Steroidogenic Factor-1 Binding Site Research Articles

NR5A1/SF-1 Collaborates with Inhibin α and the Androgen Receptor.

Steroidogenic factor 1 (SF-1) is a nuclear receptor that regulates steroidogenesis and reproductive development. NR5A1/SF-1 variants are associated with a broad spectrum of phenotypes across individuals with disorders of sex development (DSDs). Oligogenic inheritance has been suggested as an explanation. SF-1 interacts with numerous partners. Here, we investigated a constellation of gene variants identified in a 46,XY severely undervirilized individual carrying an ACMG-categorized 'pathogenic' NR5A1/SF-1 variant in comparison to the healthy carrier father. Candidate genes were revealed by whole exome sequencing, and pathogenicity was predicted by different in silico tools. We found variants in NR1H2 and INHA associated with steroidogenesis, sex development, and reproduction. The identified variants were tested in cell models. Novel SF-1 and NR1H2 binding sites in the AR and INHA gene promoters were found. Transactivation studies showed that wild-type NR5A1/SF-1 regulates INHA and AR gene expression, while the NR5A1/SF-1 variant had decreased transcriptional activity. NR1H2 was found to regulate AR gene transcription; however, the NR1H2 variant showed normal activity. This study expands the NR5A1/SF-1 network of interacting partners, while not solving the exact interplay of different variants that might be involved in revealing the observed DSD phenotype. It also illustrates that understanding complex genetics in DSDs is challenging.

A mutation inactivating the distal SF1 binding site on the human anti-Müllerian hormone promoter causes persistent Müllerian duct syndrome Acknowledgments and Funding

The persistent Mullerian duct syndrome (PMDS) is a 46,XY disorder of sexual development characterized by the persistence of Mullerian duct derivatives, uterus and tubes, in otherwise normally masculinized males. The condition, transmitted as a recessive autosomal trait, is usually due to mutations in either the anti-Mullerian hormone (AMH) gene or its main receptor. Many variants of these genes have been described, all targeting the coding sequences. We report the first case of PMDS due to a regulatory mutation. The AMH promoter contains two binding sites for steroidogenic factor 1 (SF1), one at -102 and the other at -228. Our patient carries a single base deletion at -225, significantly decreasing its capacity for binding SF1, as measured by electrophoresis mobility shift assay. Furthermore, by linking the AMH promoter to the luciferase gene, we show that the transactivation capacity of the promoter is significantly decreased by the mutation, in contrast to disruption of the -102 binding site. To explain the difference in impact we hypothesize that SF1 could partially overcome the lack of binding to the -102 binding site by interacting with a GATA4 molecule linked to a nearby response element. We show that disruption of both the -102 SF1 and the -84 GATA response elements significantly decreases the transactivation capacity of the promoter. In conclusion, we suggest that the distance between mutated SF1 sites and potentially rescuing GATA-binding motifs might play a role in the development of PMDS.

Expression and regulation of GnRHR2 gene and testosterone secretion mediated by GnRH2 and GnRHR2 within porcine testes

Gonadotropin-releasing hormone 2 receptor (GnRHR2) together with its cognate ligand involves in regulating reproductive behavior. However, little is known concerning the effect of transcription factor steroidogenic factor1 (SF-1) regulation on porcine GnRHR2 gene expression and GnRH2 regulation mechanism in testosterone secretion through GnRHR2. Our study demonstrated that GnRHR2 transcription levels were high in porcine testis. Immunohistochemistry analyses showed that GnRHR2 immunoreactivity was strong in the Leydig cells in boar testes. Two SF-1 binding sites were predicted in GnRHR2 promoter and the second site (-159/-149) was considered to be important for GnRHR2 promoter activity through site-directed mutagenesis. The binding of SF-1 to GnRHR2 promoter was confirmed by electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP). Overexpression and knockdown experiments revealed that SF-1 could up-regulate porcine GnRHR2 expression. DNA methylation of GnRHR2 promoter CpG island also specifically regulated GnRHR2 expression. Meanwhile, our study also demonstrated GnRH2 treatment promoted the expression of SF-1 and steroidogenic acute regulatory protein (StAR), and that this treatment stimulated cAMP responsive element binding protein (CREB) phosphorylation, regulated the expression of GnRHR2, especially that of GnRHR2-X1, and promoted testosterone secretion in porcine Leydig cells. We speculated that testosterone secretion mediated by GnRH2 and GnRHR2 (mainly GnRHR2-X1) was regulated by phosphorylated CREB interacting with SF-1 to control StAR expression. Taken together, the present study indicates that SF-1 and GnRH2 are the essential regulatory factors for GnRHR2 expression. This study also explores the regulation mechanism of testosterone secretion mediated by GnRH2 and GnRHR2 in porcine Leydig cells.

