Publication Date

2010-08-06

Availability

Open access

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Biochemistry and Molecular Biology (Medicine)

Date of Defense

2010-07-16

First Committee Member

T.K. Harris - Committee Chair

Second Committee Member

Terace Fletcher - Committee Member

Third Committee Member

Sharon J. Elliot - Committee Member

Fourth Committee Member

Zafar Nawaz - Mentor

Fifth Committee Member

Kerry Burnstein - Outside Committee Member

Abstract

The Estrogen Receptor alpha (ER alpha) is a multi-domain transcription factor that has been extensively studied due to its known involvement in breast cancer treatment and progression. Subsequent studies have shown coregulators are extensively involved in modulating the transcriptional activation of ER alpha and many of these proteins possess enzymatic functions. Coregulators are divided into two categories, coactivators which enhance transcriptional output and corepressors which decrease transcriptional output. One protein responsible for Angelman syndrome, E6-associated protein (E6-AP) was found to be a coactivator of ER alpha and possessed ubiquitin ligase activity; however, the ubiquitin ligase activity has been shown not to be essential to E6-AP coactivation ability. The current work was undertaken to explore the role of E6-AP in the regulation of ER alpha. E6-AP was found to play a role in a unique ligand-independent degradation pathway. Because the degradation effect was ligand-independent, it was proposed that the degradation signal mediating the event occurred through phosphorylation of E6-AP. In silico analysis of E6-AP indicated several potential phosphorylation sites on the E6-AP protein. Numerous phosphorylation sites of E6-AP were confirmed by western blot and mass spec indicating a possible phosphorylation signal mediating E6-AP/ER alpha interaction. Because it has been shown that the ligase function of E6-AP is not required for its coactivation, we then examined E6-AP coactivation of ERα in the presence of ligand. One well studied gene TFF-1 (pS2) was examined as a model ERα target gene. Estrogen-mediated transcription from TFF-1 was decreased with knockdown of E6-AP in both MCF-7 and T47D cell lines. Furthermore, under E6-AP knockdown conditions, ChIP of p300, a known histone acetyl transferase (HAT), indicated a reduced recruitment to the TFF-1 promoter in both cell lines. Interestingly, the reduced recruitment of p300 had a cell specific effect on phosphorylated RNA polymerase II (pRNA pol II) recruitment indicating cell specific functions of E6-AP. Further investigation also found a gene specific effect for E6-AP on pRNA pol II recruitment. The current work provides a new role for E6-AP as a coactivator of ER alpha in the form of a scaffold allowing creation of fully functional transcription complexes in a gene and cell specific manner.

Keywords

Degradation; Ubiquitination; Transcription

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