Molecular basis of mammalian sexual determination: activation of Mullerian inhibiting substance gene expression by SRY

Haqq, Christopher M.; King, Chih Yen; Ukiyama, Etsuji; Falsafi, Sassan; Haqq, Tania N.; Donahoe, Patricia K.; Weiss, Michael A.

doi:10.1126/science.7985018

Cited by 257 publications

(197 citation statements)

References 63 publications

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“…No in vivo targets for SRY have been defined to date, and thus the mechanism by which SRY initiates testis differentiation remains to be elucidated. Both weak repression and weak activation by human SRY have been reported in model transactivation assays in cultured cells (5)(6)(7)(8). Many sex-reversing SRY mutations affect DNAbinding͞bending activities of the mutant SRY proteins in vitro.…”

mentioning

confidence: 99%

Defective importin β recognition and nuclear import of the sex-determining factor SRY are associated with XY sex-reversing mutations

Harley

Layfield

Mitchell

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

142

132

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The architectural transcription factor SRY (sex-determining region of the Y chromosome) plays a key role in sex determination as indicated by the fact that mutations in SRY are responsible for XY gonadal dysgenesis in humans. Although many SRY mutations reduce DNA-binding͞bending activity, it is not clear how SRY mutations that do not affect interaction with DNA contribute to disease. The SRY high-mobility group domain harbors two nuclear localization signals (NLSs), and here we examine SRY from four XY females with missense mutations in these signals. In all cases, mutant SRY protein is partly localized to the cytoplasm, whereas wild-type SRY is strictly nuclear. Each NLS can independently direct nuclear transport of a carrier protein in vitro and in vivo, with mutations in either affecting the rate and extent of nuclear accumulation. The N-terminal NLS function is independent of the conventional NLS-binding importins (IMPs) and requires unidentified cytoplasmic transport factors, whereas the C-terminal NLS is recognized by IMP␤. The SRY-R133W mutant shows reduced IMP␤ binding as a direct consequence of the sex-reversing C-terminal NLS mutation. Of the N-terminal NLS mutants examined, SRY-R62G unexpectedly shows a marked reduction in IMP␤ binding, whereas SRY-R75N and SRY-R76P show normal IMP␤ binding, suggesting defects in the IMP-independent pathway. We conclude that SRY normally requires the two distinct NLS-dependent nuclear import pathways to reach sufficient levels in the nucleus for sex determination. This study documents cases of human disease being explained, at a molecular level, by the impaired ability of a protein to accumulate in the nucleus.

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confidence: 99%

Defective importin β recognition and nuclear import of the sex-determining factor SRY are associated with XY sex-reversing mutations

Harley

Layfield

Mitchell

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

142

132

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“…This postmitotic somatic cell, which is in intimate contact with developing germ cells in seminiferous tubules in the testis, is crucial for all phases of male gametogenesis, including germ cell proliferation, meiosis, and differentiation (15). Genes necessary for the Sertoli cell to support male gametogenesis have been identified, and some progress has been made in identifying regulatory mechanisms that control the transcription of some of these genes, including follicle-stimulating hormone receptor (Fshr), cathepsin L, the inhibin-␤ B subunit, transferrin, Mullerian inhibitory substance, androgen-binding protein, Dnmrt 1, GATA1, GATA4, and tissue plasminogen activator (12,13,15,17,18,21,35,41,43,53). Regulatory regions and specific cis elements that participate in the transcriptional regulation of these genes have been identified in primary Sertoli cells and Sertoli cell lines.…”

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confidence: 99%

GATA Factors and Androgen Receptor Collaborate To Transcriptionally Activate theRhox5Homeobox Gene in Sertoli Cells

