The sequence of the putative DNA-binding domain for steroidogenic factor 1 (SF-1) exactly the corresponding sequence of the mouse homolog of the Drosophila transcription factor Fushi tarazu-factor I. SF1 coordinately regulates the expression of three enzymes that are required for the biosynthesis of corticosteroids: cholesterol side chain cleavage enzyme, steroid 21-hydroxylase, and the aldosterone synthase isozyme of steroid 11 beta-hydroxylase. SF-1 interacts with the related promoter elements from these steroidogenic enzymes. SF-1 binds six steroidogenic regulatory elements. This strongly supports the model that a steroidogenic cell-selective protein interacts with related promoter elements from three steroidogenic enzymes to regulate their coordinate expression. The recognition sequence of SF-1 closely resembles the sequences of nuclear hormone receptor family members, suggesting that SF-1 may belong to this supergene family (Lala, 1992).

The cytochrome P450 steroid hydroxylases are coordinately regulated by SF-1. SF-1 coexpression increases promoter activity of the 21-hydroxylase 5'-flanking region in transfection experiments. A second FTZ-F1 homolog, embryonal long terminal repeat-binding protein (ELP), was recently isolated from embryonal carcinoma cells. SF-1 and ELP cDNAs are virtually identical for 1017 base pairs, including putative DNA-binding domains, but diverge at their 5'- and 3'-ends. One genomic clone contains both SF-1- and ELP-specific sequences, confirming their origin from a single gene. Characterization of this gene defines shared exons encoding common regions and alternative promoters and 3'-exons leading to difference ELP transcripts. Transcripts are not detected from embryonic day 8 to adult, consistent with its previous isolation from embryonal carcinoma cells and its postulated role in early embryonic development (Ikeda, 1993).

SF-1, a nuclear receptor that regulates gene expression of the cytochrome P450 steroid hydroxylases, and ELP, an embryonal protein that suppresses expression of the Moloney murine leukemia virus LTR, are isoforms transcribed from the same gene by alternative promoter usage and splicing. SF-1 is the mammalian homolog of the Drosophila fushi-tarazu factor 1 (FTZ-F1) gene. The mouse and human genes are located in the homologous regions of mouse Chr 2 and human Chr 9, respectively (Taketo, 1995).

An E box is found in the transcriptional element in the 5'-upstream region of the rat SF-1 gene. There is also a steroidogenic cell-specific transcriptional element in the first intron of the gene. SF-1 itself binds to the intronic element (SF-1 site). Thus, two elements are essential for the full transcriptional activity of the SF-1 gene. The chromatin structure around the SF-1 site and the E box is "open up" in the adrenal glands and Y-1 cells. In the liver, the same structure is "closed down." These observations indicate that the mftz-f1 gene is controlled by an autoregulatory loop in the steroidogenic tissues. The autoregulatory mechanism seems to be necessary to keep the mftz-f1 gene activated and thus maintain the tissue differentiation (Nomura, 1996).

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. A hepatic-specific transcription factor that binds to the promoter of the human CYP7A gene has been identified. This factor has been designated CPF, for CYP7A promoter binding factor. Mutation of the CPF binding site within the CYP7A promoter abolishes 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 Ftz-F1. Cotransfection of a CPF expression plasmid and a CYP7A reporter gene results in specific induction of CYP7A-directed transcription. These observations suggest that CPF is a key regulator of human CYP7A gene expression in the liver (Nitta, 1999).

The high density lipoprotein (HDL) receptor mediates the uptake of cholesterol and cholesteryl esters, substrates for steroidogenesis, from an HDL particle in the adrenal gland and gonads. Treatment of rat luteal cells with 1 mM (Bu)2cAMP for 24 h dramatically induces (118-fold) HDL receptor messenger RNA levels. The rat HDL receptor promoter contains a steroidogenic factor-1 (SF-1)-binding site (SFBd; 5'-TCAAGGCC-3') through which SF-1 protein binds and activates transcription of this gene in both human HTB9 bladder carcinoma and mouse Y1 tumor cells, an effect that is enhanced by cAMP. These observations demonstrate that this motif is required for both basal and cAMP-induced regulation of the HDL receptor gene. Cotransfection studies in Kin 8 cells, a Y1 cell line resistant to cAMP activation as a result of a mutation in the protein kinase A (PKA) regulatory subunit, shows that a functional PKA is required for cAMP induction of HDL receptor gene transcription. Deleting the activation function-2 domain (amino acids 448-461) or mutating Ser430, a potential consensus phosphorylation site for PKA in the SF-1 protein, decreases both basal and cAMP-induced activation of the HDL receptor promoter. These data suggest that these regions within the SF-1 protein are required for both basal and cAMP-induced regulation of the HDL receptor gene. The mediation of cAMP responsiveness of the HDL receptor gene by SF-1 suggests how important this trans-acting factor is in steroid hormone synthesis by assuring that all required elements (substrate and enzymes) are present when they are needed for maximal steroid production (Lopez, 1999).

