Regions flanking exon 1 regulate constitutive expression of the human antithrombin gene.
We have identified cis-acting elements and trans-acting factors that regulate constitutive expression of the human antithrombin gene. The activity of the sequences flanking the first exon of the gene was investigated using a luciferase-based reporter assay in transiently transfected HepG2, COS1, BSC40, and HeLa cells. Deletion analysis allowed the mapping of two elements able to promote antithrombin gene transcription in HepG2 and COS1 cells. The first element is located upstream of the first exon (-150/+68 nucleotides). The second element is in the first intervening sequence (+300/+700 nucleotides) and functions in an orientation opposite to that of the first. Footprint analysis showed three protected areas in the 5' upstream element at -92/-68 (element A), -14/+37 (element B), and -126/-100 nucleotides (element C). These elements acted as enhancers in luciferase reporter assays. Gel retardation analysis demonstrated that two liver-enriched transcription factors, hepatocyte nuclear factor 4 (HNF4) and CCAAT enhancer-binding protein (C/EBPa), bound to the 5' upstream element. HNF4 bound to elements A and C, whereas C/EBPa bound to element B. Element A also interacted with the ubiquitous nuclear hormone receptors chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF1), thyroid hormone receptor alpha (TRalpha), peroxisome proliferator-activated receptor alpha(PPARalpha), and retinoid X receptor alpha (RXRalpha). In HepG2 and BSC40 cells, HNF4, C/EBPalpha, and RXRalpha activated luciferase expression from a reporter construct containing the 5'-upstream minimal antithrombin gene promoter, while COUP-TF1, TRalpha, and HNF3 (alpha or beta) repressed such expression. Our results show that constitutive expression of the human antithrombin gene depends in part upon the interplay of these transcription factors and suggest that signaling pathways regulated by these factors can modulate antithrombin gene transcription.
Department of Pathology, McMaster University, Hamilton, Ontario L8N 3Z5, Canada.
F A
FA
Fernandez-Rachubinski
J H
JH
Weiner
M A
MA
Blajchman
D000818:Animals; D000990:Antithrombin III / Q000235:genetics*; D022762:CCAAT-Enhancer-Binding Proteins; D002460:Cell Line; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000235:genetics; D005091:Exons; D005786:Gene Expression Regulation; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051381:Rats; D011956:Receptors, Cell Surface / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011956:Receptors, Cell Surface; D014157:Transcription Factors; D000990:Antithrombin III