HNF 4 and COUP - TFII interact to modulate transcription of the cholesterol 7 a - hydroxylase gene ( CYP 7 A 1 )

The gene for cholesterol 7 a -hydroxylase ( CYP7A1 ) contains a sequence at nt 2 149 to 2 118 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 2 146 to 2 134 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 2 139 to 2 128 (DR0), but COUP-TFII interacted with this region at a much lower affinity than to the COUPTFII-site at nt 2 72 to 2 57 (DR4). Mutations at nt 2 139 to 2 128 or nt 2 72 to 2 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 7 a -hydroxylase gene ( CYP7A1 ). J. Lipid Res. 2000. 41: 1–11. Supplementary key words bile acid synthesis • hepatocyte nuclear factor 4 • chicken ovalbumin upstream promoter transcription factor II • orphan receptors • transcriptional regulation • cholesterol metabolism The catabolism of cholesterol to bile acids in the liver is the main mechanism for cholesterol elimination from the body, and thus plays an important role in maintaining cholesterol homeostasis (1). Cholesterol 7 a -hydroxylase (EC 1.14.13.17) catalyzes the first and rate-limiting step in the neutral bile acid synthesis pathway. Transcription of the gene ( CYP7A1 ) for this enzyme is specific to the postpartum liver and regulated by a number of factors, including bile acids, hormones, and circadian rhythm (2–6). Mice in which Cyp7A1 has been inactivated have a severe phenotype; 85% of the pups do not survive past 18 days and exhibit impaired bile, lipid, and vitamin metabolism (7, 8). Because this tightly regulated gene is so important for health and is expressed in a strictly liver-specific manner, the CYP7A1 promoter has been the subject of intense study. Transcription factor binding sites for BTEB (9), C/ EBP a , C/EBP b , DBP (5, 10), COUP-TFII (11), HNF3 a (12), and HNF4 a (13) have been mapped. Of particular interest are HNF4 and COUP-TFII because of the important roles they play in regulating gene transcription in eukaryotic cells, such as defining tissue-specific expression, embryogenesis, and response to certain effectors (14). HNF4 is expressed in the liver, intestine, and kidney (15, 16), and the homodimer recognizes a direct repeat (DR) of 5 9 AGGTCA 3 9 separated by one nucleotide (DR1) as a binding site. Transcripts for this factor are detected very early in development (17) and death in utero results from disruption of HNF4 (18). For these reasons and because HNF4 is known to regulate the expression of other liver-enriched transcription factors, such as HNF1 (19), it is believed that HNF4 activity is required very early in the cascade of transcriptional events that lead to the differentiation of endodermal cells. Defects in the HNF4 gene locus have been associated with maturity-onset diabetes of the young (MODY1) (20) and late-onset non-insulindependent diabetes mellitus (NIDDM) (21). Like HNF4, COUP-TF-gene disruptions are lethal early Abbreviations: BTEB, basal transcription element binding protein; C/EBP, CAAT/enhancer binding protein; COUP-TFII, chicken ovalbumin upstream promoter transcription factor II; CYP7A1 , cholesterol 7 a -hydroxylase gene; DMEM, Dulbecco’s modified Eagle’s medium; DBP, D-site binding protein; DR, direct repeat of the hormone response element; EMSA, electrophoretic mobility shift assay; HEPES, N-(2-hydroxyethyl)piperazine-N 9 -(2-ethanesulfonic acid); HNF, hepatocyte nuclear factor; HRE, hormone response element; LSM, linker scanning mutation; LUC, luciferase; nt, nucleotide; PEPCK, phosphoenol pyruvate carboxykinase; RXR, retinoid X receptor. 1 To whom correspondence should be addressed. at P E N N S T A T E U N IV E R S IT Y , on F ebuary 3, 2013 w w w .j.org D ow nladed fom