- Email: [email protected]
The ins and outs of transcriptional control: nucleocytoplasmic shuttling in development and disease
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a level sufficient to maintain its function. This would enable the Alu exon to diversify in such a way that the Aluderived protein could acquire new functionality that might benefit evolution [25]. In this way, exonization of Alu elements might have played an important role in the speciation of humans. Nonetheless, it is an important finding that the ‘junk’ sequence of the human genome could have a significant, and yet underrated, role in affecting gene expression and, in turn, genetic diversity. Acknowledgements We thank members of the Graveley laboratory for critical comments and discussions. Work in our laboratory is supported by NIH grants GM-62516, GM-67842 and AR-46026 to B.R.G.
References 1 Graveley, B.R. (2001) Alternative splicing: increasing diversity in the proteomic world. Trends Genet. 17, 100– 107 2 Schmucker, D. et al. (2000) Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity. Cell 101, 671 – 684 3 Lander, E.S. et al. (2001) Initial sequencing and analysis of the human genome. Nature 409, 860 – 921 4 Venter, J.C. et al. (2001) The sequence of the human genome. Science 291, 1304 – 1351 5 Pennisi, E. (2000) Human genome project. And the gene number is? Science 288, 1146 – 1147 6 Kan, Z. et al. (2001) Gene structure prediction and alternative splicing analysis using genomically aligned ESTs. Genome Res. 11, 889 – 900 7 Modrek, B. and Lee, C. (2002) A genomic view of alternative splicing. Nat. Genet. 30, 13 – 19 8 Brett, D. et al. (2002) Alternative splicing and genome complexity. Nat. Genet. 30, 29 – 30 9 Sorek, R. et al. (2002) Alu-containing exons are alternatively spliced. Genome Res. 12, 1060 – 1067 10 Lev-Maor, G. et al. (2003) The birth of an alternatively spliced exon: 30 splice-site selection in Alu exons. Science 300, 1288 – 1291 11 Waterston, R.H. et al. (2002) Initial sequencing and comparative analysis of the mouse genome. Nature 420, 520 – 562 12 Thanaraj, T.A. et al. (2003) Conservation of human alternative splice events in mouse. Nucleic Acids Res. 31, 2544 – 2552
13 Modrek, B. and Lee, C.J. (2003) Alternative splicing in the human, mouse and rat genomes is associated with an increased frequency of exon creation and/or loss. Nat. Genet. 34, 177 – 180 14 Pattanakitsakul, S. et al. (1992) Aberrant splicing caused by the insertion of the B2 sequence into an intron of the complement C4 gene is the basis for low C4 production in H-2k mice. J. Biol. Chem. 267, 7814– 7820 15 Chu, J.L. et al. (1993) The defect in Fas mRNA expression in MRL/lpr mice is associated with insertion of the retrotransposon, ETn. J. Exp. Med. 178, 723– 730 16 Rowold, D.J. and Herrera, R.J. (2000) Alu elements and the human genome. Genetica 108, 57 – 72 17 Walter, P. and Blobel, G. (1982) Signal recognition particle contains a 7S RNA essential for protein translocation across the endoplasmic reticulum. Nature 299, 691 – 698 18 Deininger, P.L. and Batzer, M.A. (1999) Alu repeats and human disease. Mol. Genet. Metab. 67, 183 – 193 19 Jurka, J. and Smith, T. (1988) A fundamental division in the Alu family of repeated sequences. Proc. Natl. Acad. Sci. U. S. A. 85, 4775 – 4778 20 Chua, K. and Reed, R. (2001) An upstream AG determines whether a downstream AG is selected during catalytic step II of splicing. Mol. Cell. Biol. 21, 1509– 1514 21 Lai, F. et al. (1997) Editing of glutamate receptor B subunit ion channel RNAs by four alternatively spliced DRADA2 double-stranded RNA adenosine deaminases. Mol. Cell. Biol. 17, 2413 – 2424 22 Mitchell, G.A. et al. (1991) Splice-mediated insertion of an Alu sequence inactivates ornithine delta-aminotransferase: a role for Alu elements in human mutation. Proc. Natl. Acad. Sci. U. S. A. 88, 815– 819 23 Vervoort, R. et al. (1998) A mutation (IVS8 þ 0.6kbdelTC) creating a new donor splice site activates a cryptic exon in an Alu-element in intron 8 of the human beta-glucuronidase gene. Hum. Genet. 103, 686– 693 24 Knebelmann, B. et al. (1995) Splice-mediated insertion of an Alu sequence in the COL4A3 mRNA causing autosomal recessive Alport syndrome. Hum. Mol. Genet. 4, 675 – 679 25 Makalowski, W. (2003) Genomics. Not junk after all. Science 300, 1246– 1247
0168-9525/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tig.2003.11.001
The ins and outs of transcriptional control: nucleocytoplasmic shuttling in development and disease James M. Smith1 and Peter A. Koopman1,2 1
Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia Australian Research Council Centre of Excellence in Biotechnology and Development, The University of Queensland, Brisbane, Queensland 4072, Australia 2
Recent findings relating to SOX transcription factors indicate that defects in organogenesis can be caused not only by impairment of the biochemical properties of transcription factors but also, in some cases, by deficient nuclear import. In addition, experimentally Corresponding author: Peter A. Koopman ([email protected]). http://tigs.trends.com
interfering with the nuclear export signals of some SOX factors has now been found to cause developmental defects. Controlling the balance of nuclear import and export might be a common means by which transcription factor activity can be regulated during development, and defects in these processes might underlie a broader spectrum of inherited developmental disorders.
