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|Title:||Biochemical and biophysical investigations of the dependences of chromatin/nucleosome structure on linker histone and histone H4K20-trimethylation||Authors:||Wang, Sai||Keywords:||DRNTU::Science::Biological sciences||Issue Date:||2018||Source:||Wang, S. (2018). Biochemical and biophysical investigations of the dependences of chromatin/nucleosome structure on linker histone and histone H4K20-trimethylation. Doctoral thesis, Nanyang Technological University, Singapore.||Abstract:||The dynamic structure of chromatin plays critical roles in genomic activities within living cells. Chromatin has different levels of compaction to cope with various cellular activities. The structure of chromatin could be loose euchromatin to expose DNA for the access of molecular machines during DNA repair, replication or gene transcription, or condensed heterochromatin in non-coding regions and chromosomes during cell mitosis. Various factors contribute to the formation and maintenance of the diversity of chromatin structures. This thesis investigates structural and biophysical properties of the primary building blocks of eukaryotic chromatin: nucleosome, chromatosome (complex of nucleosome and linker histone), and nucleosome array with techniques of cryo-electron microscopy single particle analysis (cryo-EM), X-Ray diffraction, small-angle X-ray scattering (SAXS), precipitation assay (PA), and analytical ultracentrifugation (AUC). Cryo-EM method is used to determine the structure of 177 bp chromatosome at 11 Å resolution. The two 15 bp linker DNA arms are positioned close to each other, together with the nearby nucleosome dyad region, forming a triangle area. The densities in the middle of this area are suggested to be from the bound linker histone. The linker histone C-terminal domain (CTD) is proposed to localize in the vicinity of the two linker DNA ends. The data supports the idea that the highly positively charged CTD acts as an “electrostatic bridge” to screen the negative charges of the two linker DNA leading to a more rigid and compacted overall structure. Self-association and resolubilization of the 177 bp chromatosome and 177 bp nucleosome in the presence of Mg2+ and cobalt (III) hexammine (CoHex3+) was studied by precipitation assay. Aggregation of the chromatosome proceeds at lower concentrations of Mg2+ and CoHex3+ in agreement with electrostatic rules (presence of linker histone decreases chromatosome negative charge by 25 %). Resolubilization of 177 bp nucleosome and chromatosome was observed at high concentration of Mg2+. Binding of linker histone increased stability of the aggregated state, thus higher amount of Mg2+ is required for resolubilization. The linker histone and Mg2+ coordinated regulation delivers possible biological significance in control of chromatin accessibility. X-Ray diffraction of the chromatosome precipitates allowed determination of dimensions of the 177 bp chromatosome (thickness 58.7 Å, diameter 111.2 Å). The phase of precipitated chromatosomes showed presence of bilayer organization with bilayer-bilayer distance dramatically different from the one reported in literature for the bilayers of 145-148 bp nucleosomes. The difference in bilayer-bilayer distance reflects distinct features of chromatosome shape and binding of the linker histone. Solution SAXS is applied to calculate the radius of gyration (Rg) of 177 bp nucleosome and chromatosome and to evaluate the degree of unwrapping of nucleosomal DNA. The larger Rg value of 177 bp chromatosome compare to the nucleosome corresponds to the increase of the chromatosome size caused by binding of linker histone. The deeper characteristic unwrapping dip in the chromatosome spectrum compared to that of the nucleosome suggests full wrapping of DNA in chromatosome and some degree of unwrapping in nucleosome. The results indicate that repulsion of the linker DNAs in the nucleosome might cause partial DNA unwrapping. Presence of the linker histone reduces DNA-DNA repulsion and increases DNA wrapping. AUC was applied to study the influence of heterochromatin-specific histone modification, trimethylation of Lys20 of the histone H4, on folding of the 12-177 nucleosome arrays. Both unmodified and modified (H4K20Me3) arrays achieved maximal compaction to ~50 S-value, and showed a tendency of self-association. Similar folding properties of the unmodified and H4K20Me3 arrays support the idea that while H4K20Me3 promotes the compaction of nucleosome arrays with longer NRLs, it has no additive effect on the salt-dependent folding of nucleosome arrays with shorter NRLs. Self-association of the modified (H4K20Me3) and unmodified 12-177 nucleosome array in the presence of Mg2+ and CoHex3+ was studied by precipitation assay. No difference was observed in the salt-induced precipitation between these two arrays. In coincidence with 177 bp nucleosome and chromatosome, Mg2+ induced resolubilization was observed for both modified and unmodified nucleosome arrays. The resolubilization processes were almost the same between the two arrays. Together, the results indicate that H4K20Me3 has no influence in the 12-177 nucleosome array self-association. In the electrostatic view, the similar polyelectrolyte properties of the modified and unmodified 12-177 nucleosome arrays provide explanations to the observations above.||URI:||http://hdl.handle.net/10356/73338||DOI:||10.32657/10356/73338||Fulltext Permission:||open||Fulltext Availability:||With Fulltext|
|Appears in Collections:||SBS Theses|
Updated on May 7, 2021
Updated on May 7, 2021
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