Hidden heterogeneity of transcription factor binding sites: A case study of SF-1

Steroidogenic factor 1 (SF-1) belongs to a small group of the transcription factors that bind DNA only as a monomer. Three different approaches—Sitecon, SiteGA, and oPWM—constructed using the same training sample of experimentally confirmed SF-1 binding sites have been used to recognize these sites. The appropriate prediction thresholds for recognition models have been selected. Namely, the thresholds concordant by false positive or negative rates for various methods were used to optimize the discrimination of steroidogenic gene promoters from the datasets of non-specific promoters. After experimental verification, the models were used to analyze the ChIP-seq data for SF-1. It has been shown that the sets of sites recognized by different models overlap only partially and that an integration of these models allows for identification of SF-1 sites in up to 80% of the ChIP-seq loci. The structures of the sites detected using the three recognition models in the ChIP-seq peaks falling within the [–5000, +5000] region relative to the transcription start sites (TSS) extracted from the FANTOM5 project have been analyzed. The MATLIGN classified the frequency matrices for the sites predicted by oPWM, Sitecon, and SiteGA into two groups. The first group is described by oPWM/Sitecon and the second, by SiteGA. Gene ontology (GO) analysis has been used to clarify the differences between the sets of genes carrying different variants of SF-1 binding sites. Although this analysis in general revealed a considerable overlap in GO terms for the genes carrying the binding sites predicted by oPWM, Sitecon, or SiteGA, only the last method elicited notable trend to terms related to negative regulation and apoptosis. The results suggest that the SF-1 binding sites are different in both their structure and the functional annotation of the set of target genes correspond to the predictions by oPWM+Sitecon and SiteGA. Further application of Homer software for de novo identification of enriched motifs in ChIP-Seq data for SF-1ChIP-seq dataset gave the data similar to oPWM+Sitecon.

Bcrp1 transcription in mouse testis is controlled by a promoter upstream of a novel first exon (E1U) regulated by steroidogenic factor-1

Alternative promoter usage is typically associated with mRNAs with differing first exons that contain or consist entirely of a 5′ untranslated region. The murine Bcrp1 (Abcg2) transporter has three alternative promoters associated with mRNAs containing alternative untranslated first exons designated as E1A, E1B, and E1C. The E1B promoter regulates Bcrp1 transcription in mouse intestine. Here, we report the identification and characterization of a novel Bcrp1 promoter and first exon, E1U, located upstream from the other Bcrp1 promoters/first exons, which is the predominant alternative promoter utilized in murine testis. Using in silico analysis we identified a putative steroidogenic factor-1 (SF-1) response element that was unique to the Bcrp1 E1U alternative promoter. Overexpression of SF-1 in murine TM4 Sertoli cells enhanced Bcrp1 E1U mRNA expression and increased Bcrp1 E1U alternative promoter activity in a reporter assay, whereas mutation of the SF-1 binding site totally eliminated Bcrp1 E1U alternative promoter activity. Moreover, expression of Bcrp1 E1U and total mRNA and Bcrp1 protein was markedly diminished in the testes from adult Sertoli cell-specific SF-1 knockout mice, in comparison to the testes from wild-type mice. Binding of SF-1 to the SF-1 response element in the E1U promoter was demonstrated by chromatin immunoprecipitation assays. In conclusion, nuclear transcription factor SF-1 is involved with the regulation of a novel promoter of Bcrp1 that governs transcription of the E1U mRNA isoform in mice. The present study furthers understanding of the complex regulation of Bcrp1 expression in specific tissues of a mammalian model.

