CPF: an orphan nuclear receptor that regulates liver-specific expression of the human cholesterol 7alpha-hydroxylase gene

Abstract

Cholesterol 7alpha-hydroxylase is the first and rate-limiting enzyme in a pathway through which cholesterol is metabolized to bile acids. The gene encoding cholesterol 7alpha-hydroxylase, CYP7A, is expressed exclusively in the liver. Overexpression of CYP7A in hamsters results in a reduction of serum cholesterol levels, suggesting that the enzyme plays a central role in cholesterol homeostasis. Here, we report the identification of a hepatic-specific transcription factor that binds to the promoter of the human CYP7A gene. We designate this factor CPF, for CYP7A promoter binding factor. Mutation of the CPF binding site within the CYP7A promoter abolished hepatic-specific expression of the gene in transient transfection assays. A cDNA encoding CPF was cloned and identified as a human homolog of the Drosophila orphan nuclear receptor fushi tarazu F1 (Ftz-F1). Cotransfection of a CPF expression plasmid and a CYP7A reporter gene resulted in specific induction of CYP7A-directed transcription. These observations suggest that CPF is a key regulator of human CYP7A gene expression in the liver.

Figure 1

A hepatic-specific DNase I-hypersensitive site in the CYP7A promoter region. (A) DNAs prepared from DNase I-treated nuclei (0, 0.6, 1.7, or 5.0 units/ml) from HepG2, HEK293, and Caco2 cells were separated by 1.0% agarose gel electrophoresis and subjected to Southern hybridization. A radio-labeled fragment corresponding to base pairs −944 to −468 of the CYP7A gene was used as a probe. A 5.0-kb PstI fragment was observed in each lane. A 2.8-kb hepatic-specific fragment (lanes 3–5) is indicated by the double arrow. The lane that displays molecular size markers is indicated by M and contains the 1-kb ladder (GIBCO/BRL). The sizes (in kb) of bands of the 1-kb ladder are indicated at the left of the gel. Below the gel is diagrammed a partial restriction map of the promoter region. The vertical bars represent exons 1–3 of the CYP7A gene. The arrow between the XbaI site and the first exon represents the location of the hepatic-specific DNase I-hypersensitive site. The heavy horizontal bar denotes the location of the probe used in this experiment. The 5.0-kb PstI fragment is also diagrammed. (B) A schematic diagram indicating the location of double-stranded oligonucleotides CL1–CL7 within the CYP7A promoter region that were used as radio-labeled probes. +1 denotes the transcription start site. (C–E) Formation of hepatic-specific DNA-protein complexes. EMSAs were performed with the indicated nuclear extracts and radio-labeled, double-stranded CL1–CL7 oligonucleotide probes diagrammed in B. (C) EMSAs were performed by using nuclear extracts prepared from HepG2 and 293 cells. The arrow indicates the hepatic-specific DNA-protein complex. (D) Nuclear extracts from the indicated cell lines were used in EMSAs with CL1 as the radio-labeled probe. The arrow indicates the hepatic-specific DNA-protein complex. (E) Competition EMSAs using unlabeled CL1–CL7 fragments as competitors. An EMSA was performed by using HepG2 nuclear extracts and radio-labeled CL1 with the addition of either a 30- or 60-fold molar excess of unlabeled CL1–CL7 (indicated by triangles at the top of the gel lanes). The DNA-protein complexes are indicated by the arrows. 0, no competitor.

Figure 2

Sequences (+ strand only) of oligonucleotides containing the overlapping region of CL1 and CL2, and mapping of the binding site of CPF. The DR1 site is indicated by the tandem overlining arrows (labeled repeats A and B). The 9-bp CPF binding site is indicated by the underlining arrow (labeled repeat C). The wild-type (WT) sequence is shown above the mutant oligonucleotide sequences (M1–M19). Bases that are underlined are those that were mutated. + denotes the formation of a DNA-protein complex, and − the absence of such a complex. The CPF-binding site within the CYP7A gene promoter and the Ftz-F1 consensus binding site are listed at the bottom, along with a sequence comparison of the binding site from human, rat, and hamster CYP7A promoters.

Figure 3

Effect of mutations in the CPF-binding site on CYP7A gene expression. The indicated cell lines were transfected with reporter constructs by using the calcium phosphate method (19). Luciferase assays were performed as described in Materials and Methods. Relative luciferase units were normalized to β-galactosidase activity to control for variations in transfection efficiency. The cross-out refers to the presence of two point mutations (as described in Materials and Methods). The pGL3 reporter is a vector-only control.

