Amanda Gefroh

Date of Award

January 2013

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biomedical Sciences

First Advisor

Barry Milavetz

Abstract

Some of the most rapidly-evolving fields of study in the medical and research communities presently are those which investigate our genetic code, the elements and factors that control the expression of our genetic code, as well as fields of study which analyze theories about an organisms' physiology and phenotype from the information that both of the previous topics concomitantly provide. The work of the Milavetz lab, among many others, is striving to fill the gaps in knowledge about epigenetic regulation of biological processes - here by using the well-suited model virus, Simian Virus 40. It has for some time now been agreed upon that epigenetic modifications of histone proteins not only exists, but play a crucial role in biological processes related to the availability or condensation of genetic material. Certain modifications have continuously been shown to either silence or expose genes1,2. It has also been shown that these modifications are not static but in a dynamic state3. However, the details of how, when, and why changes in the epigenetic modifications on the histone tails occurs is still being investigated. This project is intended to explore the effect of novel sequence inserts into the SV40 genome related to the enhancer region, Sp1 binding site, and late promoter. Specific variants of SV40 were created to contain either one or two copies of the enhancer region (T-129 and T-143 respectively), two copies in succession of the Sp1 site (RH-43), or a full late promoter region (T-165). Chromatin immunoprecipitation (ChIP) and real-time quantitative polymerase chain reaction (q-PCR) of the histone modifications H3K4me1/2/3, H3K9me1/2/3, H4K20me1 and RNAPII were analyzed. We found that certain modifications were affected by each sequence addition - some by a notable increase and some by a notable decrease. While more data is needed to more wholly characterize this phenomenon, these are some of the first results indicating an association between the DNA sequence present and the regulation of epigenetic histone modifications.

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