Date of Graduation

8-2016

Document Type

Dissertation (PhD)

Program Affiliation

Epigenetics and Molecular Carcinogenesis

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Sharon Y.R. Dent

Committee Member

Mark Bedford

Committee Member

Taiping Chen

Committee Member

Rick A. Finch

Committee Member

Xiaobing Shi

Abstract

Molecular profiling has identified 5 distinct subtypes of breast cancer, luminal A, luminal B, HER2-enriched, basal-like, and claudin-low breast cancer. These 5 subtypes correlate with hormone response, patient prognosis, and response to therapy. Although steady state gene expression patterns have been explored using expression microarrays, very little is known about the initial, disease-driving transcriptional changes in these cancers or epigenetic changes associated with the differential gene expression signatures. Defining these changes may provide new insights into the mechanisms by which these subtypes arise, as well as new avenues for breast cancer prevention, diagnosis, and treatment. Using Chromatin Immunoprecipitation sequencing and immunoblot technology, we investigated the genome wide profiling and global levels of eleven histone modifications localizing at promoter, gene body, and enhancer in distinct subtypes of breast cancer cell lines. With this knowledge, we will address how changes in chromatin modification landscape contribute to differential breast cancer subtypes, and identify the subtype-specific features and responses to gene regulation.

By examining the global levels of histone modifications in distinct subtypes, I found that H2Bub1 level is lower in claudin-low breast cancer cell lines compared with basal-like breast cancer cell lines. However, the mRNA levels of deubiquitinases and E3 ligases targeting H2Bub1 have no significant difference between basal-like and claudin-low subtypes. By analyzing the differential H2Bub1-enriched genes in claudin-low and basal-like subtypes, we found that miR200 family is highly enriched for H2Bub1 and has higher levels of expression in basal-like cells. miR200b overexpression decreases cell proliferation and colony formation specifically in claudin-low cells.

The main deubiquitinase for H2Bub1 is USP22 in the DUB module of the SAGA complex. USP22 is the catalytic subunit of the DUB module. Two adaptor proteins, ATXN7L3 and ENY2, are necessary for DUB activity of USP22 towards histone H2Bub1 and other substrates. ATXN7L3B shares 74% identity with the N-terminal region of ATXN7L3, but the functions of ATXN7L3B are not known. Here we report that ATXN7L3B interacts with ENY2 and USP22, but not other SAGA components. Even though ATXN7L3B localizes in the cytoplasm, ATXN7L3B overexpression increases H2Bub1 levels, while overexpression of ATXN7L3 decreases H2Bub1 levels. In vitro, ATXN7L3B competes with ATXN7L3 to bind ENY2, and in vivo, knockdown of ATXN7L3B leads to concomitant loss of ENY2. Unlike the ATXN7L3 DUB complex, the USP22-ATXN7L3B-ENY2 complex cannot deubiquitinate H2Bub1 efficiently in vitro. Moreover, ATXN7L3B knockdown inhibits migration of breast cancer cells in vitro and limits expression of ER target genes. Collectively our studies suggest that ATXN7L3B may influence breast cancer cell behavior through effects on H2Bub1 levels and SAGA DUB activity.

From analyzing the histone modification ChIP-seq result, we found that specific histone modification patterns that are characteristic of five distinct breast cancer subtypes. We also identified subtype specific genes with unique chromatin signatures and expression patterns. For example, the AFAP1-AS1 gene locus is highly enriched in H3K9ac and H3K79me2 active transcription markers in TNBC cell lines. In addition, AFAP1-AS1 expression levels are higher in triple negative cells and tumors. Moreover, silencing of AFAP1-AS1 decreases cell proliferation and colony formation of TNBC cells.

Overall, these studies confirm and extend the importance of histone modifications to gene regulation in breast cancer and may provide new avenues for therapy development in the future.

Keywords

breast cancer; TNBC; H2Bub1; claudin-low; ATXN7L3B; ENY2; SAGA; epigenetics;

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