Molecular and Biochemical Studies of Several Novel Estrogen Receptor Alpha-Interacting Proteins in Breast Cancer Cells

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. Acknowledgments Firstly, I would gratefully like to thank Dr. Yuchun Du, my academic advisor, for providing me an opportunity to join his lab. Dr. Du has taught and guided me in all the time of research and writing of this dissertation more than I could ever give him credit for here. His support and encouragement helped me a lot to overcome the issues and difficulties that I encountered in this project. I also would especially like to thank the rest of my dissertation committee members Dr. David S. McNabb, Dr. Gisela F. Erf, and Dr. Ralph Henry for their support, advice, and guidance. My sincere thanks go to former and current Dr. Du’s laboratory members, including Jianhong Zhou and Zhenqi Zhou who taught and helped me to learn some techniques, and without their precious support it would be so hard to conduct this research. And, I would like to thank undergrad student Hunter, Anna, Susana, and Abbi and all my friends for their supports. I would like to thank Biological Sciences and CEMB program at the University of Arkansas, my sponsor HCED, NIH, and ABI for all supports including financial support. Last but not the least, I would like to deeply thank my family: my parents, brothers, and sisters for endless supports and encouragements. Very special thanks go to my great wife Muna who always supports and stands beside me throughout all my life aspects no matter how things are going. I also thank my wonderful kids: Noor and Jaafar.