ABSTRACT
Phosphorylation of histone H3 threonine 118 (H3 T118ph) weakens histone DNA-contacts, disrupting the nucleosome structure. We show that Aurora-A mediated H3 T118ph occurs at pericentromeres and chromosome arms during prophase and is lost upon chromosome alignment. Expression of H3 T118E or H3 T118I (a SIN mutation that bypasses the need for the ATP-dependent nucleosome remodeler SWI/SNF) leads to mitotic problems including defects in spindle attachment, delayed cytokinesis, reduced chromatin packaging, cohesion loss, cohesin and condensin I loss in human cells. In agreement, overexpression of Aurora-A leads to increased H3 T118ph levels, causing cohesion loss, and reduced levels of cohesin and condensin I on chromatin. Normal levels of H3 T118ph are important because it is required for development in fruit flies. We propose that H3 T118ph alters the chromatin structure during specific phases of mitosis to promote timely condensin I and cohesin disassociation, which is essential for effective chromosome segregation.
Subject(s)
Adenosine Triphosphatases/metabolism , Aurora Kinase A/metabolism , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA-Binding Proteins/metabolism , Histones/metabolism , Mitosis , Multiprotein Complexes/metabolism , Protein Processing, Post-Translational , Threonine/metabolism , Animals , Cell Line , DNA/metabolism , Drosophila , Humans , Phosphorylation , CohesinsABSTRACT
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
Nucleosomes are stable DNA-histone protein complexes that must be unwrapped and disassembled for genome expression, replication, and repair. Histone posttranslational modifications (PTMs) are major regulatory factors of these nucleosome structural changes, but the molecular mechanisms associated with PTM function remains poorly understood. Here we demonstrate that histone PTMs within distinct structured regions of the nucleosome directly regulate the inherent dynamic properties of the nucleosome. Precise PTMs were introduced into nucleosomes by chemical ligation. Single molecule magnetic tweezers measurements determined that only PTMs near the nucleosome dyad increase the rate of histone release in unwrapped nucleosomes. In contrast, FRET and restriction enzyme analysis reveal that only PTMs throughout the DNA entry-exit region increase unwrapping and enhance transcription factor binding to nucleosomal DNA. These results demonstrate that PTMs in separate structural regions of the nucleosome control distinct dynamic events, where the dyad regulates disassembly while the DNA entry-exit region regulates unwrapping. These studies are consistent with the conclusion that histone PTMs may independently influence nucleosome dynamics and associated chromatin functions.
Subject(s)
DNA/metabolism , Histones/metabolism , Lysine/metabolism , Nucleosomes/metabolism , Acetylation , Algorithms , Animals , DNA/chemistry , DNA/genetics , Electrophoretic Mobility Shift Assay , Fluorescence Resonance Energy Transfer , Histones/chemistry , Histones/genetics , Kinetics , Lysine/chemistry , Lysine/genetics , Microscopy, Atomic Force , Models, Molecular , Mutation , Nucleic Acid Conformation , Nucleosomes/genetics , Protein Binding , Protein Multimerization , Protein Processing, Post-Translational , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolismABSTRACT
Nucleosomes, the fundamental units of chromatin structure, are regulators and barriers to transcription, replication and repair. Post-translational modifications (PTMs) of the histone proteins within nucleosomes regulate these DNA processes. Histone H3(T118) is a site of phosphorylation [H3(T118ph)] and is implicated in regulation of transcription and DNA repair. We prepared H3(T118ph) by expressed protein ligation and determined its influence on nucleosome dynamics. We find H3(T118ph) reduces DNA-histone binding by 2 kcal/mol, increases nucleosome mobility by 28-fold and increases DNA accessibility near the dyad region by 6-fold. Moreover, H3(T118ph) increases the rate of hMSH2-hMSH6 nucleosome disassembly and enables nucleosome disassembly by the SWI/SNF chromatin remodeler. These studies suggest that H3(T118ph) directly enhances and may reprogram chromatin remodeling reactions.