Defense Date

2012

Document Type

Thesis

Degree Name

Master of Science

Department

Microbiology & Immunology

First Advisor

Shirley Taylor

Abstract

Epigenetics is the study of heritable gene expression due to alterations in the DNA structure other than the underlying DNA sequence. DNA methylation is one of the three types of epigenetic modifications found in the eukaryotic system. It involves the incorporation of a methyl group at the 5-position of cytosine residues in the DNA. DNA methylation is associated with several notorious disorders and diseases including Fragile X Syndrome, neurodegenerative disease (Parkinson’s, Alzhiemer, etc), diabetes and cancer. Cytosine methylation of mitochondrial DNA (mtDNA) was first demonstrated several decades ago but the mechanism of generating cytosine modification and its functional importance remain elusive. Our laboratory recently demonstrated that the enzyme involved in cytosine modification of mtDNA is a novel mitochondrial isoform of DNA Methyltransferase 1, mtDNMT1. This protein is encoded in the nucleus and targeted to the mitochondria via a N-terminal targeting sequence. Bioinformatic analysis of the DNMT1 coding sequence showed a consensus NRF1 binding site that coincidently overlaps a p53 binding site within the promoter region, previously shown by this group to repress DNMT1 expression. Previous studies in the Taylor laboratory showed that mtDNMT protein expression was regulated by the transcription factor NRF1 as well as its coactivator PGC1α. PGC1α and NRF1 stimulate a large body of genes that are involved in mitochondrial biogenesis and cellular respiration in response to environmental stress. Considering the previous findings in our laboratory regarding mtDNMT1 regulation and the importance of PGC1α and NRF1 in oxidative homeostasis, we asked whether there is a mitochondrial epigenetic component in the cell’s response to cellular stress and whether up-regulation of mtDNMT1 might be part of the general response to this stress. To investigate the relationship between mtDNA methylation and oxidative homeostasis we examined the regulation of mtDNMT1 by transcription factors that respond to oxidative stress. Conditions that induced oxidative stress were applied to HCT 116 and SH-SY5Y cell lines and the protein expression of DNMT1 was observed. Ethanol and hypoxia- induced oxidative stress were observed to increase to protein level of mtDNMT1 while total DNMT1 level either remained constant or decreased. The protein level of PGC1α and NRF1 remained low in HCT 116 cells exposed to hypoxic stress, despite elevated mtDNMT1 protein level. ChIP analysis of HCT 116 cells exposed to hypoxic stress demonstrated that NRF1 and PGC1α are not regulating the transcription of DNMT1i in the mitochondria. However, we observed that p53 dissociated from the DNMT1 promoter upon hypoxic stress, indicating that the up-regulation of mtDNMT1 is through the relief of p53 suppression. The findings of this investigation proved that mtDNMT1 is receptive to oxidative stress through the regulation by p53 and suggested that mitochondrial epigenetics may be playing an integral role in the cellular stress response toward hypoxia.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

July 2012

Share

COinS