Identification of Novel Transcription Start Sites and Distinct Proximal Promoter Regions for Pregnane X Receptor (PXR) Isoforms PXR 1 and PXR 2

Abstract

TOMPKINS, LESLIE MEREDITH. Identification of Novel Transcription Start Sites and Alternative Proximal Promoters for Pregnane X Receptor (PXR) Isoforms PXR 1 and PXR 2. (under the direction of Dr. Robert C. Smart and Dr. Andrew D. Wallace.)

The identification and characterization of the orphan group of nuclear receptors has revolutionized our collective understanding of signaling pathways responsible for xenobiotic metabolism. Discovery of the pregnane X receptor (PXR) provided the mechanistic explanation to decades of research that documented xenobiotic metabolic regulation and hepatic protection through enzyme induction without a known mediator. PXR, known as the xenosensing receptor, coordinately regulates metabolic genes in response to diverse xenobiotic challenges. In particular, the ability of PXR to regulate CYP3A4, the enzyme capable of metabolizing more than 60% of all pharmaceuticals, defines its metabolic importance. Currently, the list of PXR ligands and target genes is extensive, yet investigations into the regulation and expression of PXR itself are few. After an initial review of available sequence data, we discovered discrepancies in the 5? untranslated region (UTR) and transcription start site (TSS) characterizations of the PXR gene, so we endeavored to define TSSs and proximal promoters for the well-known PXR 1 isoform and the lesser-known PXR 2 isoform. The PXR 2 isoform is the product of a unique first exon responsible for the 39 amino acid amino-terminal addition and is therefore regulated by a distinct proximal promoter. RT-PCR and primer extension experiments performed using total RNA from human liver samples identified two TSSs for each receptor isoform. These results extended the 5?UTR sequence of each isoform and defined new proximal promoters for both PXR isoforms. Separate promoter regions for receptor isoforms suggests the possibility that PXR isoforms are differential regulated or expressed in a tissue-specific fashion. Candidate response elements for liver-enriched transcription factors and other nuclear receptors were found in both promoters. Quantitative PCR illustrated PXR 2 expression as a modest but consistent 2-5% of total PXR expression with much less variability among individuals. In addition, transfection experiments demonstrated comparable transcriptional activity of PXR 2 when compared to that of PXR 1. In total, these data present a starting point from which to investigate the regulation of multiple isoforms of PXR and evaluate individual isoform contributions to the cumulative function of this important nuclear receptor in xenobiotic metabolism.