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TWO FACED COREGULATOR: COACTIVATION AND COREPRESSION OF TRANSCRIPTION BY HISTONE METHYLTRANSFERASE G9A
by
Daniel Purcell
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETIC, MOLECULAR AND CELLULAR BIOLOGY)
December 2011
Copyright 2011 Daniel Purcell
Object Description
| Title | Two faced coregulator: coactivation and corepression of transcription by histone methyltransferase G9a |
| Author | Purcell, Daniel J. |
| Author email | dpurcell@usc.edu;daniel.purcell.phd@gmail.com |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Genetic, Molecular and Cellular Biology |
| School | Keck School of Medicine |
| Date defended/completed | 2011-10-04 |
| Date submitted | 2011-10-20 |
| Date approved | 2011-10-21 |
| Restricted until | 2012-04-20 |
| Date published | 2012-04-20 |
| Advisor (committee chair) | Stallcup, Michael R. |
| Advisor (committee member) |
An, Woojin Dubeau, Louis |
| Abstract | Histone methyltransferase G9a has been primarily understood as a corepressor of gene expression, but we showed previously that G9a positively regulates nuclear receptor-mediated transcription in reporter gene assays. Here we show that endogenous G9a contributes to the estradiol (E2)-dependent induction of some endogenous target genes of estrogen receptor (ER) in MCF-7 breast cancer cells while simultaneously limiting the E2-induced expression of other ER target genes. Thus G9a has a dual and selective role as a coregulator for ER target genes. The ERα binding regions associated with the pS2 gene, which requires G9a for E2-induced expression, are transiently occupied by G9a at 15 min after beginning E2 treatment, suggesting that G9a coactivator function is by direct interaction with ERα target genes. Using transient reporter gene assays with deletion mutants of G9a, we demonstrated that domains previously associated with the corepressor functions of G9a (C-terminal SET domain, ankyrin repeat domain, and cysteine-rich domain) were unnecessary for G9a coactivator function in ERα mediated transcription. In contrast, our study shows that the N-terminal domain of G9a was necessary and sufficient for enhancement of ERα-mediated transcription and for E2-induced occupancy of G9a on ERα binding sites associated with the pS2 gene. In addition to a previously identified activation domain, we discovered a previously uncharacterized ligand-dependent ER binding function in this region, indicating how G9a is recruited to the target genes. Therefore, the coactivator and corepressor functions of G9a involve different G9a domains and different molecular mechanisms. ❧ We also show that G9a acts as a transcriptional coactivator of Runx2 mediated transcription. This activation of Runx2 transcription does not require the methyltransferase activity of G9a, and Runx2 is associated with G9a in vitro. In prostate cancer cells metastasized to bone (C4-2B), we depleted G9a and revealed that various Runx2 inducible genes displayed G9a dependence, G9a independence and G9a repression. We demonstrated Runx2 colocalization with G9a and the ability to mobilize formerly immobile fractions of G9a in the cell nucleus. However, the effect of G9a depletion did not appear to reduce Runx2 dependent invasion or migration of C4-2B cells. Occupancy by G9a on the Runx2 binding site of the CSF2 gene during Runx2 induction indicates a direct role for G9a in Runx2 mediated transcription. |
| Keyword | estrogen receptor; breast cancer; coregulator; G9a; transcription; chromatin |
| Language | English |
| Part of collection | University of Southern California dissertations and theses |
| Publisher (of the original version) | University of Southern California |
| Place of publication (of the original version) | Los Angeles, California |
| Publisher (of the digital version) | University of Southern California. Libraries |
| Provenance | Electronically uploaded by the author |
| Type | texts |
| Legacy record ID | usctheses-m |
| Rights | Purcell, Daniel J. |
| Access conditions | The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright. The original signature page accompanying the original submission of the work to the USC Libraries is retained by the USC Libraries and a copy of it may be obtained by authorized requesters contacting the repository e-mail address given. |
| Repository name | University of Southern California Digital Library |
| Repository address | USC Digital Library, University of Southern California, University Park Campus MC 7002, 106 University Village, Los Angeles, California 90089-7002, USA |
| Repository email | cisadmin@usc.edu |
| Archival file | uscthesesreloadpub_Volume71/etd-PurcellDan-353.pdf |
Description
| Title | Page 1 |
| Full text | TWO FACED COREGULATOR: COACTIVATION AND COREPRESSION OF TRANSCRIPTION BY HISTONE METHYLTRANSFERASE G9A by Daniel Purcell A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (GENETIC, MOLECULAR AND CELLULAR BIOLOGY) December 2011 Copyright 2011 Daniel Purcell |
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