Transcriptional regulation of human ferredoxin 1 in ovarian granulosa cells

Ferredoxin 1 (FDX1; adrenodoxin) is an iron–sulfur protein that is involved in various metabolic processes, including steroid hormone synthesis in mammalian tissues. We investigated the transcriptional regulation of FDX1 in ovarian granulosa cells. Previously, we reported that the NR5A family, including steroidogenic factor-1 (SF-1) and liver receptor homolog-1 could induce differentiation of human mesenchymal stem cells (hMSCs) into steroidogenic cells. A ChIP assay showed that SF-1 could bind to the FDX1 promoter in differentiated hMSCs. Luciferase reporter assays showed that transcription of FDX1 was synergistically activated by the NR5A family and 8Br-cAMP treatment through two SF-1 binding sites and a CRE-like sequence in a human ovarian granulosa cell line, KGN. Knockdown of FDX1 attenuated progesterone production in KGN cells. These results indicate transcription of FDX1 is regulated by the NR5A family and cAMP signaling, and participates in steroid hormone production in ovarian granulosa cells.

StarD7 Gene Expression in Trophoblast Cells: Contribution of SF-1 and Wnt-β-Catenin Signaling

Steroidogenic acute regulatory protein-related lipid transfer domain containing 7 (StarD7) is a poorly characterized member of the steroidogenic acute regulatory protein-related lipid transfer proteins, up-regulated in JEG-3 cells, involved in intracellular transport and metabolism of lipids. Previous studies dealing with the mechanisms underlying the human StarD7 gene expression led us to define the cis-acting regulatory sequences in the StarD7 promoter using as a model JEG-3 cells. These include a functional T cell-specific transcription factor 4 (TCF4) site involved in Wnt-β-catenin signaling. To understand these mechanisms in more depth, we examined the steroidogenic factor 1 (SF-1) contribution to StarD7 expression. Cotransfection experiments in JEG-3 cells point out that the StarD7 promoter is activated by SF-1, and this effect is increased by forskolin. EMSA using JEG-3 nuclear proteins demonstrated that SF-1 binds to the StarD7 promoter. Additionally, chromatin immunoprecipitation analysis indicated that SF-1 and β-catenin are bound in vivo to the StarD7 promoter. Reporter gene assays in combination with mutations in the SF-1 and TCF4 binding sites revealed that the StarD7 promoter is synergistically activated by SF-1 and β-catenin and that the TCF4 binding site (-614/-608) plays an important role in this activation. SF-1 amino acid mutations involved in the physical interaction with β-catenin abolished this activation; thus demonstrating that the contact between the two proteins is necessary for an efficient StarD7 transcriptional induction. Finally, these data suggest that β-catenin could function as a bridge between SF-1 and TCF4 forming a ternary complex, which would stimulate StarD7 expression. The SF-1 and β-catenin pathway convergence on StarD7 expression may have important implications in the phospholipid uptake and transport, contributing to the normal trophoblast development.

Differential regulation of the human CYP11A1 promoter in mouse brain and adrenals

CYP11A1 encodes the first enzyme of steroid biosynthesis, cytochrome P450scc. The expression of CYP11A1 in the nervous system allows neurosteroids to be synthesized de novo. In the classic steroidogenic tissues, adrenals and gonads, the key regulator controlling CYP11A1 expression is steroidogenic factor-1 (SF-1), but the transcriptional regulation of CYP11A1 in the brain is unclear. We recently used the 4.4-kb regulatory region of the human CYP11A1 gene to drive Cre recombinase expression in the diencephalon and midbrain. In this study, we characterized the regional-specific expression of Cre reporter in the SCC-Cre transgenic brain using a transient Cre/ROSA26R transgenic system. Mutation of either the upstream or proximal SF-1 binding site did not affect brain CYP11A1 promoter activity. The upstream SF-1 binding site, however, is required for CYP11A1 promoter function in the embryonic adrenals. The 3.8-kb promoter, like the 4.4-kb length promoter, directed Cre expression in the diencephalon, midbrain and olfactory epithelium, whereas Cre expression controlled by the 2.7-kb promoter was only observed in the caudal part of midbrain. This suggests that the 5'-flanking region between 3.8 and 2.7 kb contains a crucial element for activation of CYP11A1 promoter in the diencephalon, olfactory epithelium and the anterior part of midbrain. Thus we have identified regions of the promoter that control CYP11A1 expression in the brain and embryonic adrenals.

