Date of Award

4-18-2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Barry Milavetz

Abstract

Compaction of DNA by histone proteins helps to package the DNA into the nucleus at the expense of acting as a bather to biological processes that require access to the DNA. Addition and removal of acetyl groups from the histone tails with the help of enzymes known as histone acetyltransferases (HATs) and histone deacetylases (HDACs) appear to play a critical role in maintaining the structure of chromosomes and the regulation of gene expression. In order to better understand the role of histone hyperacetylation in the regulation of gene expression I studied histone hyperacetylation in Simian Virus 40 (SV40) chromosomes during the course of a lytic infection using modified chromatin immunoprecipitation techniques.Histone hyperacetylation in transcribing SV40 chromosomes was characterized utilizing a strategy in which the SV40 chromosomes undergoing transcription (operationally defined by the presence of RNAPII) were immune-selected with antibody to RNAPII and subjected to secondary chromatin immunoprecipitation with antibodies to hyperacetylated or unacetylated H4 or H3, Immune Selection Fragmentation and Immunoprecipitation (ISFIP) was used to determine the hyperacetylation status of histones independent of the location of the RNAPII and ReChromatin lmmunoprecipitation (ReChIP) was used to determine their hyperacetylation status when associated with RNAPII. While hyperacetylated H4 and H3 were found in the coding regions regardless of the location of RNAPII, unacetylated H4 and H3 were only found at sites lacking RNAPII. The absence of unacetylated H4 and H3 at sites containing RNAPII correlated with the specific association of the Histone Acetyl Transferase (HAT) p300 with RNAPII. In contrast, the presence of unacetylated H4 and H3 at sites lacking RNAPII was shown to result from the action of a histone deacetylase (HDAC) based upon the effects of the inhibitor sodium butyrate. These results suggest that the extent of hyperacetylation of H4 and H3 during transcription alternates between hyperacetylation directed by an RNAPII-associated HAT and deacetylation directed by an HDAC at other sites. By blocking translocation of RNA Polymerase II using specific inhibitors I have shown that there is a significant increase in the amount of unmodified H4 and H3 and in the case of inhibition by alpha amanitin also the loss of p300 from the RNAPII complex. In order to confirm that p300 plays a critical role in SV40 transcription, I used siRNA to knockdown the expression of p300 and showed that there is little if any SV40 late transcription. I believe that these results demonstrate that histone hyperacetylation is dynamic in the coding region of genes with p300 causing the hyperacetylation and an as yet unknown HDAC responsible for the observed deacetylation.

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