A direct role of SRY and SOX proteins in pre-mRNA splicing

The mammalian testis determining factor SRY and its related Sox factors are critical developmental regulators. They share significant similarity in their high mobility group (HMG) domain and display discrete patterns of tissue-specific expression. Here we show that SRY and the Sox protein SOX6 colocalize with splicing factors in the nucleus and are dynamically redistributed following the blockage of splicing in living cells. Anti-SOX6 antibodies supershift the spliceosomal complex from assembled splicing reactions and inhibit splicing in vitro of multiple pre-mRNA substrates. Most importantly, SOX6-depleted nuclear extracts have impaired splicing activity, which is efficiently restored by addition of the recombinant SOX6 HMG domain and also by recombinant SRY and the SOX9 HMG domain. These results reveal an unexpected biological function of the SRY, SOX6, and SOX9 gene products and provide a functional link to the biochemical mechanisms operating in mammalian sex determination and in other developmental processes regulated by Sox genes.     

A nuclear export signal within the high mobility group domain regulates the nucleocytoplasmic translocation of SOX9 during sexual determination

In mammals, male sex determination starts when the Y chromosome Sry gene is expressed within the undetermined male gonad. One of the earliest effect of Sry expression is to induce up-regulation of Sox9 gene expression in the developing gonad. SOX9, like SRY, contains a high mobility group domain and is sufficient to induce testis differentiation in transgenic XX mice. Before sexual differentiation, SOX9 protein is initially found in the cytoplasm of undifferentiated gonads from both sexes. At the time of testis differentiation and anti-Müllerian hormone expression, it becomes localized to the nuclear compartment in males whereas it is down-regulated in females. In this report, we used NIH 3T3 cells as a model to examine the regulation of SOX9 nucleo-cytoplasmic shuttling. SOX9-transfected cells expressed nuclear and cytoplasmic SOX9 whereas transfected cells treated with the nuclear export inhibitor leptomycin B, displayed an exclusive nuclear localization of SOX9. By using SOX9 deletion constructs in green fluorescent protein fusion proteins, we identified a functional nuclear export signal sequence between amino acids 134 and 147 of SOX9 high mobility group box. More strikingly, we show that inhibiting nuclear export with leptomycin B in mouse XX gonads cultured in vitro induced a sex reversal phenotype characterized by nuclear SOX9 and anti-Müllerian hormone expression. These results indicate that SOX9 nuclear export signal is essential for SOX9 sex-specific subcellular localization and could be part of a regulatory switch repressing (in females) or triggering (in males) male-specific sexual differentiation.     

An SRY-related sequence on the marsupial X chromosome: implications for the evolution of the mammalian testis-determining gene.

The SRY gene on the human, mouse, and marsupial Ychromosomes is the testis-determining gene that initiates male development inmammals. The SRY protein has a DNA-binding domain (high mobility group or HMGbox) similar to those found in the high-mobility-group proteins. SRY is specificfor the Y chromosome, but many autosomal genes have been identified that possessa similar HMG box region; those with the most closely SRY-related box regionsform a gene family now referred to as SOX genes. We have identified a sequenceon the marsupial X chromosome that shares homology with SRY. Sequencecomparisons show near-identity with the mouse and human SOX3 gene (formerlycalled a3), the SOX gene which is the most closely related to SRY. We suggesthere that the highly conserved X chromosome-linked SOX3 represents the ancestralSOX gene from which the sex-determining gene SRY was derived. In this modelSOX3/SRY divergence and the acquisition of a testis-determining role by SRYmight have preceded (and initiated) sex chromosome differentiation or,alternatively, might have been a consequence of X chromosome-Y chromosomedifferentiation initiated at the locus of an original sex-determining gene(s),later superseded by SRY.     

Sex-determining genes.

Recently, a number of genes have been identified that are associated with a failure of human sex determination, including WT1, DAX-1, SOX9, ATRX, and the Y-linked testis determination gene, SRY. Most cases of human sex reversal, XY females and XX males, do not, however, appear to be caused by mutations in these genes. This review highlights recent advances in this field and discusses the prospects of identifying genes in the sex-determining pathway. (Trends Endocrinol Metab 1997;9:342-345). (c) 1997, Elsevier Science Inc.     