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The proper development of an embryo depends on the careful orchestration of gene activity by a network of transcription factors. Genes encoding these factors must themselves be regulated in precise temporal and tissuespecific patterns; each transcription factor must bind to its proper target and act together with appropriate cofactors and partner proteins to bring about a specific effect on expression of a defined subset of the genome. Although the importance of DNA binding, trans-activation or repression, and protein interaction domains have been well documented in transcription factor function, accumulating data suggest that shuttling of transcription factors between the nucleus and the cytoplasm – nucleocytoplasmic shuttling – is a further mechanism by which their activity can be regulated. The activities of several transcription factors or cofactors, including HIV-1 Rev protein [1], signal transducer and activator of transcription 1 (STAT1) [2], adenomatosis polyposis coli protein (APC) [3], tumour protein p53 (p53) [4], breast cancer 1 early onset (BRCA1) [5] and SMAD4 [6] are modulated by shuttling in and out of the nucleus. Mutations affecting nucleocytoplasmic shuttling of some of these factors have been identified in various cancers [7]. Recent studies relating to the SOX family of developmental transcription factors have underscored the significance of this shuttling for regulating the normal development of the embryo. Defects in the nuclear import of two SOX factors, SRY [8] and SOX9 [9], have been associated with human sex reversal syndromes, whereas the blockade of nuclear export was found to interfere with the biological and molecular function of SOX9 [10] and a third SOX factor, SOX10 [11]. These studies suggest that not only import but also active export might be an important adjunct to more commonly studied mechanisms for regulating the activity of tissue-specific transcription factors during normal development. They also provide the first association of primary nucleocytoplasmic shuttling defects with syndromes affecting organogenesis in humans. Of great import Mutations in the sex determining region on the Y chromosome (SRY) account for , 10% of all cases of XY sex reversal in humans [12,13]. The only functional domain of the SRY protein known to be conserved between species is the high-mobility group (HMG) domain, a 79-amino acid region involved in the specific binding and sharp bending of DNA [14,15]. The majority of SRY mutations resulting in XY sex reversal occur within the HMG domain, predominantly causing defects in binding to target DNA sequences or impairing the ability of the SRY protein to bend DNA [16]. Aside from its functions in DNA binding and bending, the HMG domain is also responsible for nuclear localization of the SRY protein. Two independent nuclear localization signals (NLS) are situated at either end of the HMG domain (Figure 1a), an N-terminal bipartite NLS and a C-terminal basic cluster NLS [17]. Forwood and colleagues [18] recently examined the nuclear import of SRY in vitro and noted a requirement for importins in this process, specifically importin b. This is significant http://tigs.trends.com
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because in conventional NLS-dependent nuclear import, it is usually importin a rather than b that mediates the interaction with the NLS of the target protein; importin b then docks with this complex at the nuclear pore complex (NPC) where transfer across the nuclear membrane occurs [19]. In the case of SRY, however, it appears that importin b directly recognizes the C-terminal basic cluster NLS of the SRY HMG domain, and nuclear import is mediated via this mechanism. However, it should be noted that the N-terminal NLS of SRY is capable of targeting carrier proteins to the nucleus independently of the C-terminal NLS [17]. How the importin-independent function of this N-terminal NLS is established remains a mystery, but it might involve binding of calmodulin to these sequences [20], as discussed in the following sections. Is nuclear localization of SRY significant for its function in male sex determination? Clinical data from human XY sex reversal patients suggests that it is. Li and colleagues [8] reported a case of 46,XY pure gonadal dysgenesis caused by a point mutation (R133W) (Figure 1a) in the basic cluster of residues that comprises the C-terminal NLS. This mutation appears to inhibit nuclear localization while not demonstrably impairing the specific binding of SRY target sequences, or its ability to bend DNA. More recently, the R133W mutant has been shown [21] to be defective in importin b recognition. Given the phenotype of the patient, it appears that nuclear localization is a crucial and necessary step in testis development, and represents an example of a defect in organogenesis that is caused by a mutation leading to improper nuclear import of a protein. Nuclear family In the dozen years since the discovery of SRY, a gene family has sprung up around it – the SOX or SRY-like HMG box genes [22]. These genes, as the name implies, encode proteins that contain an HMG domain related to that of SRY (usually with sequence identity at the amino acid level of . 50% [23]. All of the well-characterized SOX genes, like their founding member SRY, are involved in embryonic development. SOX9 is involved in chondrocyte formation during skeletal development [24,25] and is coexpressed with SRY in the developing testis. Campomelic dysplasia (CD), a human skeletal dysgenesis syndrome resulting from deficiency of SOX9, is associated with sex reversal, with , 75% of XY campomelic dysplasia patients developing as females [26]. Sox9 is believed to act downstream of Sry in the regulatory cascade leading to maleness, based on their relative timing of expression in the mouse gonad [27,28]. Experiments in mice have demonstrated that transgenic XX embryos expressing Sox9 in their gonads develop as males [29], and have a similar sex reversal phenotype to transgenic XX mice carrying Sry [30]. These observations indicate that SOX9 is both necessary and sufficient for male sex determination in mammals. Unlike SRY, SOX9 possesses a conserved pair of C-terminal trans-activation domains in addition to its HMG domain, consisting of PQA-rich (i.e. rich in proline, glutamine and alanine) and PQS-rich elements (i.e. rich in proline, glutamine and serine) [31]. (Figure 1b) More recently, a conserved domain has been identified at the
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(a)
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Human SRY protein
DRVKRPMNAFIVWSRDQRRKMALENPRMRNSEISKQLGYQWKMLTEAEKWPFFQEAQKLQAMHREKYPNYKYRPRRKAK
1
(b)
58
136
204aa
Human SOX9 protein
Leucine-rich nuclear export signal N-terminal basic nuclear localization signal C-terminal bipartite nuclear localization signal
HMG domain
PHVKRPMNAFMVWAQAARRKLADQYPHLHNAELSKTLGKLWRLLNESEKRPFVEEAERLRVQHKKDHPDYKYQPRRRKS
1
Dim HMG domain 61 103 181
PQS PQA 339 379 509aa C-terminal activation domains TRENDS in Genetics
Figure 1. The structure of human SRY and SOX9 proteins. (a) The 204 amino acid (aa) human SRY protein contains a 79aa HMG domain (marked in black), implicated in binding and sharp bending of DNA. The HMG domain of SRY contains two NLS, an N-terminal bipartite NLS (marked in blue) and a C-terminal basic NLS (marked in red). It also contains a putative Rev-type leucine-rich NES, marked in green, although this NES has been identified solely on the basis of sequence homology and its function is yet to be determined by experiment. The position of the R133W sex reversing mutation, which inhibits nuclear localization of SRY is noted on the figure. (b) The 509aa human SOX9 protein contains several functional domains, including a 79aa HMG domain (marked in black); a dimerization (Dim) domain (marked in dark grey) positioned N-terminal to the HMG box; and two C-terminal PQA and PQS-rich transactivation domains, marked in light grey. Like that of SRY, the HMG domain of SOX9 contains both an N-terminal bipartite NLS (marked in blue) and a C-terminal basic NLS (marked in red), as well as a leucine-rich NES, marked in green. The position of the A158T sex reversing mutation, which inhibits nuclear localization of SOX9 is noted on the figure. Abbreviations: HMG, High Mobility Group; NES, nuclear export signal; NLS, nuclear localization signals; PQA, proline-glutamine-alanine; PQS, proline-glutamine-serine; SOX9, SRY-like HMG box 9; SRY, sex-determining region on the Y chromosome.
