Year of Publication

2017

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department

Molecular and Cellular Biochemistry

First Advisor

Dr. Yvonne Fondufe-Mittendorf

Abstract

Eukaryotic organisms contain their entire genome in the nucleus of their cells. In order to fit within the nucleus, genomic DNA wraps into nucleosomes, the basic, repeating unit of chromatin. Nucleosomes wrap around each other to form higher order chromatin structures. Here we study many factors that affect, or are effected by, chromatin structure including: (1) how low-dose inorganic arsenic (iAs) changes chromatin structures and their relation to global transcription and splicing patterns, and (2) how chromatin architectural proteins (CAPs) bind to and change nucleosome dynamics and DNA target site accessibility.

Despite iAs’s non-mutagenic nature, chronic exposure to low doses of iAs is associated with a higher risk of skin, lung, and bladder cancers. We sought to identify the genome-wide changes to chromatin structure and splicing profiles behind the cell’s adaptive response to iAs and its removal. Furthermore, we extended our investigation into cells that had the iAs insult removed. Our results show that the iAs-induced epithelial to mesenchymal transition and changes to the transcriptome are coupled with changes to the higher order chromatin structure and CAP binding patterns. We hypothesize that CAPs, which bind the entry/exit and linker DNA of nucleosomes, regulate DNA target site accessibility by altering of the rate of spontaneous dissociation of DNA from nucleosome.

Therefore, we investigated the effects of the repressive CAP histone H1, the activating CAP high mobility group D1 (HMGD1), and the neural CAP methyl CpG binding protein 2 (MeCP2) on the dynamics of short chromatin arrays and mononucleosomes and their effect on nucleosomal DNA accessibility. Using biochemical and biophysical analyses we show that all CAP-chromatin structures tested were susceptible to chromatin remodeling by ISWI and created more stable higher order structures than if CAPs were absent. Additionally, histone H1 and MeCP2 hinder model transcription factor Gal4 from binding its cognate DNA site within nucleosomal DNA.

Overall, we show that chromatin structure is dynamic and changes in response to environmental signals and that CAPs change nucleosome dynamics that help to regulate chromatin structures and impact transcriptional profiles.

Digital Object Identifier (DOI)

https://doi.org/10.13023/ETD.2017.185

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