Identification of a Novel Distal Control Region Upstream of the Human Steroidogenic Acute Regulatory Protein (StAR) Gene That Participates in SF-1-dependent Chromatin Architecture

StAR (steroidogenic acute regulatory protein) mediates the transport of cholesterol from the outer to the inner mitochondrial membrane, the process of which is the rate-limiting step for steroidogenesis. Transcriptional regulation of the proximal promoter of the human StAR gene has been well characterized, whereas analysis of its distal control region has not. Recently, we found that SF-1 (steroidogenic factor 1) induced the differentiation of mesenchymal stem cells (MSCs) into steroidogenic cells with the concomitant strong induction of StAR expression. Here, we show, using differentiated MSCs, that StAR expression is regulated by a novel distal control region. Using electrophoretic mobility shift (EMSA) and chromatin immunoprecipitation (ChIP) assays, we identified novel SF-1 binding sites between 3,000 and 3,400 bp upstream of StAR. A luciferase reporter assay revealed that the region worked as a strong regulator to exert maximal transcription of StAR. ChIP analysis of histone H3 revealed that upon SF-1 expression, nucleosome eviction took place at the SF-1 binding sites, not only in the promoter but also in the distal SF-1 binding sites. Chromosome conformation capture analysis revealed that the region upstream of StAR formed a chromatin loop both in the differentiated MSCs and in KGN cells, a human granulosa cell tumor cell line, where SF-1 is endogenously expressed. Finally, SF-1 knockdown resulted in disrupted formation of this chromatin loop in KGN cells. These results indicate that the novel distal control region participate in StAR activation through SF-1 dependent alterations of chromatin structure, including histone eviction and chromatin loop formation.

X-Linked Congenital Adrenal Hypoplasia with Hypogonadotropic Hypogonadism Caused by an Inversion Disrupting a Conserved Noncoding Element Upstream of theNR0B1(DAX1) Gene

X-linked congenital adrenal hypoplasia with hypogonadotropic hypogonadism (AHCH) is known to be caused by coding mutations in the nuclear receptor subfamily 0, group B, member 1 (NR0B1) gene, encoding the transcriptional repressor dosage-sensitive sex-reversal adrenal hypoplasia critical region on the X chromosome protein 1 (DAX1). Four males in a family were affected by AHCH. Our aim was to locate the genetic cause of their disease, knowing that they had no mutation in the obvious candidate gene, NR0B1. Linkage analysis of the X chromosome and mutational screening of conserved noncoding regions upstream of NR0B1 were performed. To functionally characterize the genetic defect, studies of transcription and expression of DAX1 and steroidogenic factor 1 (SF-1) were done. A 60 Mb inversion on the X chromosome with one of the inversion breakpoints located in a conserved noncoding region 4 kb upstream of NR0B1 was detected. The inversion causes relocation of a putative SF-1 binding site implicated in murine gonadal development. A reporter construct lacking this enhancer element upstream of NR0B1 was unresponsive to SF-1 transcriptional activation. Immunohistochemistry suggested that the inversion leads to SF-1 silencing in the patients' testes both in childhood and in adult life. We report a noncoding mutation causing AHCH, an inversion resulting in a phenotype similar to what is caused by intragenic NR0B1 null mutations. The inversion seems to disrupt and/or relocate regulatory sites crucial in DAX1 expression.

Conservation of mechanisms mediating gonadotrophin-releasing hormone 1 stimulation of human luteinizing hormone β subunit transcription

Gonadotrophin-releasing hormone (GNRH1) regulates pituitary luteinizing hormone (LH). Previous studies have delineated a mechanism for GNRH1-induced LHbeta subunit gene (Lhb) transcription, the rate-limiting step in LH production. GNRH1 induces expression of early growth response 1 (EGR1), which interacts with steroidogenic factor 1 (SF1) and paired-like homeodomain transcription factor 1 (PITX1) to regulate Lhb promoter activity. Though the cis-elements for these factors are conserved across species, regulation of human LHB transcription has not been thoroughly investigated. We therefore characterized LHB transcriptional regulation by GNRH1 using promoter-reporter analyses in LbetaT2 cells. GNRH1 stimulated LHB transcription via an extracellular signal-regulated kinase 1/2 pathway. EGR1 bound to two binding sites on the LHB promoter and this binding was increased by GNRH1. Mutation of either site or knockdown of endogenous EGR1 decreased basal and/or GNRH1-regulated promoter activity. The human LHB promoter also contains low and high affinity SF1 binding sites. Mutation of these elements or depletion of endogenous SF1 impaired basal and ligand-induced transcription. Knockdown of PITX1 or PITX2 isoforms impaired GNRH1 induction, and endogenous PITX1 bound to the candidate PITX binding site on the LHB promoter. Thus, the mechanism described for GNRH1 regulation of Lhb in other species is largely conserved for human LHB. We also uncover a previously unappreciated role for PITX2 isoforms in this process.

