Abstract
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.
Highlights
Identification of Factors Acting in Trans with Elements A, B, and C—Incubation of double-stranded oligonucleotides corresponding to elements A, B, or C with nuclear extracts from COS1, HeLa and HepG2 cells resulted in electrophoretic mobility shifts (Fig. 6)
Mutant A3, which combines the mutations in mutants A1 and A2, and mutant A4 (TcAgg modification at Ϫ78/ Ϫ76/Ϫ75 nt) abolished COUP-TF1 binding (Fig. 7A). These results suggest that the Ϫ79/Ϫ75 nt half-site is critical for COUP-TF1 binding and that the nucleotides immediately upstream of this motif influence COUP-TF1 binding, to a lesser degree
As RXR␣ alone was unable to bind element A, the results suggest that a complex of hepatocyte nuclear factor 4 (HNF4)-RXR␣ can form on element A
Summary
Plasmids and AT Gene Deletion Constructs—To detect AT gene promoter activity, a 6900-base pair HindIII fragment of a normal genomic human AT clone, starting 4800 nt upstream of the first exon and ending 2100 nt downstream of the first exon within the first IVS (Fig. 1), was subcloned into pUC19 (Stratagene, La Jolla, CA). This subclone and deletions derived therefrom were inserted into pSVOA-L⌬5Ј, a luciferase reporter plasmid lacking eukaryotic regulatory sequences (5). Extracts containing 10 g of protein were incubated for 15 min with
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