Cloning and Characterization of a Novel Human Hepatocyte Transcription Factor, hB1F, Which Binds and Activates Enhancer II of Hepatitis B Virus

Abstract

Enhancer II (ENII) of hepatitis B virus (HBV) is one of the essential cis-elements for the transcriptional regulation of HBV gene expression. Its function is highly liver-specific, suggesting that liver-enriched transcriptional factors play critical roles in regulating the activity of ENII. In this report, a novel hepatocyte transcription factor, which binds specifically to the B1 region (AACGACCGACCTTGAG) within the major functional unit (B unit) of ENII, has been cloned from a human liver cDNA library by yeast one-hybrid screening, and demonstrated to trans-activate ENII via the B1 region. We named this factor hB1F, for human B1-binding factor. Amino acid analysis revealed this factor structurally belongs to nuclear receptor superfamily. Based on the sequence similarities, hB1F is characterized to be a novel human homolog of the orphan receptor fushi tarazu factor I (FTZ-F1). Using reverse transcriptionpolymerase chain reaction, a splicing isoform of hB1F (hB1F-2) was identified, which has an extra 46 amino acid residues in the A/B region. Examination of the tissue distribution has revealed an abundant 5.2-kilobase transcript of hB1F is present specifically in human pancreas and liver. Interestingly, an additional transcript of 3.8 kilobases was found to be present in hepatoma cells HepG2. Fluorescent in situ hybridization has mapped the gene locus of hB1F to the region q31-32.1 of human chromosome 1. Altogether, this study provides the first report that a novel human homolog of FTZ-F1 binds and regulates ENII of HBV. The potential roles of this FTZ-F1 homolog in tissue-specific gene regulation, in embryonic development, as well as in liver carcinogenesis are discussed. Hepatitis B virus (HBV)1 is the major cause of acute and chronic hepatitis, also closely associated with the development of hepatocellular carcinoma (1). HBV predominantly infects hepatocytes, which is a prominent characteristic of hepadnaviruses. The genome of HBV is a small, circular, partially doublestranded DNA of about 3.2 kb, which contains four partially overlapping open reading frames (ORF) encoding the surface antigens (preS/S), the core antigen/e antigen (preC/C), the polymerase (P) and the X protein (X), respectively (2, 3). These HBV genes express specifically in liver, controlled by the combinatorial action of the promoters and enhancers. To date, four promoters (Sp1, Sp2, Cp, and Xp) (4) have been identified to be responsible for the transcription of the viral mRNAs, and they are regulated by two HBV enhancers. Enhancer I (ENI) functions in a relatively tissue independent manner (5, 6), while enhancer II (ENII) shows strong hepatocyte specificity. Enhancer II of HBV is located within the X ORF, about 600 bp downstream of ENI (7-9). In our previous study, ENII was mapped in a 148-bp region from nt 1627 to 1774 (HBV adr1 subtype), partially overlapped with the core promoter (Cp) (7). According to the functional analyses, it can be divided into two parts, A and B, with part B as the basal functional unit, which could be further subdi...

LRH-1/hB1F and HNF1 synergistically up-regulate hepatitis B virus gene transcription and DNA replication

“…The reporter pABCDCAT containing the HBV ENII/Cp region located upstream from the CAT reporter gene has been described [3].…”

“…The liver-specific activity. of ENII is regulated by multiple liver-enriched transcription factors, including hB1F, HNF1, HNF3β, HNF4, and C/EBP [3][4][5][6][7][8][9]. The interaction bewteen each of these factors and ENII has been studied in great detail, but less is known on the interplay of these important regulators, which we think is crucial for the function of ENII as well as the life cycle of HBV.…”

“…In previous studies, we showed that the orphan nuclear receptor hB1F and homeoprotein HNF1 can bind to the B1 and B2 elements of ENII respectively and stimulate its liver-specific activity, which consequently regulates hepatic viral gene transcription and DNA replication [3,9,10]. hB1F (also known as LRH-1, FTF, CPF) is a human homolog of the Drosophila fushi tarazu factor I (FTZ-F1) [3,11].…”

“…hB1F (also known as LRH-1, FTF, CPF) is a human homolog of the Drosophila fushi tarazu factor I (FTZ-F1) [3,11]. It shares a common modular structure with other members of the nuclear receptor superfamily, including the N-terminal A/B domain, a zinc finger DNA binding domain (DBD), a hinge region and a ligand binding domain (LBD).…”

The conserved nuclear receptor Ftz‐F1 is required for embryogenesis, moulting and reproduction in Caenorhabditis elegans

“…NHR-25 is most similar to insect Ftz-F1 from Drosophila and the silkworm Bombyx mori in both the zinc ®ngers and the Ftz-F1 box. The vertebrate sequences are products of three distinct types of genes: the steroidogenic factors (SF-1) and two groups of liver-speci®c transcription factors, one activating the a-fetoprotein gene (FTF; Galarneau et al 1996), and the other regulating a cytochrome hydroxylase gene (CPF; Nitta et al 1999) and hepatitis B virus (B1F; Li et al 1998) FTF and CPF/B1F show a greater similarity to NHR-25 in the conserved regions II and III of the ligandbinding domain (LBD) than does SF-1 (Fig. 1B).…”