Sulfonation is an important conjugation reaction involved in the biomodulation of many endogenous compounds including steroids and neurotransmitters. It also plays a role in the phase II metabolic pathway of various xenobiotics, such as paracetamol, and can in certain cases result in the bioactivation of exogenous compounds such as minoxidil and N-hydroxy aromatic amines. Sulfonation is catalysed by a gene family of enzymes called sulfotransferases (SULTs). The focus of this study was on the human SULT1A subfamily, members of which are encoded by three genes that are found in close proximity to each other on chromosome 16. SULT1A1 has been shown to have a major role in xenobiotic sulfonation. SULT1A2 has a similar substrate profile to SULT1A1, whereas SULT1A3 is the dominant sulfotransferase involved in catecholamine metabolism. Although these three SULT1A members share >90% amino acid sequence identity, their tissue-specific expression varies. This is particularly evident when comparing SULT1A1 and SULT1A3. SULT1A1 has a wide tissue distribution, with high abundance in the liver, whereas SULT1A3 is expressed at negligible levels in the adult liver, but is present in the fatal liver and in adult extrahepatic tissues such as the gastrointestinal tract.
This study aimed to identify the promoters of the SULT1A genes, using mammalian transfection systems, and to elucidate the molecular mechanisms controlling their regulation. The primary mammalian cell systems used to investigate the regulation of the SULT1A enzymes were the liver-like environments of the hepatocarcinoma cell lines HepG2 and Hep3B, as well as cultures of primary human hepatocytes. In previous studies, several different SULT1A mRNA species with varying 5'untranslated regions (5'UTRs) were identified, suggesting the use of alternate transcriptional start sites. Using 5'rapid amplification of cDNA ends and reverse transcriptase PCR, the most distal 5'UTR of SULT1A1 was identified as the major mRNA found in the above experimental system. The sequence flanking this region also contained the most active promoter of the SULT1A1 gene. Likewise, the homologous sequences in SULT1A2 and SULT1A3 proved to be the most active promoters of these SULT1A genes.
Although the promoters of the SULT1A genes lack canonical TATA-box elements, deletion constructs revealed the presence of a crucial regulatory element between bases -112 to -68 and -100 to -56 from the transcriptional start site of SULT1A1 and SULT1A2, respectively. This region contains a triplicate repeat of the Ets transcription factor binding site (EBS) core sequence GGAA. Comparison with the homologous sequence of the SULT1A3 gene (-125/+43), which displayed two thirds less promoter activity than SULT1A1, revealed that the former has a 2 base pair mutation in one of these sites (EBS1). Exchanging these two bases between the two sequences effectively converted their promoter activities.
Electrophoretic mobility shift assays using the triplicate SULT1A1 EBS motif as the probe revealed the formation of specific DNA: protein complexes when incubated with nuclear extracts from HepG2, Hep3B or mouse liver nuclear extracts. Antibodies to the ubiquitously expressed Ets transcription factors GA binding protein α/β (GABPα/β) heterodimer and Elf1 were able to supershift two of these DNA: protein complexes. Furthermore, the recombinant GABPα/β and Elf proteins were able to bind the probe and cause transcriptional activation of the SULT1A1 and SULT1A3 promoters in Drosophila melanogaster S2 cells. Interestingly, Sp1 could synergistically enhance the induction of the SULT1A1 promoter observed with GABPα/β 10-fold. This synergy was not observed when the EBS1 motif of SULT1A1 was mutated and with the SULT1A3 promoter, which differs from the SULT1A1 promoter by two base pairs in the EBS motif. This suggests that the decreased activity observed for the SULT1A3 wild type promoter in hepatocarcinoma cells is due to the inability of GABPα/β and Sp1 to act in a synergistic manner on this sequence.
Several rodent sulfotransferases have previously been reported to be under the influence of nuclear receptor activators. Although sequence analysis of the SULT1A promoters identified potential nuclear receptor response elements, co-transfection of these together with nuclear receptors and treatment with activators did not result in significant changes in promoter activities in HepG2 cells. In addition, when cultured primary human hepatocytes were treated with ligands such as the glucocorticoid receptor agonist dexamethasone or the constitutive androstane receptor activator phenobarbital, no changes in SULT1A mRNA could be observed using real-time quantitative PCR analysis, suggesting little role for nuclear receptors in the regulation of the human SULT1A subfamily.
This thesis represents the first insights into the molecular regulatory mechanisms of the human SULT1A
sulfotransferase genes. The lack of responsiveness of the SULT1A
genes to nuclear receptors and the ability of ubiquitously expressed Ets and Spl transcription factors to activate their transcriptional regulation suggests that SULT1A1, in particular, is controlled in a constitutive/ubiquitous manner. The constitutive expression pattern of these enzymes may provide a protective mechanism for the body when dealing with exposure to xenobiotics.