Abstract
The gene for cholesterol 7α-hydroxylase (CYP7A1) contains a sequence at nt −149 to −118 that was found to play a large role in determining the overall transcriptional activity and regulation of the promoter. Hepatocyte nuclear factor 4 (HNF4) and chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) synergistically activate transcription of the CYP7A1 promoter. Transactivation of CYP7A1 by HNF4 in the human hepatoma cell line, HepG2, was enhanced by cotransfection with COUP-TFII or the basal transcription element binding protein (BTEB). HNF4 prepared from rat liver nuclear extracts bound to oligomers homologous to the nt −146 to −134 sequences in electrophoretic mobility shift assays (EMSA), which corresponded to a conserved region containing a direct repeat of hormone response elements spaced by one nucleotide (DR1). The sequences surrounding this DR1 were found to be essential for the HNF4 transactivation. In vitro-translated COUP-TFII was found to bind the adjacent sequences from nt −139 to −128 (DR0), but COUP-TFII interacted with this region at a much lower affinity than to the COUP-TFII-site at nt −72 to −57 (DR4). Mutations at nt −139 to −128 or nt −72 to −57 reduced the COUP-TFII and HNF4 synergy; however, these COUP-TFII-binding sequences were not absolutely required for the cooperative effect of HNF4 and COUP-TFII on transactivation. These results indicated that the observed transactivation was the result of protein/protein interactions facilitated by the juxtaposition of the binding elements. —Stroup, D., and J. Y. L. Chiang. HNF4 and COUP-TFII interact to modulate transcription of the cholesterol 7α-hydroxylase gene (CYP7A1).
Highlights
The gene for cholesterol 7␣-hydroxylase (CYP7A1) contains a sequence at nt ؊149 to ؊118 that was found to play a large role in determining the overall transcriptional activity and regulation of the promoter
A number of cholesterol 7␣-hydroxylase gene (CYP7A1) promoter constructs with mutations were tested for their ability to be transactivated by Hepatocyte nuclear factor 4 (HNF4), COUP-TFII and basal transcription element binding protein (BTEB) at ratios found to stimulate transcription of the reporter constructs proportional to the mass of expression plasmid used, as determined previously [13] for HNF4, [11] for COUP-TFII and [9] for BTEB
This cooperative effect could have been due to protein/protein interactions between HNF4 and COUP-TFII without the need for COUPTFII binding to the promoter, as was described previously [19], or to other sequences being able to substitute for the strong COUP-TFII-activation site at nt Ϫ74 to Ϫ54
Summary
The gene for cholesterol 7␣-hydroxylase (CYP7A1) contains a sequence at nt ؊149 to ؊118 that was found to play a large role in determining the overall transcriptional activity and regulation of the promoter. The wild-type reporter construct, p Ϫ416/ϩ32, encoding the rat CYP7A1 sequences from nt Ϫ416 to ϩ32, showed greater luciferase activity with both HNF4 and COUP-TFII expressed together than either transcription factor separately (Fig. 1A).
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