Direct regulation of adult brain function by the male-specific factor SRY.

The central dogma of mammalian brain sexual differentiation has contended that sex steroids of gonadal origin organize the neural circuits of the developing brain. Recent evidence has begun to challenge this idea and has suggested that, independent of the masculinizing effects of gonadal secretions, XY and XX brain cells have different patterns of gene expression that influence their differentiation and function. We have previously shown that specific differences in gene expression exist between male and female developing brains and that these differences precede the influences of gonadal hormones. Here we demonstrate that the Y chromosome-linked, male-determining gene Sry is specifically expressed in the substantia nigra of the adult male rodent in tyrosine hydroxylase-expressing neurons. Furthermore, using antisense oligodeoxynucleotides, we show that Sry downregulation in the substantia nigra causes a statistically significant decrease in tyrosine hydroxylase expression with no overall effect on neuronal numbers and that this decrease leads to motor deficits in male rats. Our studies suggest that Sry directly affects the biochemical properties of the dopaminergic neurons of the nigrostriatal system and the specific motor behaviors they control. These results demonstrate a direct male-specific effect on the brain by a gene encoded only in the male genome, without any mediation by gonadal hormones.

Defective calmodulin-mediated nuclear transport of the sex-determining region of the Y chromosome (SRY) in XY sex reversal.

The sex-determining region of the Y chromosome (SRY) plays a key role in human sex determination, as mutations in SRY can cause XY sex reversal. Although some SRY missense mutations affect DNA binding and bending activities, it is unclear how others contribute to disease. The high mobility group domain of SRY has two nuclear localization signals (NLS). Sex-reversing mutations in the NLSs affect nuclear import in some patients, associated with defective importin-beta binding to the C-terminal NLS (c-NLS), whereas in others, importin-beta recognition is normal, suggesting the existence of an importin-beta-independent nuclear import pathway. The SRY N-terminal NLS (n-NLS) binds calmodulin (CaM) in vitro, and here we show that this protein interaction is reduced in vivo by calmidazolium, a CaM antagonist. In calmidazolium-treated cells, the dramatic reduction in nuclear entry of SRY and an SRY-c-NLS mutant was not observed for two SRY-n-NLS mutants. Fluorescence spectroscopy studies reveal an unusual conformation of SRY.CaM complexes formed by the two n-NLS mutants. Thus, CaM may be involved directly in SRY nuclear import during gonadal development, and disruption of SRY.CaM recognition could underlie XY sex reversal. Given that the CaM-binding region of SRY is well-conserved among high mobility group box proteins, CaM-dependent nuclear import may underlie additional disease states.

SOX9 regulates expression of the male fertility gene Ets variant factor 5 (ETV5) during mammalian sex development.

In humans, dysregulation of the sex determining gene SRY-box 9 (SOX9) leads to disorders of sex development (DSD). In mice, knock-out of Sox9 prior to sex determination leads to XY sex reversal, while Sox9 inactivation after sex determination leads to spermatogenesis defects. SOX9 specifies the differentiation and function of Sertoli cells from somatic cell precursors, which then orchestrate the development and maintenance of other testicular cell types, largely through unknown mechanisms. Here, we describe a novel testicular target gene of SOX9, Ets variant factor 5 (ETV5), a transcription factor responsible for maintaining the spermatogonial stem cell niche. Etv5 was highly expressed in wild-type XY but not XX mouse fetal gonads, with ETV5 protein localized in the Sertoli cells, interstitial cells and germ cells of the testis. In XY Sox9 knock-out gonads, Etv5 expression was strongly down-regulated. Similarly, knock-down of SOX9 in the human Sertoli-like cell line NT2/D1 caused a decrease in ETV5 gene expression. Transcriptomic analysis of NT2/D1 cells over-expressing SOX9 showed that ETV5 expression was increased in response to SOX9. Moreover, chromatin immunoprecipitation of these cells, as well as of embryonic mouse gonads, showed direct binding of SOX9 to ETV5 regulatory regions. We demonstrate that SOX9 was able to activate ETV5 expression via a conserved SOX site in the 5' regulatory region, mutation of which led to loss of activation. In conclusion, we present a novel target gene of SOX9 in the testis, and suggest that SOX9 regulation of ETV5 contributes to the control of male fertility.

