Date of Graduation
12-2015
Document Type
Dissertation (PhD)
Program Affiliation
Genes and Development
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
Michelle Barton, PhD
Committee Member
Jae-Il Park, PhD
Committee Member
Min Gyu Lee, PhD
Committee Member
Mong-Hong Lee, PhD
Committee Member
Xiaobing Shi, PhD
Committee Member
Mark Bedford, PhD
Abstract
In this dissertation, I discovered that TRIM28 interacts with EZH2 and regulates its activity in breast cancer cells. EZH2, the major H3K27 tri-methyltransferase, is over expressed in multiple subtypes of breast cancer; however, whether EZH2 plays an active role in breast cancer progression or treatment resistance is unknown. Here, we determined EZH2-interacting proteins in MCF7 cells by immunoprecipitation/mass spectrometry and detected TRIM28 with a high confidence score. TRIM28 is a transcription co-regulator with an N-terminal tri-partite RING finger, B-boxes and a coiled-coil domain (RBCC) and a C-terminal PHD/Bromo domain. Domain mapping revealed that the RBCC domain of TRIM28 interacts with EZH2, and the pre-SET region (Cysteine-rich domain) of EZH2 interacts with TRIM28. Transcriptome profiling and intersection of EZH2- and TRIM28-regulated genes indicated co-regulation of differentiation and morphogenesis pathways, as well as co-activation of the cytokine-cytokine receptor interaction pathway (CXCR4, CXCL12, EGFR, KIT). TRIM28 depletion in MCF7 cells decreased EZH2 recruitment and CXCR4 gene expression, in parallel with decreased mammosphere formation. TRIM28 regulation of EZH2 association with SWI/SNF complex might contribute to this process. Collectively these data indicate that EZH2 and TRIM28 regulate breast cancer stem cell maintenance by transcriptional regulation of CXCR4 gene expression.
In part two, TRIM28 is overexpressed in a human mammary epithelial cells (HMEC) model of breast cancer progression; interestingly, TRIM28 is heavily phosphorylated (pS824/pS473) in the more aggressive stages of this model. Furthermore, ectopic expression of TRIM28 WT and phosphorylation defective forms in HMEC cells indicates that TRIM28 promotes cell proliferation in a phosphorylation-dependent manner. Genome-wide TRIM28 binding profile analysis revealed transcription factor binding motifs, including TCF4 and RBP-J (a Notch effector), at TRIM28 binding sites. The promoter of AXIN2, which encompasses a TCF4 binding motif, is a direct transcriptional activation target of TRIM28. Collectively, we demonstrated a mechanism of regulation of breast cancer cell proliferation by TRIM28 in a phosphorylation dependent manner via a TCF4 binding motif.
Keywords
EZH2, TRIM28, Breast Cancer