XX Maleness and XX true hermaphroditism in SRY-negative monozygotic twins: additional evidence for a common origin

CONTEXT: Differentiation of testicular tissue in 46,XX individuals is seen either in XX males, the majority of them with SRY gene, or in individuals, usually SRY(-), with ovotesticular disorder of sex development (OT-DSD). Although they are sporadic cases, there are some reports on familial recurrence, including coexistence of XX maleness and OT-DSD in the same family. OBJECTIVE: We report on a case of SRY(-) 46,XX monozygotic twins with genital ambiguity. METHODS: Hormonal evaluation included testosterone, FSH, and LH measurements. SRY gene was investigated by PCR and two-step PCR in peripheral leukocytes and gonadal tissues, respectively. Direct DNA sequencing of the DAX-1 coding sequence was performed. Real-time PCR for SOX9 region on chromosome 17 was obtained. RESULTS: Both twins had a 46,XX karyotype. Twin A had a 1-cm phallus with chordee, penoscrotal hypospadias, and palpable gonads. Serum levels of FSH (2.34 mIU/ml), LH (8.8 mIU/ml), and testosterone (1.6 ng/ml) were normal, and biopsies revealed bilateral testes. Twin B had a 0.5-cm phallus, perineal hypospadias, no palpable gonad on the right, and a left inguinal hernia. Hormonal evaluation revealed high FSH (8.2 mIU/ml) and LH (15 mIU/ml) and low testosterone (0.12 ng/ml). Upon herniotomy, a right testis (crossed ectopia) and a small left ovotestis were found. SRY gene was absent in both peripheral leukocytes and gonadal tissue samples. Neither DAX-1 mutations nor SOX9 duplication was identified. CONCLUSIONS: This case provides evidence that both XX maleness and XX OT-DSD are different manifestations of the same disorder of gonadal development.     

Testis determination in mammals: more questions than answers.

In humans, testis development depends on a regulated genetic hierarchy initiated by the Y-linked SRY gene. Failure of testicular determination results in the condition termed 46,XY gonadal dysgenesis (GD). Several components of the testis determining pathway have recently been identified though it has been difficult to articulate a cascade with the known elements of the system. It seems, however, that early gonadal development is the result of a network of interactions instead of the outcome of a linear cascade. Accumulating evidence shows that testis formation in man is sensitive to gene dosage. Haploinsufficiency of SF1, WT1 and SOX9 is responsible for 46,XY gonadal dysgenesis. Besides, data on SRY is consistent with possible dosage anomalies in certain cases of male to female sex reversal. 46,XY GD due to monosomy of distal 9p and 10q might also be associated with an insufficient gene dosage effect. Duplications of the locus DSS can lead to a failure of testicular development and a duplication of the region containing SOX9 has been implicated in XX sex reversal. Transgenic studies in mouse have shown, however, that this mammal is less sensitive to gene dosage than man. Here, we will try to put in place the known pieces of the jigsaw puzzle that is sex determination in mammals, as far as current knowledge obtained from man and animal models allows. We are certain that from this attempt more questions than answers will arise.     

Osteopontin is a novel downstream target of SOX9 with diagnostic implications for progression of liver fibrosis in humans

Osteopontin (OPN) is an important component of the extracellular matrix (ECM), which promotes liver fibrosis and has been described as a biomarker for its severity. Previously, we have demonstrated that Sex-determining region Y-box 9 (SOX9) is ectopically expressed during activation of hepatic stellate cells (HSC) when it is responsible for the production of type 1 collagen, which causes scar formation in liver fibrosis. Here, we demonstrate that SOX9 regulates OPN. During normal development and in the mature liver, SOX9 and OPN are coexpressed in the biliary duct. In rodent and human models of fibrosis, both proteins were increased and colocalized to fibrotic regions in vivo and in culture-activated HSCs. SOX9 bound a conserved upstream region of the OPN gene, and abrogation of Sox9 in HSCs significantly decreased OPN production. Hedgehog (Hh) signaling has previously been shown to regulate OPN expression directly by glioblastoma (GLI) 1. Our data indicate that in models of liver fibrosis, Hh signaling more likely acts through SOX9 to modulate OPN. In contrast to Gli2 and Gli3, Gli1 is sparse in HSCs and is not increased upon activation. Furthermore, reduction of GLI2, but not GLI3, decreased the expression of both SOX9 and OPN, whereas overexpressing SOX9 or constitutively active GLI2 could rescue the antagonistic effects of cyclopamine on OPN expression. Conclusion: These data reinforce SOX9, downstream of Hh signaling, as a core factor mediating the expression of ECM components involved in liver fibrosis. Understanding the role and regulation of SOX9 during liver fibrosis will provide insight into its potential modulation as an antifibrotic therapy or as a means of identifying potential ECM targets, similar to OPN, as biomarkers of fibrosis. (HEPATOLOGY 2012;56:1108-1116).     