Translin Coactivates Steroidogenic Factor-1-Stimulated Transcription

Transcription of the rat P450c17 gene in Leydig cells requires steroidogenic factor-1 (SF-1) (NR5A1), nerve growth factor-inducible protein B (nurr77), COUP-TF, and SET. The -447/-419 region of this promoter contains two binding sites for orphan nuclear receptors that are required for activation by SF-1, nerve growth factor-inducible protein B, and cAMP. We identified a novel factor, steroidogenic factor-inducer of transcription-2, that binds to this -447/-419 region. We have now purified steroidogenic factor-inducer of transcription-2 from mouse Leydig MA-10 cells and identified it by mass spectrometry as translin, a 27-kDa protein that exerts many functions. By itself, translin cannot activate a P450c17-promoter/reporter construct in HeLa cells; however, translin increased SF-1-stimulated transcription 2-fold, indicating cooperativity between SF-1 and translin. Mutation of both SF-1 binding sites in the -447/-419 sequence eliminated activation by SF-1 and translin. Translin did not augment SF-1-stimulated transcription from all SF-1-responsive elements, suggesting that the activation is specific for the sequence of the SF-1 response element. Gel shift analysis of double- and single-stranded DNA showed that translin binds to single-stranded DNA, but its transcriptional activation is independent of DNA binding. The hinge region of SF-1 is necessary for activation by translin; deletion of hinge amino acids 170-225 in SF-1 eliminates translin's ability to augment SF-1-dependent transcription. A translin-like protein, called translin-associated factor X, can substitute for a translin moiety; translin homomers and translin/translin-associated factor X heteromers activated SF-1-stimulated transcription equally. Thus, we have identified a new factor that works together with SF-1 to augment gene transcription in a DNA-specific fashion.

Evidence for activation of Amh gene expression by steroidogenic factor 1

The anti-Müllerian hormone gene ( Amh) is responsible for regression in males of the Müllerian ducts. The molecular mechanism of regulation of chicken Amh expression is poorly understood. To investigate the regulation of chicken Amh expression, we have cloned Amh cDNAs from quail and duck as well as the promoter regions of the gene from chicken, quail, and duck. The expression patterns of Amh during embryonic development in these three species were found to be similar, suggesting that the regulatory mechanisms of Amh expression are conserved. The sequence of the proximal promoter of Amh contains a putative binding site for steroidogenic factor 1 (SF1), the protein product of which can up-regulate Amh in mammals. We showed here that SF1 is able to activate the chicken Amh promoter and binds to its putative SF1 binding site. These results suggest that SF1 plays a role in regulation of Amh expression in avian species.

Potential binding sites for SF-1: Recognition by the SiteGA method, experimental verification, and search for new target genes

The transcription factor SF-1 (steroidogenic factor 1) regulates the expression of the steroidogenesis genes, coordinates the development and function of the hypothalamic-pituitary-gonadal and adrenal systems, and plays an important role in the development and function of the reproductive system. SF-1 belongs to the superfamily of nuclear receptors and activates gene expression via binding as a monomer to DNA. The SiteGA method was developed for recognizing the binding sites for SF-1. The method utilizes a genetic algorithm and discriminant analysis to identify the context features of extended (93-bp) regions harboring the SF-1 sites in a training sample. Recognition of the SF-1 sites showed that the SiteGA method allows more reliable predictions as compared to the common weight matrix method. Experimental verification of 18 putative SF-1 sites predicted for the regulatory regions of the steroidogenesis genes showed that 15 (83%) of them did indeed interact with SF-1. The density of putative SF-1 sites was analyzed in the regulatory regions of genes from various functional groups, and new target genes of SF-1 were sought in the human genome. The potential targets of SF-1 include the genes coding for cytokine receptors, growth factor receptors, and proteins involved in the corresponding signal transduction pathways, as well as genes expressed in the epididymis. Expression of SF-1 in the epididymis was predicted and verified experimentally.