N-terminus, which is required for DNA-dependent dimerization of SOX9 at paired binding sites within target promoters [32,33]. Sex reversing mutations occur throughout the SOX9 gene, including within the trans-activation domains [34], although mutations in the dimerization domain have been shown to cause CD without sex reversal [33]. In terms of the nuclear localization of SOX9, two independent nuclear localization signals have been detected. These are positioned at either end of the HMG domain, as in the case of SRY [17]. Both NLS motifs are conserved in several other HMG domain proteins, including many if not all of the SOX proteins. It could be that this mechanism of nuclear localization is common to the entire SOX family [17,23]. As with SRY, cases of XY sex reversal related to deficiencies in the nuclear import of SOX9 have been reported. Preiss and colleagues [9] identified a sexreversing point mutation (A158T) (Figure 1b) that causes a twofold reduction in nuclear import efficiency in vitro compared to wild-type SOX9. Curiously, the A158T mutation affects neither of the NLSs directly but DNA binding is affected by this mutation, so it is unclear to what degree this reduction in nuclear import is involved in the observed sex reversal phenotype. These researchers characterized the mechanism of nuclear import of SOX9, showing a requirement for importin b (as observed with http://tigs.trends.com
SRY), and predicted that all SOX proteins would be found to use this mechanism. The A158T mutant protein interacts normally with importin b but was subsequently found to manifest abnormal conformation on binding of calmodulin, showing increased sensitivity to calmodulin antagonists [35]. One would predict that other CD or sex reversal-associated mutations in SOX9 would be found to correlate with defects in nuclear import. In through the out door Intuitively, a mechanism by which a transcription factor can be shunted into the nucleus would seem to be a basic requirement for its function once it is produced in the cell cytoplasm. It is perhaps not surprising that interference with this process could cause disruptions to proper cellular development. However, the story is not this simple. Recent data [10] suggest that active mechanisms for nuclear export and import are required for SOX9 function. Prior to sexual differentiation, SOX9 protein is found in the cytoplasm of undifferentiated gonads of both sexes. As differentiation of the testis proceeds following the expression of SRY, SOX9 becomes localized to the nucleus in the male gonad, whereas expression of SOX9 is downregulated in the developing ovary [36]. This observation of nucleocytoplasmic shuttling of SOX9 led to the search for a nuclear export signal (NES) in the SOX9
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protein. Accordingly, a functional leucine-rich NES was identified within the HMG domain (amino acid residues 134– 147), situated between the two NLS sites [10]. Of the nuclear export signals found in eukaryotes, the best characterized are these small hydrophobic leucine-rich NESs, first described in the HIV-1 REV protein [1]. These REV-type NESs function via interaction with the export factor chromosome region maintenance 1 (CRM1). Inhibiting the action of the leucine-rich NES of SOX9 in organ culture (by the action of leptomycin B, which specifically inhibits CRM1-dependent nuclear export) induced sex reversal in mouse XX gonads. This was characterized by sole nuclear localization of SOX9 and expression of SOX9-induced genes, such as anti-Mu¨llerian hormone (AMH), which induces the regression of the female-specific Mu¨llerian duct in the developing testis. Gasca et al. [10] proposed that the balance between nuclear import and export of SOX9 might therefore act as a switch mechanism triggering the male pathway in the developing testis. Interestingly, these findings were complemented by work on another SOX gene, SOX10, which is essential for neural crest development [37]. Although this gene plays no discernible role in sex determination, SOX10 is closely related to SOX9 on the basis of sequence similarities within and outside the HMG box. Both genes, along with SOX8, belong to subgroup E of the SOX gene family [38]. Similar to SOX9, SOX10 possesses a REV-type leucinerich NES between the two NLS sequences of its HMG domain [11], and has been shown to actively shuttle between the nucleus and cytoplasm. Rehberg and colleagues [11] demonstrated the function of this NES and showed that its mutational inactivation, or inhibition via the action of leptomycin B, was sufficient to prevent nucleocytoplasmic shuttling of SOX10. Strikingly, this led to decreased trans-activation of endogenous target genes of SOX10, as well as transfected reporter constructs. Therefore, continuous nucleocytoplasmic shuttling appears to be essential to the function of SOX10. Any protein with both an NLS and an NES can shuttle back and forth between the nucleus and cytoplasm as required. If such a protein is a transcription factor, this has clear implications for post-translational regulation, with spatial separation from its DNA target (by exclusion from the nucleus) acting as a potent inhibitor of function. Additionally, post-translational modifications, such as phosphorylation and dephosphorylation, might regulate nucleocytoplasmic shuttling by masking or exposing signals for nuclear import or export within the protein [39]. Conserved phosphorylation sites flanking the HMG domains of SRY and SOX9 [40] might be implicated in the regulation of nuclear import and export of these proteins. Significantly for the SOX gene family, the leucine-rich NES sequence found in SOX9 and SOX10 is not limited to subgroup E of the SOX family. Conserved hydrophobic residues associated with REV-type nuclear export signals have been found in the corresponding HMG-domain regions of a variety of SOX proteins across various subgroups, including SRY [10,11]. However, whether these sequences can mediate nuclear export of SOX proteins other than SOX9 and SOX10 have to be determined. http://tigs.trends.com
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Conclusion Active, continuous shuttling between nucleus and cytoplasm might prove to be important to the function of the majority of SOX proteins in development, and their malfunction in human disease. Certainly the importance of nuclear import and export of a range of SOX proteins can be tested experimentally using targeted mutation strategies in mice. In broader terms, the regulation of nucleocytoplasmic shuttling is clearly another level of control over how, when and where transcription factors operate during development, and another point at which the function of developmental transcription factors might be compromised in the aetiology of human disease. Acknowledgements We thank Josephine Bowles, Megan Wilson and Annemiek Beverdam of the Koopman laboratory for critical reading of the manuscript. J.M.S. is the recipient of an Australian Postgraduate Award and a supplementary scholarship from the Institute for Molecular Bioscience. P.A.K. is a Professorial Research Fellow of the Australian Research Council.
