The role of post translational modifications in the regulation of binding of linker histone Hho1 to chromatin in Saccharomyces cerevisiae

[摘要] The eukaryotic genome is functionally organized into a highly ordered nucleoproteinstructure, chromatin. Apart from the four nucleosomal core histones, the linker histone ispivotal to fluidity and compaction of chromatin. Yeast, a single cell eukaryote, has a shortnucleosomal repeat length, and possesses a structurally unique H1. This thesis is an attemptto study the influence of post translational modifications in regulating the interactions ofyeast linker histone with chromatin. Chapter 1 gives a broad overview of available literature.Chapter 2 presents the results from cloning, expression, purification and partialcharacterization of Hho1p. The physiochemical characteristics of recombinant protein werestudied with a view to perform subsequent purification of native Hho1p from yeast strains.The solubility and precipitation of rHho1p were explored under several conditions. Unlikecanonical linker histone, Hho1p was found to be insoluble in Perchloric acid. Chapter 3comprises generation and characterization of polyclonal antibody against Hho1p. Affinitypurified monospecific antibodies were used to optimize semi-quantitative Western blotting.Preliminary results from Western blotting analysis suggest that Hho1p is capable ofoligomeric interactions. Chapter 4 presents results for optimization of conditions for Hho1pextraction from yeast cells or nuclei. For nuclear extract preparation, a standard methodand its rapid variation, developed to limit Hho1p proteolysis, were used. The acid extractionof Hho1p from nuclei followed by its purification by reverse phase chromatographyprovided low yield for downstream analysis. However, sufficient quantities of native Hho1pcould be extracted from cell lysate using affinity matrices prepared with the monospecificαHho1p antibodies generated in-house. Several novel post translational modification sitesand binding partners of Hho1p were identified from both logarithmic and stationary phaseof yeast cell growth, providing an insight into the stage specific regulation of Hho1pchromatin binding.

[发布机构] University of the Free State