Thesteroidogenic factor 1 (SF-1)protein is atranscription factor involved insex determination by controlling the activity of genes related to the reproductive glands orgonads andadrenal glands.[5] This protein is encoded by theNR5A1 gene, a member of thenuclear receptor subfamily, located on the long arm of chromosome 9 at position 33.3. It was originally identified as a regulator of genes encodingcytochrome P450steroid hydroxylases, however, further roles in endocrine function have since been discovered.[6]
TheNR5A1 gene encodes a 461-amino acid protein that shares severalconserved domains consistent with members of the nuclear receptor subfamily.[6] The N-terminal domain includes two zinc fingers and is responsible for DNA binding via specific recognition of target sequences. Variations of AGGTCA DNA motifs allows SF-1 to interact with the major groove of the DNA helix and monomerically bind.[7] Following binding, trans-activation of target genes depends on recruitment of co-activators such asSRC-1,GRIP1,PNRC, orGCN5. Other critical domains of SF-1 include a proline-rich hinge region, ligand-binding domain, and a C-terminal activation domain for transcriptional interactions. A 30-amino acid extension of the DNA-binding domain known as the A-box stabilizes monomeric binding by acting as a DNA anchor. The hinge region can undergo post-transcriptional and translational modifications such as phosphorylation bycAMP-dependent kinase, that further enhance stability and transcriptional activity.[8]
SF-1 is considered an orphan receptor as high-affinity naturally occurring ligands have yet to be identified.
Analysis of mouse SF-1cDNA revealed sequence similarities withDrosophilafushi tarazu factor I (FTZ-F1) which regulates the fushi tarazuhomeobox gene.[9] Several other FTZ-F1homologs have been identified that implicate high level ofsequence conservation among vertebrates and invertebrates. For example, SF-1 cDNA shares an identical 1017 base-pair sequence with embryonal long terminal repeat-binding protein (ELP) cDNA isolated fromembryonal carcinoma cells, differing only in their terminal ends.[9]
SF-1 expression is localized to adult steroidogenic tissues correlating with known expression profiles of steroid hydroxylases. Usingin situ hybridization with SF-1 cRNA specific probe detected gene transcripts inadrenocortical cells, Leydig cells, and ovarian theca andgranulosa cells.[9] SF-1 specific antibody studies confirmed expression profile of SF-1 in rats[10] and humans[11] corresponding to sites of transcript detection.
Genetic sex in mammals is determined by the presence or absence of theY chromosome at fertilization. Sexually dimorphic development of embryonic gonads into testes or ovaries is activated by theSRY gene product.[12] Sexual differentiation is then directed by hormones produced by embryonic testes, the presence of ovaries, or complete absence of gonads. SF-1 transcripts initially localize to the urogenital ridge before SF-1 expressing cells resolve into distinct adrenocortical and gonadal precursors that ultimately give rise to adrenal cortex and gonads.
SF-1 transcripts precede the onset of SRY expression in the fetal testes hinting at gonadal developmental role. SRY influences the differentiation of the fetal testes into distinct compartments: testicular cords and interstitial region containing Leydig cells.[12] Increase in SF-1 protein and detection in the steroidogenic Leydig cells and testicular cords coincides with development.
However, in the ovaries, gonadal sexual differentiation is facilitated by reductions in SF-1 transcript and protein. SF-1 levels is strongly expressed at the onset of follicular development intheca and granulosa cells which precedes expression of thearomatase enzyme responsible forestrogen biosynthesis.
Embryonic mouse SF-1 transcripts have been discovered to localize within regions of the developing diencephalon and subsequently in theventromedial hypothalamic nucleus (VMH) suggesting roles beyond steroidogenic maintenance.[9]
RT-PCR approaches have detected transcripts of mice FTZ-F1 gene in the placenta and spleen; and SF-1 transcripts in the human placenta.[13]
Transcription capacity of SF-1 can be influenced by post-translational modification. Specifically, phosphorylation ofserine 203 is mediated bycyclin-dependent kinase 7. Mutations to CDK7 prevent interaction with the basal transcription factor,TFIIH, and formation of CDK-activating kinase complex. This inactivity has shown to repress phosphorylation of SF-1 and SF-1-dependent transcription.[14]
SF-1 is a critical regulator of reproduction, regulating the transcription of key genes involved in sexual development and reproduction, most notablyStAR andP450SCC. It can form a transcriptional complex withTDF to up-regulate transcription of theSox9 gene. Its targets includegenes at every level of thehypothalamic-pituitary-gonadal axis, as well as many genes involved ingonadal andadrenalsteroidogenesis.[15]
SF-1 has been identified as a transcriptional regulator for an array of different genes related to sex determination and differentiation,reproduction, andmetabolism via binding to their promoters. For example, SF-1 controls expression ofAmh gene inSertoli cells, whereby the presence or absence of the gene product affects development ofMüllerian structures. Increased AMH protein levels leads to regression of such structures.[6]Leydig cells express SF-1 to regulate transcription ofsteroidogenesis andtestosterone biosynthesis genes causing virilization in males.
First identified as a regulator of steroid hydroxylases within adrenocortical cells, studies aimed to define localization and expression of SF-1 have since revealed enzyme activity within other steroidogenic cells.[6]
Table 1. Example of genes regulated by SF-1 in steroidogenic cells
TheMüllerian inhibiting substance (MIS orAMH) gene within Sertoli cells contains a conserved motif identical to the optimal binding sequence for SF-1. Gel mobility shift experiments and use of SF-1-specificpolyclonal antibodies established binding complexes of SF-1 to MIS,[16] however, other studies suggest the MIS promoter is repressed and not activated by SF-1 binding.
