Author: Dione Anne Gardner-Stephen
Gardner-Stephen, Dione Anne, 2008 Transcriptional Regulation of Human UDP-Glucuronosyltransferases, Flinders University, School of Medicine
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The UDP-glucuronosyltransferases (UGTs) are a superfamily of enzymes that glucuronidate small, lipophilic molecules, thereby altering their biological activity and excretion. In humans, important examples of UGT substrates include molecules of both endogenous and xenobiotic origin; thus, UGTs are considered essential contributors to homeostatic regulation and an important defence mechanism against chemical insult. In keeping with both roles, UGTs are most strongly expressed in the liver, a predominant organ involved in detoxification. Rates of glucuronidation in humans are neither uniform among individuals, nor constant in an individual over time. Genetic determinants and non-endogenous signals are both known to influence the expression of UGTs, which in turn may affect the efficacy of certain pharmaceutical treatments or alter long-term risk of developing disease. Thus, this thesis focuses on the transcriptional regulation of UGT genes in humans, particularly on mechanisms that are likely to be relevant to their expression and variation in the liver. Two major approaches were used: firstly, extensive studies of several UGT promoters were performed to identify and characterise transcriptional elements that are important for UGT expression; and secondly, important hepatic transcription factors were investigated as potential regulators of UGT genes. UGT1A3, UGT1A4 and UGT1A5 are a subset of highly related, but independently regulated, genes of the human UGT1 subfamily. UGT1A3 and UGT1A4 are expressed in the liver, whereas UGT1A5 is not. The presented analysis of the UGT1A3, UGT1A4 and UGT1A5 proximal promoters demonstrates that a hepatocyte nuclear factor (HNF)1-binding site common to all three promoters is important for UGT1A3 and UGT1A4 promoter activity in vitro, but is insufficient to drive UGT1A5 expression. Two additional elements required for the maximal activity of the UGT1A3 promoter were also identified that may distinguish this gene from UGT1A4. UGT1A3 was investigated further, focusing on mechanisms that may contribute to interindividual variation in UGT1A3 expression. Polymorphisms in the UGT1A3 proximal promoter were identified and their functional consequences tested. Known variants of HNF1alpha were also tested for altered activity towards the UGT1A3 gene. UGT1A9 is the only hepatic member of the UGT1A7-1A10 subgroup of UGT1 enzymes. Previous work had identified HNF1-binding sites in all four genes, and HNF4alpha as an UGT1A9-specific regulator. The work presented herein extends these findings to show that HNF1 factors and HNF4alpha synergistically regulate UGT1A9, and that HNF4alpha is not the only transcription factor responsible for the unique presence of UGT1A9 in the liver. Liver-enriched transcription factors screened as potential UGT regulators were chosen from the HNF1, HNF4, HNF6, FoxA and C/EBP protein families. Functional interactions newly identified by this work were HNF4alpha with UGT1A1 and UGT1A6, HNF6 with UGT1A4 and UGT2B11, FoxA1 and FoxA3 with UGT2B11, UGT2B15 and UGT2B28 and C/EBPalpha with UGT2B17. Observations were also made regarding different patterns of interaction between each UGT and the transcription factors tested, particularly HNF1alpha.
Keywords: UDP-glucuronosyltransferase,hepatocyte nuclear factor,gene expression,transcriptional regulation
Subject: Medicine thesis
Thesis type: Doctor of Philosophy
School: School of Medicine
Supervisor: Professor Peter Mackenzie