Binding of Steroidogenic Factor-1 to the Regulatory Region Might Not Be Critical for Transcriptional Regulation of the Human CYP1B1 Gene

Cytochrome P450 (CYP) 1B1, which catalyzes 17beta-estradiol 4-hydroxylation, is expressed in steroid-related tissues including ovary, testis, and adrenal gland. Generally, the expressions of steroidogenic CYPs are transcriptionally regulated by steroidogenic factor-1 (SF-1) and cAMP response element (CRE) binding protein (CREB). In the present study, we examined the possibility that the human CYP1B1 gene might be regulated by SF-1 and CREB. Gel shift analyses revealed that in vitro translated SF-1 can bind to the putative SF-1 binding sites, SF-1a (at -1722) and SF-1b (at -2474), on the CYP1B1 gene. In vitro translated CREB barely binds to the putative SF-1 binding sites. Luciferase analysis revealed that a reporter plasmid, pGL3 (-2623/+25), containing the SF-1a and SF-1b elements is transactivated by the concomitant co-expression of SF-1 and protein kinase A (PKA). However, the transcriptional activity is induced by PKA alone. Mutations in the SF-1a and SF-1b elements did not affect the luciferase activity. Thus, the binding of SF-1 to the putative SF-1 binding sites of the human CYP1B1 gene might not be essential for transcriptional regulation. Interestingly, deletion and mutation analyses indicated that the PKA signaling pathway is involved in the xenobiotic responsive element (XRE)-mediated transactivation of the human CYP1B1 gene.

Indirect Regulation of Human Dehydroepiandrosterone Sulfotransferase Family 1A Member 2 by Thyroid Hormones

Thyroid hormone, T(3), regulates cell metabolism, differentiation, and development. cDNA microarrays were performed to study the mechanism of target gene regulation after T(3) treatment in a thyroid hormone receptor-alpha (TRalpha)-overexpressing hepatoma cell line (HepG2-TRalpha). The differentially expressed target genes are several metabolic enzymes, including dehydroepiandrosterone-sulfotransferase family 1A member 2 (SULT2A1). Enzyme SULT2A1 was elevated roughly 5-fold at the protein level and 9-fold increase at the mRNA level after 48 h T(3) treatment in HepG2-TRalpha cells. Cycloheximide inhibited T(3)-induced SULT2A1 expression, suggesting that regulation was indirect. SULT2A1 has been reported to be regulated by the two transcription factors, steroidogenic factor 1 (SF1) and GATA, in the human adrenal gland. T(3) induced a 2.5- to 3.5-fold elevation of SF1 at the protein level and a 6.2-fold increase at the RNA level in HepG2-TRalpha cells. About seven SF1 binding sites exist on the SULT2A1 gene. To identify and localize the critical SF1 binding site, series of deletion mutants of SULT2A1 promoter fragments in pGL2 plasmid were constructed. The promoter activity of the SULT2A1 gene was enhanced about 2.8- to 7.1-fold by T(3). The -228 SF1 binding site was identified as the most critical site because deleting this region reduced T(3)-induced expression. Transcription factor SF1 application enhanced the -228 but not -117 reporter plasmid activities. SULT2A1 and SF1 up-regulation at protein and RNA levels in thyroidectomized rats occurred after T(3) application. In summary, this work demonstrated that the SULT2A1 gene was mediated by SF1 and indirectly regulated by T(3). Further study is required to elucidate the physiological importance of SULT2A1 induction mediated by T(3).

No association between primary aldosteronism and two CYP11B2 polymorphisms in a well-characterized Australian sample

Two aldosterone (aldo) synthase gene (CYP11B2) polymorphisms have been reported to be associated with undifferentiated hypertension (HT), low renin HT and HT due to primary aldosteronism (PAL); (i) a C-344T substitution at a steroidogenic factor-1 binding site in the promoter and (ii) a conversion involving transfer of intron 2 from 11β-hydroxylase (CYP11B1). However, other studies have reported no association or implicated the other allele. So far, reported studies examining these polymorphisms have been confined to European or Japanese hypertensive populations. Using similar laboratory and statistical methods, we compared frequencies of these polymorphisms in 107 predominantly white Caucasian Australian patients with PAL (confirmed by fludrocortisone suppression testing after screening using aldo/renin ratio), with those in 99 normotensive controls. No differences in frequency were found for either polymorphism, whether comparing controls to all with PAL or to those with aldo producing adenoma [n=18] or bilateral adrenal hyperplasia [n=71] (differentiated by adrenal venous sampling). The two polymorphisms were in linkage disequilibrium, as previously reported. Reported associations with HT in several different populations could be due to linkage disequilibrium of these alleles with important functional polymorphisms. The conflicting association data may be a result of the CYP11B2 variants being present on multiple different risk haplotypes. Alternatively, ethnic background might be an important determinant of allele distribution for these polymorphisms. Given the current study's sample size, a direct effect of these polymorphisms in a small subset of our patients is possible, but it excludes overall relative risks greater than 1.9-fold. (See Table 1)