References 1 Fischer, U. et al. (1995) The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell 82, 475– 483 2 Mowen, K. and David, M. (2000) Regulation of STAT1 nuclear export by Jak1. Mol. Cell. Biol. 20, 7273– 7281 3 Neufeld, K.L. et al. (2000) Ademotatous polyposis coli protein contains two nuclear export signals and shuttles between the nucleus and cytoplasm. Proc. Natl. Acad. Sci. U. S. A. 97, 12085 – 12090 4 Stommel, J.M. et al. (1999) A leucine-rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by NES masking. EMBO J. 18, 1660 – 1672 5 Rodriguez, J.A. and Henderson, B.R. (2000) Identification of a functional nuclear export signal in BRCA1. J. Biol. Chem. 275, 38589 – 38596 6 Watanabe, M. et al. (2000) Regulation of intracellular dynamics of Smad4 by its leucine-rich nuclear export signal. EMBO Rep. 1, 176 – 182 7 Fabbro, M. and Henderson, B.R. (2003) Regulation of tumour suppressors by nuclear-cytoplasmic shuttling. Exp. Cell Res. 282, 59 – 69 8 Li, B. et al. (2001) Human sex reversal due to impaired nuclear localization of SRY – a clinical correlation. J. Biol. Chem. 276, 46480 – 46484 9 Preiss, S. et al. (2001) Compound effects of point mutations causing campomelic dysplasia/autosomal sex reversal upon SOX9 structure, nuclear transport, DNA binding, and transcriptional activation. J. Biol. Chem. 276, 27864 – 27872 10 Gasca, S. et al. (2002) A nuclear export signal within the high mobility group domain regulates the nucleocytoplasmic translocation of SOX9 during sexual determination. Proc. Natl. Acad. Sci. U. S. A. 99, 11199– 11204 11 Rehberg, S. et al. (2002) Sox10 is an active nucleocytoplasmic shuttle protein, and shuttling is crucial for Sox10-mediated transactivation. Mol. Cell. Biol. 22, 5826– 5834 12 Numabe, H. et al. (1992) DNA analyses of XX and XX-hypospadiac males. Hum. Genet. 90, 211 – 214 13 Fechner, P.Y. et al. (1993) The role of the sex determining region Y gene in the etiology of 46,XX maleness. J. Clin. Endocrinol. Metab. 76, 690– 695 14 Giese, K. et al. (1994) Distinct DNA-binding properties of the high mobility group domain of murine and human SRY sex-determining factors. Proc. Natl. Acad. Sci. U. S. A. 91, 3368– 3374 15 Pontiggia, A. et al. (1994) Sex-reversing mutations affect the architecture of SRY-DNA complexes. EMBO J. 13, 6115 – 6124 16 Koopman, P.A. (2001) Sry, Sox9 and mammalian sex determination. In Genes and Mechanisms in Vertebrate Sex Determination (Scherer, G. and Schmid, M., eds), pp. 25 – 56, Birkhauser-Verlag 17 Su¨dbeck, P. and Scherer, G. (1997) Two independent nuclear
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localization signals are present in the DNA-binding high mobility group domains of SRY and SOX9. J. Biol. Chem. 272, 27848 – 27852 Forwood, J. et al. (2001) The C-terminal nuclear localization signal of the sex determining region Y (SRY) high mobility group domain mediates nuclear import through importin beta 1. J. Biol. Chem. 276, 46575 – 46582 Kohler, M. et al. (1999) Nuclear protein transport pathways. Exp. Nephrol. 7, 290 – 294 Harley, V.R. et al. (1996) The HMG box of SRY is a calmodulin binding domain. FEBS Lett. 391, 24– 28 Harley, V.R. et al. (2003) Defective importin b recognition and nuclear import of the sex-determining factor SRY are associated with XY sexreversing mutations. Proc. Natl. Acad. Sci. U. S. A. 100, 7045 – 7050 Bowles, J. et al. (2000) Phylogeny of the SOX family of developmental transcription factors based on sequence and structural indicators. Developmental Biology 227, 239 – 255 Wilson, M. and Koopman, P. (2002) Matching SOX: partner proteins and co-factors of the SOX family of transcriptional regulators. Curr. Opin. Genet. Dev. 12, 441– 446 Wright, E. et al. (1995) The Sry-related gene Sox-9 is expressed during chondrogenesis in mouse embryos. Nat. Genet. 9, 15 – 20 Bi, W. et al. (2001) Haploinsufficiency of Sox9 results in defective cartilage primordia and premature skeletal mineralization. Proc. Natl. Acad. Sci. U. S. A. 98, 6698 – 6703 Houston, C.S. et al. (1983) The campomelic syndrome: review, report of 17 cases, and follow-up on the currently 17- year old boy first reported by Maroteaux et al. in 1971. Am. J. Med. Genet. 15, 3 – 28 Kent, J. et al. (1996) A male-specific role for SOX9 in vertebrate sex determination. Development 122, 2813 – 2822 Morais da Silva, S. et al. (1996) Sox9 expression during gonadal development implies a conserved role for the gene in testis differentiation in mammals and birds. Nat. Genet. 14, 62 – 68 Vidal, V. et al. (2001) Sox9 induces testis development in XX transgenic mice. Nat. Genet. 28, 216 – 217
30 Koopman, P. et al. (1991) Male development of chromosomally female mice transgenic for Sry. Nature 351, 117 – 121 31 Clarkson, M.J. and Harley, V.R. (2002) Sex with two SOX on: SRY and SOX9 in testis development. Trends Endocrinol. Metab. 13, 106 – 111 32 Bernard, P. et al. (2003) Dimerization of SOX9 is required for chondrogenesis, but not for sex determination. Hum. Mol. Genet. 12, 1755– 1765 33 Sock, E. et al. (2003) Loss of DNA-dependent dimerization of the transcription factor SOX9 as a cause for campomelic dysplasia. Hum. Mol. Genet. 12, 1439 – 1447 34 Su¨dbeck, P. et al. (1996) Sex reversal by loss of the C-terminal transactivation domain of human SOX9. Nat. Genet. 13, 230– 232 35 Argentaro, A. et al. (2003) A SOX9 defect of calmodulin-dependent nuclear import in campomelic dysplasia/autosomal sex reversal. J. Biol. Chem. 278, 33839 – 33847 36 de Santa Barbara, P. et al. (2000) Expression and subcellular localization of SF-1, SOX9, WT1, and AMH proteins during early human testicular development. Dev. Dyn. 217, 293– 298 37 Pingault, V. et al. (1998) SOX10 mutations in patients with Waardenburg-Hirschsprung disease. Nat. Genet. 18, 171– 173 38 Bowles, J. et al. (2000) Phylogeny of the SOX family of developmental transcription factors based on sequence and structural indicators. Dev. Biol. 227, 239 – 255 39 Gasiorowski, J.Z. and Dean, D.A. (2003) Mechanisms of nuclear transport and interventions. Adv. Drug Deliv. Rev. 55, 703– 716 40 Harley, V.R. et al. (2003) The molecular action and regulation of the testis-determining factors, SRY (Sex-determining Region on the Y chromosome) and SOX9 (SRY-related high-mobility group (HMG) Box 9). Endocr. Rev. 24, 466 – 487
0168-9525/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tig.2003.11.007
Could you name the most significant papers published in life sciences this month? Updated daily, Research Update presents short, easy-to-read commentary on the latest hot papers, enabling you to keep abreast with advances across the life sciences. Written by laboratory scientists with a keen understanding of their field, Research Update will clarify the significance and future impact of this research. Our experienced in-house team is under the guidance of a panel of experts from across the life sciences who offer suggestions and advice to ensure that we have high calibre authors and have spotted the ‘hot’ papers. Visit the Research Update daily at http://update.bmn.com and sign up for email alerts to make sure you don’t miss a thing. This is your chance to have your opinion read by the life science community, if you would like to contribute, contact us at [email protected] http://tigs.trends.com
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a level sufficient to maintain its function. This would enable the Alu exon to diversify in such a way that the Aluderived protein could acquire new functionality that might benefit evolution [25]. In this way, exonization of Alu elements might have played an important role in the speciation of humans. Nonetheless, it is an important finding that the ‘junk’ sequence of the human genome could have a significant, and yet underrated, role in affecting gene expression and, in turn, genetic diversity. Acknowledgements We thank members of the Graveley laboratory for critical comments and discussions. Work in our laboratory is supported by NIH grants GM-62516, GM-67842 and AR-46026 to B.R.G.
