CPF: an Orphan Nuclear Receptor That Regulates Liver-Specific Expression of the Human Cholesterol 7␣-Hydroxylase Gene

Proc. Natl. Acad. Sci. USA Vol. 96, pp. 6660–6665, June 1999 Biochemistry CPF: An orphan nuclear receptor that regulates liver-specific expression of the human cholesterol 7a-hydroxylase gene MASAHIRO NITTA*, SHERRY KU,CHALINE BROWN,ARTHUR Y. OKAMOTO, AND BEI SHAN† Biology Department, Tularik Inc., Two Corporate Drive, South San Francisco, CA 94080 Communicated by Steven L. McKnight, University of Texas Southwestern Medical Center, Dallas, TX, April 12, 1999 (received for review February 17, 1999) ABSTRACT Cholesterol 7a-hydroxylase is the first and Although the sterol regulatory element binding protein rate-limiting enzyme in a pathway through which cholesterol pathway, which is responsible for regulating genes involved in is metabolized to bile acids. The gene encoding cholesterol cholesterol uptake and biosynthesis, is well characterized, the 7a-hydroxylase, CYP7A, is expressed exclusively in the liver. molecular basis for cholesterol catabolism is largely unknown. Overexpression of CYP7A in hamsters results in a reduction of The major catabolic pathway for cholesterol removal is the serum cholesterol levels, suggesting that the enzyme plays a production of bile acids, which occurs exclusively in the liver central role in cholesterol homeostasis. Here, we report the (10). Cholesterol 7a-hydroxylase (Cyp7a), a member of the identification of a hepatic-specific transcription factor that cytochrome P450 family, is the first and rate-limiting enzyme binds to the promoter of the human CYP7A gene. We designate in a major bile acid biosynthetic pathway (11). The expression this factor CPF, for CYP7A promoter binding factor. Mutation of CYP7A is tightly regulated. The CYP7A gene is expressed of the CPF binding site within the CYP7A promoter abolished exclusively in the liver where it is induced by dietary cholesterol hepatic-specific expression of the gene in transient transfec- and suppressed by bile acids (11–13). Several independent lines tion assays. A cDNA encoding CPF was cloned and identified of evidence indicate that cholesterol catabolism plays a central as a human homolog of the Drosophila orphan nuclear recep- role in cholesterol homeostasis. Treatment of laboratory an- tor fushi tarazu F1 (Ftz-F1). Cotransfection of a CPF expres- imals with colestipol or cholestyramine, two bile acid binding sion plasmid and a CYP7A reporter gene resulted in specific induction of CYP7A-directed transcription. These observa- resins, decreases serum cholesterol levels (11, 12, 14, 15). In tions suggest that CPF is a key regulator of human CYP7A gene addition, overexpression of the CYP7A gene in hamsters expression in the liver. reduces total cholesterol and low density lipoprotein cholesterol levels (16). Thus, regulating the production of Cyp7a represents a potential therapeutic strategy for the discovery of In mammalian cells, cholesterol is an essential membrane new cholesterol-lowering drugs. component and is required for the synthesis of both sterols and HepG2 cells, a hepatoma-derived cell line, were used as a nonsterols necessary for normal cell function. Excess cholesterol causes the formation of toxic precipitates in cells, which model system to investigate the molecular mechanisms under- may accumulate on arterial walls and eventually lead to lying hepatic-specific expression of the human CYP7A gene atherosclerosis (1). It is therefore crucial that cholesterol levels (17). In this report, we used DNase I hypersensitivity mapping are maintained under tight control at all times. Three major to characterize the human CYP7A promoter. These studies led regulatory pathways are involved in the maintenance of cel- to the discovery of a hepatic-specific regulatory element within lular cholesterol homeostasis: (i) uptake of dietary cholesterol the CYP7A promoter. We then cloned the gene encoding a via the low density lipoprotein (LDL) receptor, (ii) endoge- CYP7A promoter binding protein and identified it as a human nous cholesterol biosynthesis, and (iii) metabolic conversion of homolog of the orphan nuclear receptor fushi tarazu F1 cholesterol to bile acids (2). The link among these regulatory (Ftz-F1) from Drosophila (18). This transcription factor, des- pathways is cholesterol itself. Cholesterol serves as a feedback ignated CYP7A promoter binding factor (CPF), represents a or feed-forward signal, coordinating the expression of key specific transcriptional inducer of human CYP7A gene expres- genes whose products are involved in these pathways (3). When sion. intracellular cholesterol levels are elevated, the transcription of genes encoding the LDL receptor and cholesterol biosyn- MATERIALS AND METHODS thetic enzymes [including hydroxymethyl glutaryl (HMG)- CoA synthase and HMG-CoA reductase] is suppressed. This Cells and Plasmids. HepG2 (a human hepatoma cell line), negative feedback process is mediated by a family of transcrip- HEK293 (a transformed human embryonic kidney cell line), tion factors designated sterol regulatory element binding and Caco2 (a human colon adenocarcinoma cell line) were proteins (SREBPs) (4–6). SREBPs contain an N-terminal purchased from American Type Culture Collection. SV589, a transcription factor domain, a DNA-binding basic helix–loop– transformed human fibroblast cell line, was a gift from Michael helix–leucine zipper motif, two hydrophobic transmembrane Brown and Joseph Goldstein (University of Texas Southwest- domains that anchor the protein in the endoplasmic reticulum ern Medical Center, Dallas). Cells were cultured in DMEMy (ER), and a C-terminal regulatory domain (6). When intra- Ham’s F-12 (1:1) supplemented with 10% FCS at 37°C, 5% cellular cholesterol levels are low, a two-step proteolytic CO2 in a humidified incubator. cascade releases the N-terminal transcription factor domain of SREBP from the ER membrane (7–9). The transcription Abbreviations: CYP7A, cholesterol 7a-hydroxylase; CPF, CYP7A factor then enters the nucleus and activates sterol response promoter binding factor; EMSA, electrophoretic mobility-shift assay; element-regulated genes (7–9). mLRH-1, mouse liver receptor homolog. Data deposition: The sequence reported in this paper has been The publication costs of this article were defrayed in part by page charge deposited in the GenBank database (accession no. AF146343). *Present address: Sumitomo Pharmaceuticals Research Center, 1–98, payment. This article must therefore be hereby marked ‘‘advertisement’’ in Kasugadenaka 3-chome, Konohana-ku, Osaka, Japan. accordance with 18 U.S.C. §1734 solely to indicate this fact. †To whom reprint requests should be addressed. e-mail: shan@ PNAS is available online at www.pnas.org. tularik.com. 6660 Downloaded by guest on September 24, 2021 Biochemistry: Nitta et al. Proc. Natl. Acad. Sci. USA 96 (1999) 6661 pGL3CYP7Awt was constructed by subcloning the 2716y (1 3 107) were cultured in media containing 100 mCiyml of 114 fragment of the human CYP7A gene (a gift from David [35S]methionine for 90 min. Cells were harvested and then Russell, University of Texas Southwestern Medical Center) lysed by three freeze-thaw cycles in buffer containing 50 mM into the pGL3-luciferase reporter plasmid (Promega). TriszHCl (pH 7.5), 125 mM NaCl, 5 mM EDTA, and 0.1% pGL3CYP7Am-129y130 and pGL3CYP7Am-61y62 contain (volyvol) NP-40. Cell lysates then were used for immunopre- mutations at positions 2129 and 2130 (GG to TT) and 261 cipitation with the anti-CPF antibody. Precipitated samples and 262 (AA to TC), respectively. The two base-pair substi- were resolved by 10% SDSyPAGE, and the gels were dried and tutions were introduced into pGL3CYP7Awt by using the exposed to x-ray film. ExSite mutagenesis kit (Stratagene). pfCPF contains a Flag 9 epitope-tagged sequence at the 5 end of the CPF gene cloned RESULTS into pcDNA3 (Invitrogen). Nuclear receptors used in this study were cloned by PCR using QUICK-Clone cDNA pur- DNase I-Hypersensitive Site Mapping of the Human CYP7A chased from CLONTECH. Gene. The DNase I-hypersensitive mapping technique was DNase I Hypersensitivity Mapping. HepG2, HEK293, or used to identify potential hepatic-specific regulatory regions of Caco2 cells (3 3 106) were harvested and lysed in a buffer (1.5 the human CYP7A gene. DNase I hypersensitivity is known to ml) containing 50 mM TriszHCl (pH 7.9), 100 mM KCl, 5 mM be associated with the open chromatin conformations of y MgCl2, 0.05% (vol vol) saponin, 200 mM 2-mercaptoethanol, regulatory regions near transcriptionally active genes (22). and 50% (volyvol) glycerol. Nuclei were collected by centrif- Nuclei prepared from HepG2, HEK293, and Caco2 cells were ugation and resuspended in a buffer containing 100 mM NaCl, treated with increasing amounts of DNase I. The DNAs then z 3 50 mM Tris HCl (pH 7.9), 3 mM MgCl2,1mMDTT,1 were extracted, digested with PstI, transferred onto nylon, and complete protease inhibitor mixture (Boeringer Mannheim), hybridized with a radio-labeled fragment containing nucleo- and sequentially diluted DNase I (0.6, 1.7, or 5 unitsyml). tides 2944 to 2468 of the CYP7A gene. As indicated in Fig. 1A, Nuclei suspensions were incubated at 37°C for 20 min. The this probe hybridized to the predicted 5-kb PstI fragment in reactions were terminated by addition of EDTA to a final DNA isolated from all cell types examined. However, a second concentration of 100 mM. After RNase A and Proteinase K 2.8-kb DNA fragment was detected with the same probe and treatment, genomic DNA was prepared and subjected to found only in HepG2 cells. As DNase I treatment was ex- Southern hybridization (19). tended, the intensity of the 2.8-kb DNA band increased, while Electrophoretic Mobility-Shift Assay (EMSA). Nuclear ex- the intensity of the parental 5-kb DNA band diminished tracts were prepared from cultured cells by the method of concomitantly. These observations revealed the existence of a Schreiber et al. (20), except that KCl was used instead of NaCl DNase I-hypersensitive site located roughly 200 bp upstream at the indicated concentration. In vitro transcription and from the CYP7A transcription start site. The 2.8-kb band was translation reactions were performed with the TNT system observed only in HepG2 cells and not in HEK293 or Caco2 (Promega).

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