Isolation and Characterization of Estrogen Receptor- Interacting Proteins

Author

Yun Kyoung Kang

Date of Award

2003

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Roeder Laboratory

Abstract

Estrogen fulfills a variety of physiological roles through its nuclear receptors, estrogen receptors (ERs). The action of ERs is modulated and mediated by diverse interacting cofactors. The initial aim of this thesis work was to identify ligand-, subtype- and/or cell typespecific ER-interacting proteins. I identified a number of nuclear extract-derived proteins that interact with immobilized ER ligand binding domains (LBDs) in a 17(3-estradiol (E2)-dependent manner. The most prominent of these are components of the thyroid hormone receptor associated protein (TRAP)/Mediator coactivator complex, which interacts with ERa and ER(3 both in unfractionated nuclear extracts and in purified form equally efficiently. Although some of other interacting proteins displayed subtype specificity, the main focus of the work was to characterize the function of TRAP/Mediator in ER-dependent transcription for both ERa and ER(3. Studies with extracts from TRAP220-/- mouse embryo fibroblasts (MEFs) revealed that the interactions between TRAP and ERs are dependent on TRAP22 0, a TRAP/Mediator subunit previously shown to interact with ER and other nuclear receptors in a ligand-dependent manner. The physiological relevance of the in vitro interaction was further documented by the isolation of an ERa- TRAP/Mediator complex from HeLa cells expressing an epitope-tagged ERa lacking AB domain (f:ERa,AAB). Most importantly, the complete TRAP/Mediator complex was shown to directly enhance ER function in a highly purified cell free transcription system. I further examined the roles for TRAP2 2 0 NR box in ER-TRAP/Mediator interaction. Both NR boxes were found to be important for ER-TRAP/Mediator interaction. Transient transfection assays with TRAP220-/- MEFs revealed that the absence of TRAP220 or mutations in either or both NR boxes attenuated modestly ER dependent transcription in TRAP220-/- MEFs. This indicated that TRAP220 and its NR boxes are required for the optimal transcrictipationl activity of ER. However, the observation of the significant E2 responses in TRAP220-/- MEFs suggested the existence of alternative pathways for E2 responses different from TRA/Mediator or the absence of natural constraints that impose a requirement for TRAP220 to overcome. Similarly, cell-free transcription assays also showed modest defect in function of TRAP/Mediator for ER dependent transcription when TRAP/Mediator contains TRAP2 2 0 NR mutants.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Recommended Citation

Kang, Yun Kyoung, "Isolation and Characterization of Estrogen Receptor- Interacting Proteins" (2003). Student Theses and Dissertations. 378.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/378