References 1 Graveley, B.R. (2001) Alternative splicing: increasing diversity in the proteomic world. Trends Genet. 17, 100– 107 2 Schmucker, D. et al. (2000) Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity. Cell 101, 671 – 684 3 Lander, E.S. et al. (2001) Initial sequencing and analysis of the human genome. Nature 409, 860 – 921 4 Venter, J.C. et al. (2001) The sequence of the human genome. Science 291, 1304 – 1351 5 Pennisi, E. (2000) Human genome project. And the gene number is? Science 288, 1146 – 1147 6 Kan, Z. et al. (2001) Gene structure prediction and alternative splicing analysis using genomically aligned ESTs. Genome Res. 11, 889 – 900 7 Modrek, B. and Lee, C. (2002) A genomic view of alternative splicing. Nat. Genet. 30, 13 – 19 8 Brett, D. et al. (2002) Alternative splicing and genome complexity. Nat. Genet. 30, 29 – 30 9 Sorek, R. et al. (2002) Alu-containing exons are alternatively spliced. Genome Res. 12, 1060 – 1067 10 Lev-Maor, G. et al. (2003) The birth of an alternatively spliced exon: 30 splice-site selection in Alu exons. Science 300, 1288 – 1291 11 Waterston, R.H. et al. (2002) Initial sequencing and comparative analysis of the mouse genome. Nature 420, 520 – 562 12 Thanaraj, T.A. et al. (2003) Conservation of human alternative splice events in mouse. Nucleic Acids Res. 31, 2544 – 2552
13 Modrek, B. and Lee, C.J. (2003) Alternative splicing in the human, mouse and rat genomes is associated with an increased frequency of exon creation and/or loss. Nat. Genet. 34, 177 – 180 14 Pattanakitsakul, S. et al. (1992) Aberrant splicing caused by the insertion of the B2 sequence into an intron of the complement C4 gene is the basis for low C4 production in H-2k mice. J. Biol. Chem. 267, 7814– 7820 15 Chu, J.L. et al. (1993) The defect in Fas mRNA expression in MRL/lpr mice is associated with insertion of the retrotransposon, ETn. J. Exp. Med. 178, 723– 730 16 Rowold, D.J. and Herrera, R.J. (2000) Alu elements and the human genome. Genetica 108, 57 – 72 17 Walter, P. and Blobel, G. (1982) Signal recognition particle contains a 7S RNA essential for protein translocation across the endoplasmic reticulum. Nature 299, 691 – 698 18 Deininger, P.L. and Batzer, M.A. (1999) Alu repeats and human disease. Mol. Genet. Metab. 67, 183 – 193 19 Jurka, J. and Smith, T. (1988) A fundamental division in the Alu family of repeated sequences. Proc. Natl. Acad. Sci. U. S. A. 85, 4775 – 4778 20 Chua, K. and Reed, R. (2001) An upstream AG determines whether a downstream AG is selected during catalytic step II of splicing. Mol. Cell. Biol. 21, 1509– 1514 21 Lai, F. et al. (1997) Editing of glutamate receptor B subunit ion channel RNAs by four alternatively spliced DRADA2 double-stranded RNA adenosine deaminases. Mol. Cell. Biol. 17, 2413 – 2424 22 Mitchell, G.A. et al. (1991) Splice-mediated insertion of an Alu sequence inactivates ornithine delta-aminotransferase: a role for Alu elements in human mutation. Proc. Natl. Acad. Sci. U. S. A. 88, 815– 819 23 Vervoort, R. et al. (1998) A mutation (IVS8 þ 0.6kbdelTC) creating a new donor splice site activates a cryptic exon in an Alu-element in intron 8 of the human beta-glucuronidase gene. Hum. Genet. 103, 686– 693 24 Knebelmann, B. et al. (1995) Splice-mediated insertion of an Alu sequence in the COL4A3 mRNA causing autosomal recessive Alport syndrome. Hum. Mol. Genet. 4, 675 – 679 25 Makalowski, W. (2003) Genomics. Not junk after all. Science 300, 1246– 1247
0168-9525/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tig.2003.11.001
The ins and outs of transcriptional control: nucleocytoplasmic shuttling in development and disease James M. Smith1 and Peter A. Koopman1,2 1
Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia Australian Research Council Centre of Excellence in Biotechnology and Development, The University of Queensland, Brisbane, Queensland 4072, Australia 2
Recent findings relating to SOX transcription factors indicate that defects in organogenesis can be caused not only by impairment of the biochemical properties of transcription factors but also, in some cases, by deficient nuclear import. In addition, experimentally Corresponding author: Peter A. Koopman ([email protected]). http://tigs.trends.com
interfering with the nuclear export signals of some SOX factors has now been found to cause developmental defects. Controlling the balance of nuclear import and export might be a common means by which transcription factor activity can be regulated during development, and defects in these processes might underlie a broader spectrum of inherited developmental disorders.