Figure 4

(A) cDNA and predicted amino acid sequence of CPF. A human liver cDNA library was screened with a probe based on the conserved DNA-binding domain of the Drosophila Ftz-F1 nuclear receptor. Nucleotide positions are shown on the right. The predicted amino acid sequence is written in single-letter abbreviations below the nucleotide sequence. A schematic representation of CPF, mLRH-1, and dFtz-F1 is shown below the CPF nucleotide and protein sequence. We have noted that during preparation of our manuscript, a similar but shorter, cDNA sequence with an identical coding region and slightly different 5′ and 3′ untranslated regions was reported by Li et al. (41). (B) Amino acid sequence alignment of mLRH-1 and human CPF, along with CPF variants 1 and 2. The amino acids conserved among all four clones are highlighted in bold. The structural/functional domains of CPF are illustrated to the right of the alignment with percent identity to mLRH-1 in parenthesis. A/B: N-terminal variable region; C: DNA-binding domain; D: Variable hinge region; E/F: Ligand-binding domain. Arrows above the sequence alignment indicate boundaries of the domains.

Figure 4

(A) cDNA and predicted amino acid sequence of CPF. A human liver cDNA library was screened with a probe based on the conserved DNA-binding domain of the Drosophila Ftz-F1 nuclear receptor. Nucleotide positions are shown on the right. The predicted amino acid sequence is written in single-letter abbreviations below the nucleotide sequence. A schematic representation of CPF, mLRH-1, and dFtz-F1 is shown below the CPF nucleotide and protein sequence. We have noted that during preparation of our manuscript, a similar but shorter, cDNA sequence with an identical coding region and slightly different 5′ and 3′ untranslated regions was reported by Li et al. (41). (B) Amino acid sequence alignment of mLRH-1 and human CPF, along with CPF variants 1 and 2. The amino acids conserved among all four clones are highlighted in bold. The structural/functional domains of CPF are illustrated to the right of the alignment with percent identity to mLRH-1 in parenthesis. A/B: N-terminal variable region; C: DNA-binding domain; D: Variable hinge region; E/F: Ligand-binding domain. Arrows above the sequence alignment indicate boundaries of the domains.

Figure 5

Functional similarity of in vitro-synthesized and endogenous CPF. (A) EMSAs with in vitro-synthesized CPF-1 (IVTCPF) and CL1 as the radio-labeled probe. Lanes 2–4, 0.1, 0.5 and 1.0 μl of in vitro-synthesized CPF-1 (IVTCPF), respectively, and lane 1, 1 μg of HepG2 nuclear extract (NE). The arrow indicates the CPF binding complex. (B) Competition EMSAs. EMSAs were performed as described above with the addition of unlabeled competitor oligonucleotides, including wild type (wt) and those with mutations in either the CPF binding site (M5) or a region outside of the CPF binding site (M4). A 60-fold molar excess of each competitor was used. The arrow denotes the location of the bound DNA-protein complex. NE, HepG2 nuclear extract; −, no competitor, IVTCPF, in vitro-translated CPF. (C) EMSA in the presence of CPF-specific antibodies. EMSAs were performed with HepG2 nuclear extracts and in vitro-synthesized CPF with the addition of either preimmune serum (Preimmu) or rabbit anti-CPF antibodies (Immu). Lanes 1 and 6, no antibody (0); lanes 2, 4, 7, and 9, 0.1 μl of serum; lanes 3, 5, 8, and 10, 1 μl of serum. The arrow denotes the location of the bound DNA-protein complex. (D) Immunoprecipitation of CPF in HepG2 cells. HEK293 and HepG2 cells were grown in the presence of [³⁵S]methionine. Preimmune (P) and rabbit anti-CPF antibodies (I) then were used in immunoprecipitation experiments with HEK293 (lanes 1 and 2) and HepG2 (lanes 3 and 4) extracts. ³⁵S-methionine-labeled in vitro-synthesized CPF (IVTCPF) was loaded into lane 5. The precipitated samples were separated by 10% SDS/PAGE, and gel was dried and exposed to x-ray film. The arrow denotes the immunoprecipitated and in vitro-synthesized CPF. Molecular size markers (in kd) are shown on the left.

Figure 6

Activation of CYP7A gene expression by CPF. Various expression plasmids and control plasmid pcDNA3 (0.5 μg) were cotransfected into HEK293 cells with 0.5 μg of luciferase reporter plasmid containing the human CYP7A promoter sequence (−716 to +14). (A) Full-length CPF, liver X receptor α (LXRα), retinoid X receptor α (RXRα), and hepatic nuclear factor 4α (HNF4α). (B) Full-length CPF, and its variants. (C) Control plasmid pcDNA3 or an expression plasmid encoding a flag-tagged full-length CPF (0, 0.015, 0.05, 0.15, or 0.5 μg) was cotransfected into 293 cells with the human CYP7A promoter luciferase reporter plasmid. Relative luciferase activity normalized to β-galactosidase activity is shown for each transfection.

Figure 7

Tissue blot analyses of CPF and CYP7A mRNA expression. (A) The tissue blot was hybridized with radio-labeled CPF cDNA. (B) The same blot was stripped and rehybridized either with a riboprobe derived from CYP7A cDNA or with (C) radio-labeled β-actin cDNA as a control. Molecular size markers (in kb) are shown at the left of A and B.