Activation of the Mouse TATA-less and Human TATA- Containing UDP-Glucuronosyltransferase 1A1 Promoters by Hepatocyte Nuclear Factor 1 PASCAL BERNARD, HERVÉ GOUDONNET, YVES ARTUR, BÉATRICE DESVERGNE, and WALTER WAHLI

UDP-glucuronosyltransferase (UGT) 1A1 (UGT1A1) catalyzes the glucuronidation of bilirubin in liver. Among all UGT isoforms identified to date, it is the only relevant bilirubin-glucuronidating enzyme in human. Because glucuronoconjugation is the major route of bilirubin elimination, any genetic alteration that affects bilirubin glucuronosyltransferase activity may result in a more or less severe hyperbilirubinemia. In this study, we report the cloning and characterization of the transcriptional regulation of the mouse UGT1A1 gene. Primary-structure analysis of the mouse Thymidine Adevice promoter revealed marked differences with its human homolog. First, the mouse promoter lacks the highly polymorphic thymidine/adenine repeat occurring in the human promoter, which has been associated with some forms of hyperbilirubinemia. Second, an L1 transposon element, which is absent in the human promoter, is found 480 bp upstream of the transcription start site in mouse. Using the electromobility shift and DNase I footprinting experiments, we have identified a hepatocyte nuclear factor 1-binding site in the mouse UGT1A1 promoter that confers responsiveness to both factors HNF1a and HNF1b in HEK293 cells. Furthermore, we show that this element, which is conserved in the human promoter, also confers strong HNF1 responsiveness to the human UGT1A1 gene. Together, these results provide evidence for a major regulatory function of this liver-enriched transcription factor in UGT1A1 activity in both rodents and human. In mammals, detoxification of the hydrophobic bilirubin occurs mainly in the liver via its conjugation with uridine diphosphate glucuronic acid. This reaction, which is catalyzed by microsomal UDP-glucuronosyltransferases (UGTs) (EC 2.4.1.17) produces hydrophilic bilirubin-monoand diconjugates that are excreted at high rates in the bile (Hauser and Gollan, 1990; Jansen et al., 1992). UGTs are produced by a multigene superfamily with specificity for a variety of endogenous substrates and xenobiotics. Mammalian UGTs are subdivided into two groups, the UGT1 and the UGT2 families. UGT1 family members are encoded by a particularly complex gene of about 500 kb (“the UGT1 cluster,” Mackenzie et al., 1997). It consists of multiple related homologous unique first exons (exon 1), which encode the isoform specific N terminus of the enzymes. The C-terminal part is encoded in a single set of four exons (exons 2–5), which is common to all UGT1 isoforms (Ritter et al., 1992; Emi et al., 1995; Mackenzie et al., 1997). A promoter region with its regulatory elements is thought to be located upstream of each unique first exon, but, for several of them, this region has not yet been characterized (Ritter et al., 1992). The UGT1A1 isoform is likely to be the major isoform involved in bilirubin detoxification in human (Bosma et al., 1994). Inherited disorders that decrease or suppress UGT1A1 expression result in unconjugated bilirubin accumulation. Mild forms of unconjugated hyperbilirubinemia are known as Gilbert and Crigler-Najjar type II syndromes, whereas in more severe forms, the Crigler-Najjar type I disease can be fatal (Aono et al., 1995; Bosma et al., 1995). Some of the mutations causing these disorders are localized in the common exons 2 to 5, leading to an alteration of all isoforms produced by the UGT1 locus (reviewed in Mackenzie et al., 1997). Others are in exon 1A1 and thus alter only UGT1A1 activity. Thus far, the UGT1A1*28 polymorphism is the only genetic variation reported in a regulatory region of the gene. It is responsible for most Gilbert’s disease cases (Bosma et This work was supported by grants from the Swiss National Science Foundation (B.D. and W.W.), the Etat de Vaud, and the Conseil Régional de Bourgogne. P.B. was supported by a fellowship from the Association de Recherche sur le Cancer (ARC; Villejuif, France) and the Etat de Vaud. 1 The nucleotide sequence reported in this paper has been submitted to the GenBank database with GenBank accession number AF093878. ABBREVIATIONS: UGT, UDP-glucuronosyltransferases; HEK, human embryonic kidney; EMSA, electrophoretic mobility shift assay; MLNE, mouse liver nuclear extract; PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; UTR, untranslated region, RSV, Rous sarcoma virus; TA, thymidine/adenine; WCE, whole-cell extract. 0026-895X/99/030526-11$3.00/0 Copyright © The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 56:526–536 (1999). 