Using a mouse Leydig tumor cell line, the mechanisms involved in thyroid hormone-induced steroidogenic acute regulatory (StAR) protein gene expression, and steroidogenesis has been investigated. Triiodothyronine (T3) induces an approximately 3.6-fold increase in the steady-state level of StAR mRNA, which parallels the level of the acute steroid response ( approximately 4.0-fold), as monitored by quantitative reverse transcriptase-polymerase chain reaction assay and progesterone production, respectively. The T3-stimulated progesterone production is effectively inhibited by actinomycin-D or cycloheximide, indicating the requirement of on-going mRNA and protein synthesis. T3 displays the highest affinity of [125I]iodo-T3 binding and is most potent in stimulating StAR mRNA expression. In accordance, T3 significantly increases testosterone production in primary cultures of adult mouse Leydig cells. The T3 and human chorionic gonadotropin (hCG) effects on StAR expression are similar in magnitude and additive. Cells expressing steroidogenic factor 1 (SF-1) show marginal elevation of StAR expression, but coordinately increase T3-induced StAR mRNA expression and progesterone levels. In contrast, overexpression of DAX-1, a repressor of SF-1 activity, markedly diminishes the SF-1 mRNA expression, and concomitantly abolishes T3-mediated responses. Noteworthy is the fact that T3 augments the SF-1 mRNA expression, while inhibition of the latter by DAX-1 strongly impairs T3 action. Northern hybridization analysis reveals four StAR transcripts that increase 3-6-fold following T3 stimulation. These observations clearly identify a regulatory cascade of thyroid hormone-stimulated StAR expression and steroidogenesis that provides novel insight into the importance of a thyroid-gonadal connection in the hormonal control of Leydig cell steroidogenesis (Manna, 1999).

The steroidogenic acute regulatory (StAR) protein mediates the rate-limiting step of steroidogenesis, which is the transfer of cholesterol to the inner mitochondrial membrane. In steroidogenic tissues, StAR expression is acutely regulated by trophic hormones through a cAMP second messenger pathway, leading to increased StAR mRNA levels within 30 min, reaching maximal levels after 4-6 h of stimulation. The molecular mechanisms underlying such regulation remain unknown. The StAR promoter was examined for putative transcription factor-binding sites that may regulate transcription in a developmental and/or hormone-induced context. Two putative CCAAT/enhancer binding protein (C/EBP) DNA elements have been identified at -113 (C1) and -87 (C2) in the mouse StAR promoter. C/EBP beta binds with high affinity to C1, but C2 proves to be a low-affinity C/EBP site. Functional analysis of these sites in the murine StAR promoter show that mutation of one or both of these binding sites decreases both basal and (Bu)2cAMP-stimulated StAR promoter activity in MA-10 Leydig tumor cells, without affecting the fold activation [(Bu)2cAMP-stimulated/basal] of the promoter. These two C/EBP binding sites are required for steroidogenic factor-1 (SF-1)-dependent transactivation of the StAR promoter in a nonsteroidogenic cell line. These data indicate that in addition to SF-1, C/EBP beta is involved in the transcriptional regulation of the StAR gene and may play an important role in developmental and hormone-responsive regulation of steroidogenesis (Reinhart, 1999).

Steroidogenic factor 1 (SF-1) is an orphan nuclear receptor that serves as an essential regulator of many hormone-induced genes in the vertebrate endocrine system. The apparent absence of a SF-1 ligand prompted speculation that this receptor is regulated by alternative mechanisms involving signal transduction pathways. Maximal SF-1-mediated transcription and interaction with general nuclear receptor cofactors depends on phosphorylation of a single serine residue (Ser-203) located in a major activation domain (AF-1) of the protein. Moreover, phosphorylation-dependent SF-1 activation is likely mediated by the mitogen-activated protein kinase (MAPK) signaling pathway. It is proposed this single modification of SF-1 and the subsequent recruitment of nuclear receptor cofactors couple extracellular signals to steroid and peptide hormone synthesis, thereby maintaining dynamic homeostatic responses in stress and reproduction (Hammer, 1999).