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The proper development of an embryo depends on the careful orchestration of gene activity by a network of transcription factors. Genes encoding these factors must themselves be regulated in precise temporal and tissuespecific patterns; each transcription factor must bind to its proper target and act together with appropriate cofactors and partner proteins to bring about a specific effect on expression of a defined subset of the genome. Although the importance of DNA binding, trans-activation or repression, and protein interaction domains have been well documented in transcription factor function, accumulating data suggest that shuttling of transcription factors between the nucleus and the cytoplasm – nucleocytoplasmic shuttling – is a further mechanism by which their activity can be regulated. The activities of several transcription factors or cofactors, including HIV-1 Rev protein [1], signal transducer and activator of transcription 1 (STAT1) [2], adenomatosis polyposis coli protein (APC) [3], tumour protein p53 (p53) [4], breast cancer 1 early onset (BRCA1) [5] and SMAD4 [6] are modulated by shuttling in and out of the nucleus. Mutations affecting nucleocytoplasmic shuttling of some of these factors have been identified in various cancers [7]. Recent studies relating to the SOX family of developmental transcription factors have underscored the significance of this shuttling for regulating the normal development of the embryo. Defects in the nuclear import of two SOX factors, SRY [8] and SOX9 [9], have been associated with human sex reversal syndromes, whereas the blockade of nuclear export was found to interfere with the biological and molecular function of SOX9 [10] and a third SOX factor, SOX10 [11]. These studies suggest that not only import but also active export might be an important adjunct to more commonly studied mechanisms for regulating the activity of tissue-specific transcription factors during normal development. They also provide the first association of primary nucleocytoplasmic shuttling defects with syndromes affecting organogenesis in humans. Of great import Mutations in the sex determining region on the Y chromosome (SRY) account for , 10% of all cases of XY sex reversal in humans [12,13]. The only functional domain of the SRY protein known to be conserved between species is the high-mobility group (HMG) domain, a 79-amino acid region involved in the specific binding and sharp bending of DNA [14,15]. The majority of SRY mutations resulting in XY sex reversal occur within the HMG domain, predominantly causing defects in binding to target DNA sequences or impairing the ability of the SRY protein to bend DNA [16]. Aside from its functions in DNA binding and bending, the HMG domain is also responsible for nuclear localization of the SRY protein. Two independent nuclear localization signals (NLS) are situated at either end of the HMG domain (Figure 1a), an N-terminal bipartite NLS and a C-terminal basic cluster NLS [17]. Forwood and colleagues [18] recently examined the nuclear import of SRY in vitro and noted a requirement for importins in this process, specifically importin b. This is significant http://tigs.trends.com
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because in conventional NLS-dependent nuclear import, it is usually importin a rather than b that mediates the interaction with the NLS of the target protein; importin b then docks with this complex at the nuclear pore complex (NPC) where transfer across the nuclear membrane occurs [19]. In the case of SRY, however, it appears that importin b directly recognizes the C-terminal basic cluster NLS of the SRY HMG domain, and nuclear import is mediated via this mechanism. However, it should be noted that the N-terminal NLS of SRY is capable of targeting carrier proteins to the nucleus independently of the C-terminal NLS [17]. How the importin-independent function of this N-terminal NLS is established remains a mystery, but it might involve binding of calmodulin to these sequences [20], as discussed in the following sections. Is nuclear localization of SRY significant for its function in male sex determination? Clinical data from human XY sex reversal patients suggests that it is. Li and colleagues [8] reported a case of 46,XY pure gonadal dysgenesis caused by a point mutation (R133W) (Figure 1a) in the basic cluster of residues that comprises the C-terminal NLS. This mutation appears to inhibit nuclear localization while not demonstrably impairing the specific binding of SRY target sequences, or its ability to bend DNA. More recently, the R133W mutant has been shown [21] to be defective in importin b recognition. Given the phenotype of the patient, it appears that nuclear localization is a crucial and necessary step in testis development, and represents an example of a defect in organogenesis that is caused by a mutation leading to improper nuclear import of a protein. Nuclear family In the dozen years since the discovery of SRY, a gene family has sprung up around it – the SOX or SRY-like HMG box genes [22]. These genes, as the name implies, encode proteins that contain an HMG domain related to that of SRY (usually with sequence identity at the amino acid level of . 50% [23]. All of the well-characterized SOX genes, like their founding member SRY, are involved in embryonic development. SOX9 is involved in chondrocyte formation during skeletal development [24,25] and is coexpressed with SRY in the developing testis. Campomelic dysplasia (CD), a human skeletal dysgenesis syndrome resulting from deficiency of SOX9, is associated with sex reversal, with , 75% of XY campomelic dysplasia patients developing as females [26]. Sox9 is believed to act downstream of Sry in the regulatory cascade leading to maleness, based on their relative timing of expression in the mouse gonad [27,28]. Experiments in mice have demonstrated that transgenic XX embryos expressing Sox9 in their gonads develop as males [29], and have a similar sex reversal phenotype to transgenic XX mice carrying Sry [30]. These observations indicate that SOX9 is both necessary and sufficient for male sex determination in mammals. Unlike SRY, SOX9 possesses a conserved pair of C-terminal trans-activation domains in addition to its HMG domain, consisting of PQA-rich (i.e. rich in proline, glutamine and alanine) and PQS-rich elements (i.e. rich in proline, glutamine and serine) [31]. (Figure 1b) More recently, a conserved domain has been identified at the
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(a)
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Human SRY protein
DRVKRPMNAFIVWSRDQRRKMALENPRMRNSEISKQLGYQWKMLTEAEKWPFFQEAQKLQAMHREKYPNYKYRPRRKAK
1
(b)
58
136
204aa
Human SOX9 protein
Leucine-rich nuclear export signal N-terminal basic nuclear localization signal C-terminal bipartite nuclear localization signal
HMG domain
PHVKRPMNAFMVWAQAARRKLADQYPHLHNAELSKTLGKLWRLLNESEKRPFVEEAERLRVQHKKDHPDYKYQPRRRKS
1
Dim HMG domain 61 103 181
PQS PQA 339 379 509aa C-terminal activation domains TRENDS in Genetics
Figure 1. The structure of human SRY and SOX9 proteins. (a) The 204 amino acid (aa) human SRY protein contains a 79aa HMG domain (marked in black), implicated in binding and sharp bending of DNA. The HMG domain of SRY contains two NLS, an N-terminal bipartite NLS (marked in blue) and a C-terminal basic NLS (marked in red). It also contains a putative Rev-type leucine-rich NES, marked in green, although this NES has been identified solely on the basis of sequence homology and its function is yet to be determined by experiment. The position of the R133W sex reversing mutation, which inhibits nuclear localization of SRY is noted on the figure. (b) The 509aa human SOX9 protein contains several functional domains, including a 79aa HMG domain (marked in black); a dimerization (Dim) domain (marked in dark grey) positioned N-terminal to the HMG box; and two C-terminal PQA and PQS-rich transactivation domains, marked in light grey. Like that of SRY, the HMG domain of SOX9 contains both an N-terminal bipartite NLS (marked in blue) and a C-terminal basic NLS (marked in red), as well as a leucine-rich NES, marked in green. The position of the A158T sex reversing mutation, which inhibits nuclear localization of SOX9 is noted on the figure. Abbreviations: HMG, High Mobility Group; NES, nuclear export signal; NLS, nuclear localization signals; PQA, proline-glutamine-alanine; PQS, proline-glutamine-serine; SOX9, SRY-like HMG box 9; SRY, sex-determining region on the Y chromosome.