526 at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from al., 1995). This polymorphism concerns the six-thymidine/ adenine (TA) track in the wild-type UGT1A1 promoter, which is replaced by five, seven, or eight TA repeats in the altered genes, whose activity is inversely correlated with the length of the track (Beutler et al., 1998). Expression of the human UGT1A1 mRNA is primarily in the liver but can also be detected in biliary and colon epithelia (Ritter et al., 1992; Strassburg et al., 1997, 1998). In rat, the tissue distribution is similar in liver and intestine. In addition, UGT1A1 is also expressed at low levels in kidney, which is not the case in human (Ritter et al., 1992; Emi et al., 1995). Surprisingly, only the promoter of the human UGT1A1 gene has been sequenced thus far. However, with the exception of the TA track, no functional promoter characterization has been performed (Brierley et al., 1996). The broad use of mice and rats as laboratory animals for pharmacological studies has prompted us to investigate UGT1A1 transcriptional regulation in the mouse, not least because the gene knockout technology is now well mastered for this animal and might be applied later to the UGT1 locus once characterized. Moreover, studies using this technology provided evidence for a role of liver-enriched transcription factors in UGT1A1 expression. The inactivation of the HNF1a gene resulted in a mild hyperbilirubinemia, which, according to the above-mentioned observations, might reflect an altered UGT1A1 gene expression (Pontoglio et al., 1996). Similarly, knock-out of C/EBPa in mouse liver affects UGT1A1 gene expression resulting in a severe toxic hyperbilirubinemia (Lee et al., 1997). These observations, together with UGT1A1 being the main isoform from the UGT1 locus expressed in rat liver (Emi et al., 1995), argue strongly for a crucial role of the UGT1A1 isoform in bilirubin detoxification in rodents. Recently, other studies have highlighted the important role of HNF1a and C/EBPa in regulating the transcription of the rat UGT2B1 isoform (Hansen et al., 1997, 1998). In contrast, nothing is presently known of the mechanisms of transcriptional regulation of the UGT1A1 gene by these factors, although their involvement has been suggested, as mentioned above. To better understand the physiological role of the UGT1A1 isoform in the mouse, we have isolated the UGT1A1 gene. Its promoter was then compared with that of the human gene, which revealed the lack of a TATA box in the mouse promoter. Furthermore, evidence is provided for a direct involvement of HNF1 in the regulation of the mouse and human UGT1A1 promoters. Experimental Procedures Materials. The expression plasmids pRSV-HNF1a and pRSVHNF1b were a kind gift from Dr. Moshe Yaniv (Unité des Virus Oncogènes, Institut Pasteur, Paris, France). The a-HCt-284 polyclonal antiserum directed against HNF1a (Chouard et al., 1997) was a kind gift from Dr. Marco Pontoglio (Unité des Virus Oncogènes, Institut Pasteur, Paris, France) and Dr. Moshe Yaniv. All UGT1A1 nucleotide sequences used were numbered relative to the major transcription start site determined in this study. Isolation and Characterization of Genomic and cDNA Clones. A portion (1 3 10 plaque-forming units) of a mouse L129/ SVJ genomic DNA library in lFIXII (Clontech Laboratories, Palo Alto, CA) were screened with two oligonucleotides complementary to the mouse UGT1A1 cDNA (Chu et al., 1997) as probes. Two clones (lmPGT1A1–1, lmPGT1A1–2) were isolated, plaque-purified, and characterized by polymerase chain reaction (PCR) and restriction enzyme analysis. From the lmPGT1A1–1 clone, a 2.4-kb XbaI restriction fragment containing the UGT1A1 first exon was subcloned in the pBluescript KSII vector, yielding the pKS-1A1 genomic subclone. DNA was sequenced on both strands by the dideoxy termination method using T7 DNA polymerase (T7 sequencing kit; Pharmacia Biotech Europe, Dübendorf, Switzerland). The cDNA clones were obtained by screening 1 3 10 plaque-forming units of an adult mouse liver 59-extended cDNA library in lgt10 with the same oligonucleotides as indicated above, yielding 10 independent clones. Similarly, a 15-day-old embryonic mouse 59-extended cDNA library in lgt10 was screened following the same procedure, yielding one clone. After PCR amplification using phage-specific oligonucleotides, the 11 independent clones were subcloned into the pBluescript vector for subsequent sequencing. The human UGT1A1 promoter was cloned by PCR on human genomic DNA and subcloned into the pBLCAT3 vector yielding the 2617/115 hUGT1A1-chloramphenicol acetyltransferase (CAT) construct. The construct was sequenced on both strands. Its sequence is identical with that published previously (Brierley et al., 1996) except for the length of the TA repeat in the polymorphic TATA box, which is (TA)5TAA instead of (TA)6TAA (Beutler et al., 1998). RNA Isolation and Primer Extension Analysis. Total RNA was isolated from mouse liver by the phenol and guanidine isothiocyanate method following the supplier’s instructions (Trizol reagent; Life Technologies, Basel, Switzerland). An end-labeled antisense oligonucleotide primer complementary to nucleotides 195/1120 of the UGT1A1 gene was hybridized to 50 mg of total RNA for 1 h at 42°C in 50 mM Tris-HCl (pH 8.3), 8 mM MgCl2, 30 mM KCl, 10 mM DTT in a final volume of 50 ml. The primer-annealed RNA was then subjected to extension by adding 2 mM each dNTP and 25 units of avian myeloblastosis virus-reverse transcriptase (Pharmacia Biotech Europe, Dübendorf, Switzerland) for 1 h at 42, 44, or 46°C. The reaction was stopped by addition of 50 mM EDTA and 15 mg of Rnase Avian myeloblastosis virus (Boehringer Manheim, Rotkreuz, Switzerland) and incubated for 1 h at 37°C. The reaction products were resolved on a 7 M urea, 8% polyacrylamide-13 Tris-borate-EDTA