Gonadotrope-specific element, or GSE, in the promoter of the gene encoding α-subunit ofglycoproteins (α-GSU) resembles the SF-1 binding sires. Studies have implicated SF-1 as an upstream regulator of a collection of genes required for gonadotrope function via GSE.[17]
SF-1 knockout mice displayed profound defects in the VMH suggesting potential target genes at the site. Target genes have yet to be identified due to difficulties in studying gene expression in neurons.
Several approaches usedtargeted gene disruption in mouse embryonic stem cells with the aim of identifying potential target genes of SF-1. The different targeting strategies include disruption to exons encoding for the zinc finger motif, disruption of a 3’-exon and targeted mutation of the initiator methionine. The corresponding observed phenotypic effects on endocrine development and function were found to be quite similar.[6]
Sf-1 knockout mice displayed diminishedcorticosterone levels while maintaining elevatedACTH levels. Observed morphological changes and DNA fragmentation was consistent with apoptosis and structural regression resulting in the death of all mice within 8 days after birth.[18]
Sf-1 function was determined to be necessary for development of primary steroidogenic tissue as evidenced by complete lack of adrenal and gonadal glands in the knockout. Male to female sex reversal of genitalia was also observed.[19]
Mutations in NR5A1 can produce intersex genitals, absence of puberty, and infertility. It is one cause of arrest of ovarian function in women <40 years of age, which occurs in 1% of all women.
Two SF-1 variants associated with primary adrenal failure andcomplete gonadal dysgenesis have been reported as caused byNR5A1 mutations. One reported case was found to havede novoheterozygous p.G35E change to the P-box domain.[20] The affected region allows for DNA binding specificity through interactions with regulatory response elements of target genes. This p.G35E change may have a mild competitive or dominant negative effect on transactivation resulting in severe gonadal defects and adrenal dysfunction. Similarly,homozygous p.R92Q change within the A-box interfered with monomeric binding stability and reduced functional activity.[20] This change requires mutations to both allele to display phenotypic effects as heterozygous carriers showed normal adrenal function.
HeterozygousNR5A1 changes are emerging as a frequent contributor in46, XY complete gonadal dysgenesis.[20] In affected individuals, sexual development does not match their chromosomal makeup. Males, despite having 46, XYkaryotype, develop female external genitalia, as well as uterus and fallopian tubes, along with gonadal defects rendering them nonfunctional.[22]NR5A1 mutations have also been linked to partial gonadal dysgenesis, whereby affected individuals have ambiguous genitalia, urogenital sinus, absent or rudimentary Müllerian structures, and other abnormalities.[20]
Typically, these genetic changes areframeshift,nonsense, ormissense mutations that alter DNA-binding and gene transcription. While many arede novo, one-third of cases have been maternally inherited in a similar manner asX-linked inheritance. Furthermore, one report of homozygous missense mutation p.D293N within the ligand-binding domain of SF-1 revealedautosomal recessive inheritance was also possible.[21]
Analysis ofNR5A1 in men withnon-obstructive male factor infertility found those with gene changes had more severe forms of infertility and lower testosterone levels.[23] These changes affected the hinge region of SF-1. It is important to note further studies are required to establish the relationship between SF-1 changes and infertility.
^Takayama K, Sasano H, Fukaya T, Morohashi K, Suzuki T, Tamura M, Costa MJ, Yajima A (September 1995). "Immunohistochemical localization of Ad4-binding protein with correlation to steroidogenic enzyme expression in cycling human ovaries and sex cord stromal tumors".The Journal of Clinical Endocrinology and Metabolism.80 (9):2815–21.doi:10.1210/jcem.80.9.7673429.PMID7673429.
^Shen WH, Moore CC, Ikeda Y, Parker KL, Ingraham HA (June 1994). "Nuclear receptor steroidogenic factor 1 regulates the müllerian inhibiting substance gene: a link to the sex determination cascade".Cell.77 (5):651–61.doi:10.1016/0092-8674(94)90050-7.PMID8205615.S2CID13364008.
^Luo X, Ikeda Y, Parker KL (May 1994). "A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation".Cell.77 (4):481–90.doi:10.1016/0092-8674(94)90211-9.PMID8187173.S2CID28194376.
^Sugawara T, Saito M, Fujimoto S (August 2000). "Sp1 and SF-1 interact and cooperate in the regulation of human steroidogenic acute regulatory protein gene expression".Endocrinology.141 (8):2895–903.doi:10.1210/endo.141.8.7602.PMID10919277.S2CID20567318.
^Mellgren G, Børud B, Hoang T, Yri OE, Fladeby C, Lien EA, Lund J (May 2003). "Characterization of receptor-interacting protein RIP140 in the regulation of SF-1 responsive target genes".Molecular and Cellular Endocrinology.203 (1–2):91–103.doi:10.1016/S0303-7207(03)00097-2.PMID12782406.S2CID733221.
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Oba K, Yanase T, Nomura M, Morohashi K, Takayanagi R, Nawata H (September 1996). "Structural characterization of human Ad4bp (SF-1) gene".Biochemical and Biophysical Research Communications.226 (1):261–7.doi:10.1006/bbrc.1996.1343.PMID8806624.
Crawford PA, Polish JA, Ganpule G, Sadovsky Y (October 1997). "The activation function-2 hexamer of steroidogenic factor-1 is required, but not sufficient for potentiation by SRC-1".Molecular Endocrinology.11 (11):1626–35.doi:10.1210/mend.11.11.9970.PMID9328345.S2CID35590139.
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