P300 Regulates the Synergy of Steroidogenic Factor-1 and Early Growth Response-1 in Activating Luteinizing Hormone-β Subunit Gene

Tight regulation of luteinizing hormone-beta subunit (LHbeta) expression is critical for differentiation and maturation of mammalian sexual organs and reproductive function. Two transcription factors, steroidogenic factor-1 (SF-1) and early growth response-1 (Egr-1), play a central role in activating LHbeta promoter, and the synergy between these two factors is essential in mediating gonadotropin-releasing hormone stimulation of LHbeta promoter. Here we demonstrate that the transcriptional co-activator p300 regulates this synergy. Overexpression of p300 results in strong stimulation of LHbeta promoter but only in the presence of both SF-1 and Egr-1, and not in the presence of other Egr proteins. Mutation of the binding sites for either SF-1 or Egr-1 completely abolishes the synergy between these two factors, as well as the influence of p300. Importantly, LHbeta promoter is precipitated using p300 antibodies in a chromatin immunoprecipitation assay with LbetaT2 gonadotropes, and this effect is enhanced by gonadotropin-releasing hormone. The influence of p300 on LHbeta promoter is potentiated by steroid receptor co-activator, as well as by E1A proteins, and attenuated by Smad nuclear interacting protein 1. Taken together, these results suggest that p300 is recruited to LHbeta promoter where it coordinates the functional synergy between SF-1 and Egr-1.

Analysis of zebrafish cyp19 promoters

Cyp19 encodes P450 aromatase, the key enzyme catalyzing the conversion of androgens into estrogens. Estrogens play a crucial role in the anatomical, functional and behavioral characteristics of sexually dimorphic development. In zebrafish, two cyp19 genes, cyp19a and cyp19b, expressed in ovary and brain, respectively, were found. We have isolated the promoter regions of the zebrafish cyp19 genes from a bacterial artificial chromosome library to search for regulatory sequences that bind to transcription factors. Sequences like arylhydrocarbon receptor (AhR) recognition site, estrogen receptor recognition half sites (1/2ERE) and c-AMP responsive elements were found in the 5′-flanking regions of both cyp19 genes. For ovarian-specific expression, we found binding sites for steroidogenic factor-1 (SF-1), GATA transcription factor 4 (GATA-4) and Wilm tumor 1 (WT1–KTS) on the promoter region of cyp19a but not cyp19b. For brain-specific expression of the cyp19b gene, sequences for recognition of chicken ovalbumin upstream promoter-transcription factor (COUP) and Ptx-1 were detected in the promoter. The importance of these putative control elements in ovary and brain-specific promoter has been assessed by sequence comparison among various species.

Isolation and expression analysis of the canine insulin-like factor 3 gene.

The insulin-like factor 3 (INSL3 or relaxin-like factor) is a hormone produced mainly in gonadal tissues in males and females. Deletion of INSL3 or its receptor in male mice leads to the undescended testes, or cryptorchidism. Here we describe an isolation and analysis of full-length canine INSL3 gene. The INSL3 gene is composed of two exons within a small genomic region. Putative translation of the isolated cDNA yields 132 amino acid preproINSL3 that has the domain structure characteristic for the insulin-relaxin peptide superfamily with a well-conserved receptor-binding domain. Northern blot hybridization showed stronger expression of INSL3 in testis than in ovary. Reverse transcription-polymerase chain reaction analysis of the INSL3 expression revealed a minor splice variant of INSL3 potentially encoding 105 amino acids peptide. We established that the medium, conditioned with recombinant canine INSL3, produced from the full-length cDNA, but not from the minor splice variant, activated human GREAT/LGR8 receptor in vitro. In addition to the functional allele of INSL3, genomic DNA of one of the analyzed dogs contained an intronless nonexpressed pseudogene of INSL3. We isolated canine INSL3 promoter and showed that its activity was strongly mediated by steroidogenic factor-1 in vitro. Using site-specific mutagenesis, we identified a well-conserved steroidogenic factor-1 binding site within canine INSL3 promoter.