Date of Award

2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Pharmacology

First Advisor

Mitchell A. Lazar

Abstract

Rosiglitazone (rosi) is a powerful insulin sensitizer, but serious toxicities have curtailed its widespread clinical use. Rosi functions as a high-affinity ligand for PPARgamma, the adipocyte-predominant nuclear receptor (NR). The classic model of NR action, involving binding of ligand to the NR on DNA, explains positive regulation of gene expression, but both ligand-dependent transcriptional repression and indirect regulation are not well understood. We have addressed these issues by studying the direct effects of rosiglitazone on gene transcription, using global run-on sequencing (GRO-seq). Rosi-induced changes in gene body transcription were pronounced after 10 minutes and correlated with steady-state mRNA levels as well as with transcription at nearby enhancers (eRNAs). Up-regulated eRNAs occurred almost exclusively at PPARg binding sites, to which rosi treatment recruited coactivators including MED1, p300, and CBP, without changes in binding of the corepressor NCoR. By contrast, down-regulated eRNAs fell in sites devoid of PPARg but enriched for a variety of other TFs in the C/EBP and AP-1 families. These enhancers lost coactivator binding upon rosi treatment, suggesting that rosi treatment causes redistribution of coactivators to PPARg sites and away from enhancers containing other TFs, leading to transcriptional repression at these eRNAs and their target genes. We also investigated the function of MRL-24, a compound that has been shown to lack PPARg transactivation activity and regulate a distinct subset of PPARg target genes while functioning as an equally effective insulin sensitizer as rosi. Though our goal was to identify whether MRL-24 regulates the same functional enhancers marked by eRNAs as rosi, we instead found that MRL-24 does not control a distinct subset of target genes, but rather acts as a partial agonist for PPARg. Together, these studies further our understanding of transcriptional regulation by modulation of PPARg activity, including insights into determining functional enhancers and mechanisms of transcriptional repression by activation of a NR.

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