Interactions of Steroid Hormone Receptors With Coactivators and DNA

II Steroid hormone receptors (HR) activate gene transcription through binding to ligands, locating specific hormone response elements (HREs) on DNA, recruiting coactivators and then chromatin remodeling complexes and ultimately the basal transcription machinery. In vivo data suggests that the components of transcription complexes at HREs undergo rapid exchange and replacement. Using our Fluorescence Anisotropy Microplate Assay (FAMA), we studied the interaction of HR with HRE and coactivators quantitatively and dynamically. HR dissociated from fluorescein-labeled (fl) HRE with a half time of about an hour. The dissociation processes can be accelerated dramatically and the half-life of the complex shortened to less than a minute by adding increasing concentrations of unlabeled HRE. The data indicates the movement of HR among HREs is not a passive process, in which the receptor physically dissociates from one DNA binding site, goes into solution and then rebinds to another DNA site. Instead, rapid receptor movement results from a dynamic process in which a new DNA segment actively displaces the original DNA segment. This dynamic replacement process extends to coactivator exchange. Dissociation of full-length steroid receptor coactivator1 (SRC1) from estrogen receptor (ER)-fl consensus estrogen response elements (cEREs) complex has a relatively short half time of 3-4 minutes LxxLL motif containing peptides actively displace SRC1a from ER-flcERE complex and increase the rate of dissociation of SRC1 from ER by 5-20 fold. Therefore, active displacement plays a central role in coactivator-ER interactions. Coactivator-ER interactions and ER-ERE interactions are not completely independent. Only agonist bound ER-ERE, but not antagonist bound ER-ERE, recruits SRC. Full-length SRC1, SRC2 and nuclear receptor interaction domain (NID) of SRC3 all dramatically enhance ER binding to the protease inhibitor 9 (PI-9) estrogen response unit (ERU), but not to the cERE or PS2 ERE. Excess NID reduces this increase. We propose the novel hypothesis that coactivators can act as bridging or scaffolding factors using their multiple LxxLL domains to link ER dimers bound at the two ER binding sites in the PI-9 ERU, thereby forming a larger and more stable complex and thus enhancing ER binding. In conclusion, steroid hormone receptor binding to DNA and coactivator binding to receptor are dynamic and interactive.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2015

Author dc:creator

• Zhang, Chen

Contributors dc:contributor

• Shapiro, David J.

• Chemistry, Biochemistry

Language dc:language

eng

Identifier

(MiAaPQ)AAI3347568

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/84861