Deletion of long-range regulatory elements upstream of SOX9 causes campomelic dysplasia

Campomelic dysplasia (CD) is a rare, neonatal human chondrodysplasia characterized by bowing of the long bones and often associated with male-to-female sex-reversal. Patients present with either heterozygous mutations in the SOX9 gene or chromosome rearrangements mapping at least 50 kb upstream of SOX9. Whereas mutations in SOX9 ORF cause haploinsufficiency, the effects of translocations 5′ to SOX9 are unclear. To test whether these rearrangements also cause haploinsufficiency by altering spatial and temporal expression of SOX9, we generated mice transgenic for human SOX9-lacZ yeast artificial chromosomes containing variable amounts of DNA sequences upstream of SOX9. We show that elements necessary for SOX9 expression during skeletal development are highly conserved between mouse and human and reveal that a rearrangement upstream of SOX9, similar to those observed in CD patients, leads to a substantial reduction of SOX9 expression, particularly in chondrogenic tissues. These data demonstrate that important regulatory elements are scattered over a large region upstream of SOX9 and explain how particular aspects of the CD phenotype are caused by chromosomal rearrangements 5′ to SOX9.     

Analysis of the sex-determining region of the Y chromosome (SRY) in sex reversed patients: point-mutation in SRY causing sex-reversion in a 46,XY female.

The first and essential step in normal sexual differentiation takes place during the 5th-6th week of gestation. The testis determining factor (TDF) directs the undifferentiated gonad into a testis, which secretes hormones responsible for normal male development. A new candidate for TDF has recently been reported, and it has been called the sex determining region of the Y (SRY). The hypothesis has been supported by the finding of XX individuals with SRY, and two females with 46,XY karyotype and a mutation in SRY. However, XX males without SRY has been reported, and the role of SRY still has to be determined. We have tested three human females with 46,XY karyotype and gonadal dysgenesis and two 46,XX males for the presence of SRY using the polymerase chain reaction and subsequent DNA sequencing. Both 46,XX males contained SRY, whereas one of the 46,XY females had suffered a point mutation in SRY changing a codon for lysine to a stop codon. This information supports the hypothesis that SRY is significant in normal male sex differentiation. The two remaining 46,XY individuals had an intact HMG box, but it is possible that a mutation may be found in a regulatory gene or further downstream in the gene regulatory cascade. Two patients including the one with a mutation in SRY had gonadoblastomas supporting the hypothesis that another gene on the Y-chromosome is involved in the pathogenesis of this neoplasia.     

The human sex-reversing ATRX gene has a homologue on the marsupial Y chromosome, ATRY: implications for the evolution of mammalian sex determination

Mutations in the ATRX gene on the human X chromosome cause X-linked alpha-thalassemia and mental retardation. XY patients with deletions or mutations in this gene display varying degrees of sex reversal, implicating ATRX in the development of the human testis. To explore further the role of ATRX in mammalian sex differentiation, the homologous gene was cloned and characterized in a marsupial. Surprisingly, active homologues of ATRX were detected on the marsupial Y as well as the X chromosome. The Y-borne copy (ATRY) displays testis-specific expression. This, as well as the sex reversal of ATRX patients, suggests that ATRY is involved in testis development in marsupials and may represent an ancestral testis-determining mechanism that predated the evolution of SRY as the primary mammalian male sex-determining gene. There is no evidence for a Y-borne ATRX homologue in mouse or human, implying that this gene has been lost in eutherians and its role supplanted by the evolution of SRY from SOX3 as the dominant determiner of male differentiation.