Recombinant expression, purification and characterisation of the HMG domain of human SRY.

The HMG domain is a DNA binding and bending 'architectural' motif involved in chromatin re-modelling during transcription. Recombinant SRY HMG domain protein, 88 amino acids in length, has been produced in E. coli. Using FPLC and a stirred ultra-filtration cell, this domain has been purified to homogeneity and concentrated to yield milligram quantities. Functional characterisation studies of the pure, concentrated SRY HMG domain show the recombinantly expressed protein to be active in terms of DNA binding and calmodulin binding activities.

Failure of SOX9 regulation in 46XY disorders of sex development with SRY, SOX9 and SF1 mutations.

BACKGROUND: In human embryogenesis, loss of SRY (sex determining region on Y), SOX9 (SRY-related HMG box 9) or SF1 (steroidogenic factor 1) function causes disorders of sex development (DSD). A defining event of vertebrate sex determination is male-specific upregulation and maintenance of SOX9 expression in gonadal pre-Sertoli cells, which is preceded by transient SRY expression in mammals. In mice, Sox9 regulation is under the transcriptional control of SRY, SF1 and SOX9 via a conserved testis-specific enhancer of Sox9 (TES). Regulation of SOX9 in human sex determination is however poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: We show that a human embryonal carcinoma cell line (NT2/D1) can model events in presumptive Sertoli cells that initiate human sex determination. SRY associates with transcriptionally active chromatin in NT2/D1 cells and over-expression increases endogenous SOX9 expression. SRY and SF1 co-operate to activate the human SOX9 homologous TES (hTES), a process dependent on phosphorylated SF1. SOX9 also activates hTES, augmented by SF1, suggesting a mechanism for maintenance of SOX9 expression by auto-regulation. Analysis of mutant SRY, SF1 and SOX9 proteins encoded by thirteen separate 46,XY DSD gonadal dysgenesis individuals reveals a reduced ability to activate hTES. CONCLUSIONS/SIGNIFICANCE: We demonstrate how three human sex-determining factors are likely to function during gonadal development around SOX9 as a hub gene, with different genetic causes of 46,XY DSD due a common failure to upregulate SOX9 transcription.

A SOX9 defect of calmodulin-dependent nuclear import in campomelic dysplasia/autosomal sex reversal.

During mammalian sex determination, SOX9 is translocated into the nuclei of Sertoli cells within the developing XY gonad. The N-terminal nuclear localization signal (NLS) is contained within a SOX consensus calmodulin (CaM) binding region, thereby implicating CaM in nuclear import of SOX9. By fluorescence spectroscopy and glutaraldehyde cross-linking, we show that the SOX9 HMG domain and CaM interact in vitro. The formation of a SOX9.CaM binary complex is calcium-dependent and is accompanied by a conformational change in SOX9. A CaM antagonist, calmidazolium chloride (CDZ), was observed to block CaM recognition of SOX9 in vitro and inhibit both nuclear import and consequent transcriptional activity of SOX9 in treated cells. The significance of the SOX9-CaM interaction was highlighted by analysis of a missense SOX9 mutation, A158T, identified from a XY female with campomelic dysplasia/autosomal sex reversal (CD/SRA). This mutant binds importin beta normally despite defective nuclear import. Fluorescence and quenching studies indicate that in the unbound state, the A158T mutant shows a similar conformation to that of the WT SOX9, but in the presence of CaM, the mutant undergoes unusual conformational changes. Furthermore, SOX9-mediated transcriptional activation by cells expressing the A158T mutant is more sensitive to CDZ than cells expressing WT SOX9. These results suggest first that CaM is involved in the nuclear transport of SOX9 in a process likely to involve direct interaction and second, that CD/SRA can arise, at least in part, from a defect in CaM recognition, ultimately leading to reduced ability of SOX9 to activate transcription of cartilage and testes-forming genes.