N-terminus, which is required for DNA-dependent dimerization of SOX9 at paired binding sites within target promoters [32,33]. Sex reversing mutations occur throughout the SOX9 gene, including within the trans-activation domains [34], although mutations in the dimerization domain have been shown to cause CD without sex reversal [33]. In terms of the nuclear localization of SOX9, two independent nuclear localization signals have been detected. These are positioned at either end of the HMG domain, as in the case of SRY [17]. Both NLS motifs are conserved in several other HMG domain proteins, including many if not all of the SOX proteins. It could be that this mechanism of nuclear localization is common to the entire SOX family [17,23]. As with SRY, cases of XY sex reversal related to deficiencies in the nuclear import of SOX9 have been reported. Preiss and colleagues [9] identified a sexreversing point mutation (A158T) (Figure 1b) that causes a twofold reduction in nuclear import efficiency in vitro compared to wild-type SOX9. Curiously, the A158T mutation affects neither of the NLSs directly but DNA binding is affected by this mutation, so it is unclear to what degree this reduction in nuclear import is involved in the observed sex reversal phenotype. These researchers characterized the mechanism of nuclear import of SOX9, showing a requirement for importin b (as observed with http://tigs.trends.com
SRY), and predicted that all SOX proteins would be found to use this mechanism. The A158T mutant protein interacts normally with importin b but was subsequently found to manifest abnormal conformation on binding of calmodulin, showing increased sensitivity to calmodulin antagonists [35]. One would predict that other CD or sex reversal-associated mutations in SOX9 would be found to correlate with defects in nuclear import. In through the out door Intuitively, a mechanism by which a transcription factor can be shunted into the nucleus would seem to be a basic requirement for its function once it is produced in the cell cytoplasm. It is perhaps not surprising that interference with this process could cause disruptions to proper cellular development. However, the story is not this simple. Recent data [10] suggest that active mechanisms for nuclear export and import are required for SOX9 function. Prior to sexual differentiation, SOX9 protein is found in the cytoplasm of undifferentiated gonads of both sexes. As differentiation of the testis proceeds following the expression of SRY, SOX9 becomes localized to the nucleus in the male gonad, whereas expression of SOX9 is downregulated in the developing ovary [36]. This observation of nucleocytoplasmic shuttling of SOX9 led to the search for a nuclear export signal (NES) in the SOX9
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protein. Accordingly, a functional leucine-rich NES was identified within the HMG domain (amino acid residues 134– 147), situated between the two NLS sites [10]. Of the nuclear export signals found in eukaryotes, the best characterized are these small hydrophobic leucine-rich NESs, first described in the HIV-1 REV protein [1]. These REV-type NESs function via interaction with the export factor chromosome region maintenance 1 (CRM1). Inhibiting the action of the leucine-rich NES of SOX9 in organ culture (by the action of leptomycin B, which specifically inhibits CRM1-dependent nuclear export) induced sex reversal in mouse XX gonads. This was characterized by sole nuclear localization of SOX9 and expression of SOX9-induced genes, such as anti-Mu¨llerian hormone (AMH), which induces the regression of the female-specific Mu¨llerian duct in the developing testis. Gasca et al. [10] proposed that the balance between nuclear import and export of SOX9 might therefore act as a switch mechanism triggering the male pathway in the developing testis. Interestingly, these findings were complemented by work on another SOX gene, SOX10, which is essential for neural crest development [37]. Although this gene plays no discernible role in sex determination, SOX10 is closely related to SOX9 on the basis of sequence similarities within and outside the HMG box. Both genes, along with SOX8, belong to subgroup E of the SOX gene family [38]. Similar to SOX9, SOX10 possesses a REV-type leucinerich NES between the two NLS sequences of its HMG domain [11], and has been shown to actively shuttle between the nucleus and cytoplasm. Rehberg and colleagues [11] demonstrated the function of this NES and showed that its mutational inactivation, or inhibition via the action of leptomycin B, was sufficient to prevent nucleocytoplasmic shuttling of SOX10. Strikingly, this led to decreased trans-activation of endogenous target genes of SOX10, as well as transfected reporter constructs. Therefore, continuous nucleocytoplasmic shuttling appears to be essential to the function of SOX10. Any protein with both an NLS and an NES can shuttle back and forth between the nucleus and cytoplasm as required. If such a protein is a transcription factor, this has clear implications for post-translational regulation, with spatial separation from its DNA target (by exclusion from the nucleus) acting as a potent inhibitor of function. Additionally, post-translational modifications, such as phosphorylation and dephosphorylation, might regulate nucleocytoplasmic shuttling by masking or exposing signals for nuclear import or export within the protein [39]. Conserved phosphorylation sites flanking the HMG domains of SRY and SOX9 [40] might be implicated in the regulation of nuclear import and export of these proteins. Significantly for the SOX gene family, the leucine-rich NES sequence found in SOX9 and SOX10 is not limited to subgroup E of the SOX family. Conserved hydrophobic residues associated with REV-type nuclear export signals have been found in the corresponding HMG-domain regions of a variety of SOX proteins across various subgroups, including SRY [10,11]. However, whether these sequences can mediate nuclear export of SOX proteins other than SOX9 and SOX10 have to be determined. http://tigs.trends.com
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Conclusion Active, continuous shuttling between nucleus and cytoplasm might prove to be important to the function of the majority of SOX proteins in development, and their malfunction in human disease. Certainly the importance of nuclear import and export of a range of SOX proteins can be tested experimentally using targeted mutation strategies in mice. In broader terms, the regulation of nucleocytoplasmic shuttling is clearly another level of control over how, when and where transcription factors operate during development, and another point at which the function of developmental transcription factors might be compromised in the aetiology of human disease. Acknowledgements We thank Josephine Bowles, Megan Wilson and Annemiek Beverdam of the Koopman laboratory for critical reading of the manuscript. J.M.S. is the recipient of an Australian Postgraduate Award and a supplementary scholarship from the Institute for Molecular Bioscience. P.A.K. is a Professorial Research Fellow of the Australian Research Council.
