Regulatory mechanisms controlling human hepatocyte nuclear factor 4alpha gene expression.
Hepatocyte nuclear factor 4alpha (HNF-4alpha) (nuclear receptor 2A1) is an essential regulator of hepatocyte differentiation and function. Genetic and molecular evidence suggests that the tissue-restricted expression of HNF-4alpha is regulated mainly at the transcriptional level. As a step toward understanding the molecular mechanism involved in the transcriptional regulation of the human HNF-4alpha gene, we cloned and analyzed a 12.1-kb fragment of its upstream region. Major DNase I-hypersensitive sites were found at the proximal promoter, the first intron, and the more-upstream region comprising kb -6.5, -8.0, and -8.8. By the use of reporter constructs, we found that the proximal-promoter region was sufficient to drive high levels of hepatocyte-specific transcription in transient-transfection assays. DNase I footprint analysis and electrophoretic mobility shift experiments revealed binding sites for HNF-1alpha and -beta, Sp-1, GATA-6, and HNF-6. High levels of HNF-4alpha promoter activity were dependent on the synergism between either HNF-1alpha and HNF-6 or HNF-1beta and GATA-6, which implies that at least two alternative mechanisms may activate HNF-4alpha gene transcription. Chromatin immunoprecipitation experiments with human hepatoma cells showed stable association of HNF-1alpha, HNF-6, Sp-1, and COUP-TFII with the promoter. The last factor acts as a repressor via binding to a newly identified direct repeat 1 (DR-1) sequence of the human promoter, which is absent in the mouse homologue. We present evidence that this sequence is a bona fide retinoic acid response element and that HNF-4alpha expression is upregulated in vivo upon retinoic acid signaling.
Hatzis
P
P
Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, 711 10 Herakleion, Crete, Greece.
Talianidis
I
I
D000818:Animals; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001665:Binding Sites; D019556:COS Cells; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D008024:Ligands; D051379:Mice; D008954:Models, Biological; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000096:biosynthesis* / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013997:Time Factors; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics*; D014162:Transfection; D014212:Tretinoin / Q000378:metabolism; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D002843:Chromatin; D004268:DNA-Binding Proteins; C496592:HNF4A protein, human; D051557:Hepatocyte Nuclear Factor 4; D008024:Ligands; C403128:MLX protein, human; D010750:Phosphoproteins; C501970:Tcfl4 protein, mouse; D014157:Transcription Factors; D014212:Tretinoin