Date of Graduation
Doctor of Philosophy in Cell & Molecular Biology (PhD)
Ralph L. Henry
Second Committee Member
Gisela F. Erf
Third Committee Member
David S. McNabb
Breast Cancer, Chromatin Target of PRMT1 (CHTOP), Estrogen Receptor Alpha, Heat Shock Protein (Hsp), Histone Acetyltransferase (HAT), Protein Arginine Methyltransferase 5 (PRMT5)
Breast cancer is the second leading cause of cancer-related death in women, and approximately 70% of incidences are estrogen receptor (ER)-positive breast cancer. ERα and its interacting proteins play a key role in the development and progression of breast cancer. However, how ERα regulates its target gene expression and hence cell proliferation is not fully understood. To enhance our understanding of the molecular mechanism by which ERα regulates gene expression, we used a quantitative proteomic method to identify cellular proteins that interact with ERα. The first group of proteins that were identified to associate with ERα are heat shock proteins (Hsps). We identified 21 Hsps and 3 Hsp cochaperones that were associated with ERα. Co-immunoprecipitation assay demonstrated that Hsp70-1 and Hsc70, the two most abundant ERα-associated proteins, interacted with ERα in both transcriptionally active and inactive chromatin of MCF7 cells.
A novel of protein that was identified to interact with ERα is histone acetyltransferase 1 (HAT1). We showed that HAT1 physically binds ERα through the E domain of ERα, and silencing HAT1 by shRNA significantly increased the ERα-mediated transcription in MCF7 cells. Importantly, our data suggest that HAT1 regulates ERα transcriptional activity through affecting the interactions of ERα with histone proteins around the promoter region of ERα target genes in breast cancer cells.
We also identified and confirmed that protein arginine methyltransferase 5 (PRMT5) is a new ERα interacting partner, and PRMT5 interacts with ERα preferentially in the cytoplasm of MCF7 cells. Functionally, we found that overexpression of PRMT5 in MCF7 cells significantly decreased ERα transcriptional activity.
Finally, we demonstrated that chromatin target of PRMT1 (CHTOP) directly binds to ERα through the E domain of ERα. We found that knockout of CHTOP by CRISPR-Cas9 significantly decreased ERα transcriptional activity, and the effect is potentially through decreasing protein levels of MEP50, an ERα coactivator.
In summary, we identified and characterized several novel ERα-interacting proteins that play significant roles in regulating ERα transcriptional activities. Our results provide new insight into the molecular mechanisms by which ERα controls its target gene expression and regulates cell proliferation in ERα-positive cells.
Dhamad, A. E. (2017). Molecular and Biochemical Studies of Several Novel Estrogen Receptor Alpha-Interacting Proteins in Breast Cancer Cells. Graduate Theses and Dissertations Retrieved from https://scholarworks.uark.edu/etd/2403