Transcriptional regulation of human oxysterol 7 alpha-hydroxylase gene (CYP7B1) by Sp1.
Humans; Protein Binding; Gene Expression Regulation; Cell Line; Transfection; Base Sequence; Binding Sites/genetics; Molecular Sequence Data; Cytochrome P450 Family 7; Mutagenesis; Luciferases/genetics/metabolism; Recombinant Fusion Proteins/genetics/metabolism; CpG Islands/genetics; Cytochrome P-450 Enzyme System/*genetics/metabolism; DNA/genetics; Sequence Deletion; Sp1 Transcription Factor/metabolism/*physiology; Steroid Hydroxylases/*genetics/metabolism; Cultured; Binding; Competitive; Transcription; Genetic; Enzymologic; Tumor Cells; Site-Directed; Regulatory Sequences; Nucleic Acid/genetics
Oxysterol 7 alpha-hydroxylase catalyzes hydroxylation of oxysterols and neurosterols and plays a role in the alternative bile acid synthesis pathway. This gene is widely expressed in many organs and peripheral tissues and may protect tissues from the toxicity of oxysterols. Mutation in CYP7B1 caused neonatal cholestasis. To examine the regulatory mechanisms governing CYP7B1 expression, the 5' flanking sequence of the CYP7B1 was analyzed and revealed a CpG island of about 1.2 kb. Transient transfection assays of deletion mutants of the CYP7B1 promoter-luciferase reporter gene in human liver-derived HepG2, fibroblast NT1088, and human embryonic kidney 293 cell lines revealed that the region from -291 to +189 was critical for gene transcription. Three GC box sequences located between -25 and +10 were essential for basal transcription because mutations of these sequences markedly reduced promoter activity. Sp1 and Sp3 bound to these sequences as demonstrated by DNase I footprinting assays and electrophoretic mobility shift assay. Thus, regulation of CYP7B1 transcription by Sp1 may play a pivotal role in regulating oxysterol levels, which regulate cholesterol metabolism.
Wu Z; Chiang J Y
Gene
2001
2001-07
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
Transcriptional activation of the cholesterol 7alpha-hydroxylase gene (CYP7A) by nuclear hormone receptors.
Animals; Rats; Transcription Factors/metabolism; Base Sequence; Molecular Sequence Data; DNA/metabolism; Cholesterol 7-alpha-Hydroxylase/*genetics; Hepatocyte Nuclear Factor 4; Mutagenesis; Luciferases/genetics; Retinoid X Receptors; Phosphoproteins/metabolism; COUP Transcription Factor II; COUP Transcription Factors; *Transcriptional Activation; Bile Acids and Salts/biosynthesis; DNA-Binding Proteins/metabolism; Hormones/*physiology; Oligonucleotide Probes/metabolism; Genes; Cultured; Receptors; Genetic; Cytoplasmic and Nuclear/*physiology; Tumor Cells; Reporter; Retinoic Acid/metabolism; Promoter Regions; Nucleic Acid; Site-Directed; *Receptors; Repetitive Sequences; Steroid
The gene encoding cholesterol 7alpha-hydroxylase (CYP7A), the rate-limiting enzyme in bile acid synthesis, is transcriptionally regulated by bile acids and hormones. Previously, we have identified two bile acid response elements (BARE) in the promoter of the CYP7A gene. The BARE II is located in nt -149/-118 region and contains three hormone response element (HRE)-like sequences that form two overlapping nuclear receptor binding sites. One is a direct repeat separated by one nucleotide DR1 (-146- TGGACTtAGTTCA-134) and the other is a direct repeat separated by five nucleotides DR5 (-139-AGTTCAaggccGGG TAA-123). Mutagenesis of these HRE sequences resulted in lower transcriptional activity of the CYP7A promoter/reporter genes in transient transfection assay in HepG2 cells. The orphan nuclear receptor, hepatocyte nuclear factor 4 (HNF-4)1, binds to the DR1 sequence as assessed by electrophoretic mobility shift assay, and activates the CYP7A promoter/reporter activity by about 9-fold. Cotransfection of HNF-4 plasmid with another orphan nuclear receptor, chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII), synergistically activated the CYP7A transcription by 80-fold. The DR5 binds the RXR/RAR heterodimer. A hepatocyte nuclear factor-3 (HNF-3) binding site (-175-TGTTTGTTCT-166) was identified. HNF-3 was required for both basal transcriptional activity and stimulation of the rat CYP7A promoter activity by retinoic acid. Combinatorial interactions and binding of these transcription factors to BAREs may modulate the promoter activity and also mediate bile acid repression of CYP7A gene transcription.
Crestani M; Sadeghpour A; Stroup D; Galli G; Chiang J Y
Journal of lipid research
1998
1998-11
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
Regulation of human sterol 27-hydroxylase gene (CYP27A1) by bile acids and hepatocyte nuclear factor 4alpha (HNF4alpha).
Humans; Cell Line; Transfection; Gene Expression Regulation/drug effects; Base Sequence; Binding Sites/genetics; Response Elements/genetics; Molecular Sequence Data; Mutation; Chenodeoxycholic Acid/pharmacology; Transcription Factors/genetics/*metabolism; Hepatocyte Nuclear Factor 4; Mutagenesis; *DNA-Binding Proteins; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Bile Acids and Salts/*pharmacology; Cholestanetriol 26-Monooxygenase; DNA/chemistry/genetics; Luciferases/genetics/metabolism; Phosphoproteins/genetics/*metabolism; Recombinant Fusion Proteins/genetics/metabolism; Steroid Hydroxylases/*genetics; DNA; Dose-Response Relationship; Drug; Cultured; Receptors; Tumor Cells; Cloning; Molecular; Sequence Analysis; Promoter Regions; Genetic/*genetics; Cytoplasmic and Nuclear/genetics/metabolism; Site-Directed
Mitochondrial sterol 27-hydroxylase (CYP27A1) catalyses sterol side-chain oxidation of bile acid synthesis from cholesterol, and the first reaction of the acidic bile acid biosynthetic pathway. Hydrophobic bile acids suppress human CYP27A1 gene reporter activity when assayed in human hepatocellular blastoma HepG2 cells. Bile acids also inhibit CYP27A1 reporter activity in human embryonic kidney 293 cells. A putative bile acid response element (BARE) was mapped to a region downstream of nt -147 of the human CYP27A1 gene, within which a binding site for a liver-specific nuclear receptor, HNF4alpha, is identified. HNF4alpha strongly stimulates CYP27A1 gene transcription and mutation of its binding site markedly reduced promoter activity. Results suggest that human CYP27A1 gene transcription is suppressed by bile acids and HNF4alpha plays a pivotal role in transcriptional regulation of this gene.
Chen Wenling; Chiang John Y L
Gene
2003
2003-08
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).