucleosomes. Histone H3 acetyl-lysine 56 [H3(K56ac)] and DNA sequence within the nucleosome entry-exit region additively influence nucleosomal DNA accessibility by increasing the unwrapping rate without impacting rewrapping. These combined epigenetic and genetic factors influence transcription factor (TF) occupancy within the nucleosome by at least one order of magnitude and enhance nucleosome disassembly by the DNA mismatch repair complex, hMSH2-hMSH6. Our results combined with the observation that â¼30% of Saccharomyces cerevisiae TF-binding sites reside in the nucleosome entry-exit region suggest that modulation of nucleosome unwrapping is a mechanism for regulating transcription and DNA repair.
Subject(s)
Chromatin Assembly and Disassembly , DNA/chemistry , Nucleosomes/metabolism , Animals , Base Sequence , Binding Sites , DNA/metabolism , DNA-Binding Proteins/metabolism , Histones , MutS Homolog 2 Protein/metabolism , Nucleosomes/chemistry , Saccharomyces cerevisiae/genetics , Transcription Factors/metabolism , Xenopus laevisABSTRACT
The SWI/SNF chromatin remodeling complex changes the positions where nucleosomes are bound to DNA, exchanges out histone dimers, and disassembles nucleosomes. All of these activities depend on ATP hydrolysis by the catalytic subunit Snf2, containing a DNA-dependent ATPase domain. Here we examine the role of another domain in Snf2 called SnAC (Snf2 ATP coupling) that was shown previously to regulate the ATPase activity of SWI/SNF. We have found that SnAC has another function besides regulation of ATPase activity that is even more critical for nucleosome remodeling by SWI/SNF. We have found that deletion of the SnAC domain strongly uncouples ATP hydrolysis from nucleosome movement. Deletion of SnAC does not adversely affect the rate, processivity, or pulling force of SWI/SNF to translocate along free DNA in an ATP-dependent manner. The uncoupling of ATP hydrolysis from nucleosome movement is shown to be due to loss of SnAC binding to the histone surface of nucleosomes. While the SnAC domain targets both the ATPase domain and histones, the SnAC domain as a histone anchor plays a more critical role in remodeling because it is required to convert DNA translocation into nucleosome movement.
Subject(s)
Adenosine Triphosphatases/metabolism , Chromatin Assembly and Disassembly/genetics , Histones/metabolism , Nucleosomes/genetics , Saccharomyces cerevisiae Proteins/metabolism , Transcription Factors/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphate/metabolism , Animals , Chromosome Mapping , DNA, Fungal/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Deletion , Hydrolysis , Mutagenesis, Site-Directed , Protein Interaction Domains and Motifs , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Transcription Factors/genetics , Xenopus laevisABSTRACT
Histone post-translational modifications are essential for regulating and facilitating biological processes such as RNA transcription and DNA repair. Fifteen modifications are located in the DNA-histone dyad interface and include the acetylation of H3-K115 (H3-K115Ac) and H3-K122 (H3-K122Ac), but the functional consequences of these modifications are unknown. We have prepared semisynthetic histone H3 acetylated at Lys-115 and/or Lys-122 by expressed protein ligation and incorporated them into single nucleosomes. Competitive reconstitution analysis demonstrated that the acetylation of H3-K115 and H3-K122 reduces the free energy of histone octamer binding. Restriction enzyme kinetic analysis suggests that these histone modifications do not alter DNA accessibility near the sites of modification. However, acetylation of H3-K122 increases the rate of thermal repositioning. Remarkably, Lys --> Gln substitution mutations, which are used to mimic Lys acetylation, do not fully duplicate the effects of the H3-K115Ac or H3-K122Ac modifications. Our results are consistent with the conclusion that acetylation in the dyad interface reduces DNA-histone interaction(s), which may facilitate nucleosome repositioning and/or assembly/disassembly.