Bhardwaj

Rao

Kaur

et al. 2008

Molecular and Cellular Biology

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How Sertoli-specific expression is initiated is poorly understood. Here, we address this issue using the proximal promoter (Pp) from the Rhox5 homeobox gene. Its Sertoli cell-specific expression is achieved, in part, through a negative regulatory element that inhibits Pp transcription in non-Sertoli cell lines. Complementing this negative regulation is positive regulation conferred by four androgen-response elements (AREs) that interact with the androgen receptor (AR), a nuclear hormone receptor expressed at high levels in Sertoli cells. A third control mechanism is provided by a consensus GATA-binding site that is crucial for Pp transcription both in vitro and in vivo. Several lines of evidence suggested that GATA factors and AR act cooperatively to activate Pp transcription: (i) the GATA-binding site crucial for Pp transcription is in close proximity to two of the AREs, (ii) GATA and AR form a complex with the Pp in vitro, (iii) overexpression of GATA factors rescued expression from mutant Pp constructs harboring defective AREs, and (iv) incubation of a Sertoli cell line with testosterone triggered corecruitment of AR and GATA4 to the Pp. Collectively, our results suggest that the Rhox5 gene achieves Sertoli cell-specific transcription using a combinatorial strategy involving negative and cooperative positive regulation.

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“…SRY 1 (sex-determining region Y) is a DNA-binding protein encoded on the Y chromosome and acts as a genetic switch that diverts primordial gonads from the ovarian pathway toward male differentiation to form testes (1)(2)(3). SRY activates transcription at the promoter region of the Mü llerian inhibitor substance (MIS) gene (4,5); the product of MIS is responsible for the regression of the female Mü llerian ducts, the precursor of the uterus, fallopian tubes, and upper vagina, in male embryos.…”

mentioning

confidence: 99%

The C-terminal Nuclear Localization Signal of the Sex-determining Region Y (SRY) High Mobility Group Domain Mediates Nuclear Import through Importin β1

Forwood¹,

Harley²,

Jans³

2001

Journal of Biological Chemistry

109

129

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The sex-determining factor SRY is a DNA-binding protein that diverts primordial gonads from the ovarian pathway toward male differentiation to form testes. It gains access to the nucleus through two distinct nuclear localization signals (NLSs) that flank the high mobility group (HMG) DNA-binding domain, but the mechanisms through which these NLSs operate have not been studied. In this study, we reconstitute the nuclear import of SRY in vitro, demonstrating a lack of requirement for exogenous factors for nuclear accumulation and a significant reduction in nuclear transport in the presence of antibodies to importin ␤ but not importin ␣. Using a range of quantitative binding assays including enzymelinked immunosorbent assay, fluorescence polarization, and native gel mobility electrophoresis, we assess the binding of importins to SRY, demonstrating a high affinity recognition (in the low nM range) by Imp␤ independent of Imp␣. In assessing the contribution of each NLS, we found that the N-terminal NLS was recognized poorly by importins, whereas the C-terminal NLS was bound by importin ␤ with similar affinity to SRY. We also found that RanGTP, but not RanGDP, could dissociate the SRY-importin ␤ complex in solution using FP. We describe a novel double-fluorescent label DNA binding assay to demonstrate mutual exclusivity between importin ␤ recognition and DNA binding on the part of SRY, which may represent an alternative release mechanism upon nuclear entry. This study represents the first characterization of the nuclear import pathway for a HMG domain-containing protein. Importantly, it demonstrates for the first time that recognition of SRY by Imp␤ is of comparable affinity to that with which Imp␣/␤ recognizes conventional NLS-containing substrates.

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Molecular basis of mammalian sexual determination: activation of Mullerian inhibiting substance gene expression by SRY

Cited by 257 publications

References 63 publications

Defective importin β recognition and nuclear import of the sex-determining factor SRY are associated with XY sex-reversing mutations

Defective importin β recognition and nuclear import of the sex-determining factor SRY are associated with XY sex-reversing mutations

GATA Factors and Androgen Receptor Collaborate To Transcriptionally Activate theRhox5Homeobox Gene in Sertoli Cells

The C-terminal Nuclear Localization Signal of the Sex-determining Region Y (SRY) High Mobility Group Domain Mediates Nuclear Import through Importin β1

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