Imran Hussain

Graduation Semester and Year

2012

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry

Department

Chemistry and Biochemistry

First Advisor

Subhrangsu Mandal

Abstract

Homeobox (HOX) genes are highly conserved family of genes that play vital roles during cell differentiation and embryogenesis. Of the 39 HOX genes present in human, HOXC6 is a critical players in mammary gland development, milk production and is over-expressed in breast and prostate cancer. I demonstrated that HOXC6 is transcriptionally regulated by estrogen (E2). The HOXC6 promoter contains two putative estrogen response elements (EREs). Promoter analysis using luciferase-based reporter assay demonstrated that both EREs are responsive to E2, ERE1half being more responsive than ERE2half. Estrogen receptors alpha and beta (ERα and ERβ) bind to these EREs in an E2-dependent manner and antisense-mediated knockdown of ERs suppressed the E2-dependent activation of HOXC6 expression. Similarly, knockdown of mixed lineage leukemia (MLL) histone methylases, MLL2 and MLL3, decreased E2-mediated activation of HOXC6. However, depletion of MLL1 or MLL4 showed no significant effect. MLL2 and MLL3 were bound to the HOXC6 EREs in an E2-dependent manner. In contrast, MLL1 and MLL4 that were bound to the HOXC6 promoter in the absence of E2, decreased upon exposure to E2. MLL2 and MLL3 play key roles in histone H3K4-trimethylation and recruitment of general transcription factors and RNA polymerase II (RNAPII) in the HOXC6 promoter during E2-dependent transactivation. Nuclear receptor corepressors, N-CoR and SAFB1, were bound to the HOXC6 promoter in absence of E2 and that binding were decreased upon E2 treatment indicating their critical role in suppressing HOXC6 gene expression under non-activated condition. Furthermore, I examined the expression of HOXC6 in breast cancer tissue and its gene expression status in MCF7 cells in presence of endocrine disrupting chemicals such as bisphenol-A (BPA) and diethylstilbestrol (DES). My studies demonstrated that HOXC6 is differentially over-expressed in breast cancer tissue. Similar to E2, HOXC6 expression is also transcriptionally induced by BPA and DES. MLL histone methylases MLL2 and MLL3 coordinate with ERs, bind to the HOXC6 promoter upon exposure to BPA and DES, increase histone H3K4-trimethylation and recruitment of RNAPII to the HOXC6 promoter and transcriptionally activates HOXC6 expression. In another study, roles of E2 and E2-like compounds (BPA and DES) in regulation of another HOX gene HOXA5 has been studied. HOXA5 expression is important for the development of lung, gastro-intestinal tract and vertebrae. My studies demonstrated that HOXA5 is over-expressed in breast cancer patient tissue and ER-positive breast cancer cells. HOXA5 expression is critical for cell viability as shown by flow cytometry, TUNEL and growth assay. Similar to E2, HOXA5 expression is also transcriptionally induced by BPA and DES. Promoter analysis confirms three EREhalf sites within 1000 nt upstream of the transcription start site but only ERE1half and ERE2half were found induced by E2, BPA and DES using luciferase-based reporter assay. MLL histone methylases MLL2 and MLL3 coordinate with ERs, bind to the HOXA5 promoter upon exposure to E2, BPA and DES, increase histone H3K4-trimethylation, histone acetylation and recruitment of RNAPII to the HOXA5 promoter and transcriptionally activate HOXA5 gene expression.

Disciplines

Chemistry | Physical Sciences and Mathematics

Comments

Degree granted by The University of Texas at Arlington

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