Defense Date

2016

Document Type

Thesis

Degree Name

Master of Science

Department

Molecular Biology and Genetics

First Advisor

Shirley Taylor

Second Advisor

Rita Shiang

Third Advisor

Mike McVoy

Fourth Advisor

Mike Groteweil

Abstract

The field of epigenetics is gaining popularity and speed, due in part to its capability to answer lingering questions about the root cause of certain diseases. Epigenetics plays a crucial role in regulation of the cell and cell survival, particularly by cytosine methylation. It remains controversial if DNMT’s which facilitate methylation are present in mammalian mitochondria and what the functional significance they may have on modification of mitochondrial DNA. CRISPR-Cas9 technology enabled genome editing to remove the MTS (mitochondrial targeting sequence) from DNMT1 of HCT116 cells, purposefully minimizing effects on nuclear cytosine methylation, while exclusively impacting mitochondrial modification. Removal of the DNMT1 MTS did not completely prevent the localization of this enzyme to the mitochondria according to immunoblot analysis. As well, deletion of the MTS in DNMT1 revealed only a small decline in transcription; not until removal of DNMT3B did we see a two-fold decrease in transcription from mitochondrial protein coding genes. No significant decline in transcription occurred when a DNMT3B knockout also lost the MTS of DNMT1; this study is evidencing that DNMT3B is possibly the more significant methyltransferase in the mitochondria. Our aim from this study and future research is to clearly characterize which enzymes in the mitochondria are controlling cytosine modifications and to understand the mechanistic complexities that accompany cause and consequence of epigenetic modifications.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

10-10-2016

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