The 5'-region of the murine N-methyl-d-aspartate (NMDA) receptor channel subunit NR2C (GluRepsilon3) gene has been cloned and the cis- and trans-activating regulatory elements responsible for its tissue specific activity have been characterized. By using a native epsilon3-promoter/lacZ-construct and various 5'-deletion constructs, beta-galactosidase expression in non-neuronal NIH3T3 cells and in neuronal epsilon3-gene-expressing HT-4 cells were compared. Large parts of the epsilon3 promoter are shown to be responsible for the repression of the epsilon3 gene in non-neuronal cells. Deletion of exon 1 sequences leads to an enhancement of epsilon3 transcription, suggesting a role for the 5'-untranslated region in epsilon3 gene regulation. Sequence analysis of the promoter region reveals potential binding sites for the transcription factor Sp1, the murine fushi tarazu factor1 (FTZ-F1) homologs, embryonic LTR binding proteins (ELP1,2,3) and steroidogenic factor (SF-1), as well as for the chicken ovalbumin upstream promoter transcription-factor (COUP-TF). Electrophoretic mobility shift assays confirm specific binding of Sp1, SF-1 and COUP-TFI. Whereas point mutation studies indicate that, in neuronal HT-4 cells, Sp1 is apparently not critically involved in basal epsilon3 gene transcription, SF1 is a positive regulator. This was evident from a selective enhancement of epsilon3-promoter-driven reporter gene expression upon cotransfection of an SF1-expression vector, which was reverted by deletion and point mutation of the SF1 binding site (Pieri, 1999).

The Dax-1 gene encodes a protein that is structurally related to members of the orphan nuclear receptor superfamily. Dax-1 is coexpressed with another orphan nuclear receptor, steroidogenic factor-1 (SF-1), in the adrenal, gonads, hypothalamus, and pituitary gland. Mutations in Dax-1 cause adrenal hypoplasia congenita, a disorder that is characterized by adrenal insufficiency and hypogonadotropic hypogonadism. These developmental and endocrine abnormalities are similar to those caused by disruption of the murine Ftz-F1 gene (which encodes SF-1), suggesting that these nuclear receptors act along the same developmental cascade. Cloning of the murine Dax-1 gene revealed a candidate SF-1-binding site in the Dax-1 promoter. In transient expression assays in SF-1-deficient JEG-3 cells, SF-1 stimulates expression of the Dax-1 promoter. However, deletion or mutation of the consensus SF-1-binding site does not eliminate SF-1 stimulation. Further analyses have revealed the presence of a cryptic SF-1 site that creates an imperfect direct repeat of the SF-1 element. When linked to the minimal thymidine kinase promoter, each of the isolated SF-1 sites is sufficient to mediate transcriptional regulation by SF-1. Mutation of both SF-1 sites eliminates SF-1 binding and stimulation of the Dax-1 promoter. Unexpectedly, mutation of either half of the composite SF-1 sites increases basal activity in JEG-3 cells, suggesting interaction of a repressor protein. Gel shift analyses of the composite response element reveals an additional complex that is not supershifted by SF-1 antibodies. This complex was eliminated by mutation of either half-site, and it was supershifted by antibodies against chicken ovalbumin upstream promoter-transcription factor (COUP-TF). It is proposed that Dax-1 is stimulated by SF-1, and that SF-1 and COUP-TF provide antagonistic pathways that converge upon a common regulatory site (Yu, 1998).

Enhancer II (ENII) of hepatitis B virus (HBV) is one of the essential cis-elements for the transcriptional regulation of HBV gene expression. Its function is highly liver-specific, suggesting that liver-enriched transcriptional factors play critical roles in regulating the activity of ENII. In this report, a novel hepatocyte transcription factor, which binds specifically to the B1 region (AACGACCGACCTTGAG) within the major functional unit (B unit) of ENII, has been cloned from a human liver cDNA library by yeast one-hybrid screening, and has been demonstrated to trans-activate ENII via the B1 region. This factor has been named hB1F, for human B1-binding factor. Amino acid analysis revealed this factor structurally belongs to the nuclear receptor superfamily. Based on the sequence similarities, hB1F is characterized to be a novel human homolog of the orphan receptor FTZ-F1. A splicing isoform of hB1F (hB1F-2) was identified, which has an extra 46 amino acid residues in the A/B region. Examination of the tissue distribution has revealed an abundant 5.2-kilobase transcript of hB1F is present specifically in human pancreas and liver. Interestingly, an additional transcript of 3.8 kilobases was found to be present in hepatoma cells HepG2. Fluorescent in situ hybridization has mapped the gene locus of hB1F to the region q31-32.1 of human chromosome 1. Altogether, this study provides the first report that a novel human homolog of FTZ-F1 binds and regulates ENII of HBV. The potential roles of this FTZ-F1 homolog in tissue-specific gene regulation, in embryonic development, as well as in liver carcinogenesis are discussed (Li, 1998).