Histone acetylation and nucleosome dynamics

• Main Author: Venkataraman, Shanmugasundaram
• Published: University of Edinburgh 2001
• Subjects: 572.6
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.663209

In this report, I will describe purification of core histone octamers from chicken blood, HeLa nuclei and yeast cells, along with preparation of DNA fragments containing the 208 bp 5S rDNA gene and the adult beta (b^A)-globin gene promoter. <i>In vitro</i> experiments studying the effect of histone acetylation on the positioning and mobility of nucleosomes on the sea urchin 5S rDNA gene and the chicken b^A-globin gene promoter will be described. The former provides a well studied nucleosome positioning and mobility model system, while the latter is a developmentally regulated gene, with globin gene switching through the early stages of the lifetime of the chicken, and a proposed involvement of positioned nucleosomes in its regulation. The aim was to determine the difference between hypoacetylated and hyperacetylated core histones in terms of their influence upon nucleosome positioning and mobility. In earlier studies, it was noted that there was a difference in relative positioning intensities between the two forms (<i>ie.</i> hypoacetylated core histones preferentially positioned at certain sites, while hyperacetylated core histones positioned at the same sites but with different relative affinities). Therefore, acetylation affects where a nucleosomes is able to position. I have carried on this work to further characterize nucleosome positioning and to study the implications of histone acetylation on nucleosome mobility. I have found subtle differences in the thermodynamics and kinetics of hyperacetylated nucleosomes compared to hypeoacetylated nucleosomes: hyperacetylated nucleosomes appear to have a lower threshold in both these parameters when studied using the 208 pb rDNA fragment. Experiments involving two other types of core histones, trypsinized chicken core histone octamers and chicken core histone tetramers will also be described, which will be placed into the context of the results found with the other types of core histones. Finally, I will describe the effect of reconstituting hyperacetylated core histones with methylated DNA, long known to be a mediator of transcriptional repression, in the form of the chicken b^A-globin gene promoter.