References 1 Fischer, U. et al. (1995) The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell 82, 475– 483 2 Mowen, K. and David, M. (2000) Regulation of STAT1 nuclear export by Jak1. Mol. Cell. Biol. 20, 7273– 7281 3 Neufeld, K.L. et al. (2000) Ademotatous polyposis coli protein contains two nuclear export signals and shuttles between the nucleus and cytoplasm. Proc. Natl. Acad. Sci. U. S. A. 97, 12085 – 12090 4 Stommel, J.M. et al. (1999) A leucine-rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by NES masking. EMBO J. 18, 1660 – 1672 5 Rodriguez, J.A. and Henderson, B.R. (2000) Identification of a functional nuclear export signal in BRCA1. J. Biol. Chem. 275, 38589 – 38596 6 Watanabe, M. et al. (2000) Regulation of intracellular dynamics of Smad4 by its leucine-rich nuclear export signal. EMBO Rep. 1, 176 – 182 7 Fabbro, M. and Henderson, B.R. (2003) Regulation of tumour suppressors by nuclear-cytoplasmic shuttling. Exp. Cell Res. 282, 59 – 69 8 Li, B. et al. (2001) Human sex reversal due to impaired nuclear localization of SRY – a clinical correlation. J. Biol. Chem. 276, 46480 – 46484 9 Preiss, S. et al. (2001) Compound effects of point mutations causing campomelic dysplasia/autosomal sex reversal upon SOX9 structure, nuclear transport, DNA binding, and transcriptional activation. J. Biol. Chem. 276, 27864 – 27872 10 Gasca, S. et al. (2002) A nuclear export signal within the high mobility group domain regulates the nucleocytoplasmic translocation of SOX9 during sexual determination. Proc. Natl. Acad. Sci. U. S. A. 99, 11199– 11204 11 Rehberg, S. et al. (2002) Sox10 is an active nucleocytoplasmic shuttle protein, and shuttling is crucial for Sox10-mediated transactivation. Mol. Cell. Biol. 22, 5826– 5834 12 Numabe, H. et al. (1992) DNA analyses of XX and XX-hypospadiac males. Hum. Genet. 90, 211 – 214 13 Fechner, P.Y. et al. (1993) The role of the sex determining region Y gene in the etiology of 46,XX maleness. J. Clin. Endocrinol. Metab. 76, 690– 695 14 Giese, K. et al. (1994) Distinct DNA-binding properties of the high mobility group domain of murine and human SRY sex-determining factors. Proc. Natl. Acad. Sci. U. S. A. 91, 3368– 3374 15 Pontiggia, A. et al. (1994) Sex-reversing mutations affect the architecture of SRY-DNA complexes. EMBO J. 13, 6115 – 6124 16 Koopman, P.A. (2001) Sry, Sox9 and mammalian sex determination. In Genes and Mechanisms in Vertebrate Sex Determination (Scherer, G. and Schmid, M., eds), pp. 25 – 56, Birkhauser-Verlag 17 Su¨dbeck, P. and Scherer, G. (1997) Two independent nuclear
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localization signals are present in the DNA-binding high mobility group domains of SRY and SOX9. J. Biol. Chem. 272, 27848 – 27852 Forwood, J. et al. (2001) The C-terminal nuclear localization signal of the sex determining region Y (SRY) high mobility group domain mediates nuclear import through importin beta 1. J. Biol. Chem. 276, 46575 – 46582 Kohler, M. et al. (1999) Nuclear protein transport pathways. Exp. Nephrol. 7, 290 – 294 Harley, V.R. et al. (1996) The HMG box of SRY is a calmodulin binding domain. FEBS Lett. 391, 24– 28 Harley, V.R. et al. (2003) Defective importin b recognition and nuclear import of the sex-determining factor SRY are associated with XY sexreversing mutations. Proc. Natl. Acad. Sci. U. S. A. 100, 7045 – 7050 Bowles, J. et al. (2000) Phylogeny of the SOX family of developmental transcription factors based on sequence and structural indicators. Developmental Biology 227, 239 – 255 Wilson, M. and Koopman, P. (2002) Matching SOX: partner proteins and co-factors of the SOX family of transcriptional regulators. Curr. Opin. Genet. Dev. 12, 441– 446 Wright, E. et al. (1995) The Sry-related gene Sox-9 is expressed during chondrogenesis in mouse embryos. Nat. Genet. 9, 15 – 20 Bi, W. et al. (2001) Haploinsufficiency of Sox9 results in defective cartilage primordia and premature skeletal mineralization. Proc. Natl. Acad. Sci. U. S. A. 98, 6698 – 6703 Houston, C.S. et al. (1983) The campomelic syndrome: review, report of 17 cases, and follow-up on the currently 17- year old boy first reported by Maroteaux et al. in 1971. Am. J. Med. Genet. 15, 3 – 28 Kent, J. et al. (1996) A male-specific role for SOX9 in vertebrate sex determination. Development 122, 2813 – 2822 Morais da Silva, S. et al. (1996) Sox9 expression during gonadal development implies a conserved role for the gene in testis differentiation in mammals and birds. Nat. Genet. 14, 62 – 68 Vidal, V. et al. (2001) Sox9 induces testis development in XX transgenic mice. Nat. Genet. 28, 216 – 217
30 Koopman, P. et al. (1991) Male development of chromosomally female mice transgenic for Sry. Nature 351, 117 – 121 31 Clarkson, M.J. and Harley, V.R. (2002) Sex with two SOX on: SRY and SOX9 in testis development. Trends Endocrinol. Metab. 13, 106 – 111 32 Bernard, P. et al. (2003) Dimerization of SOX9 is required for chondrogenesis, but not for sex determination. Hum. Mol. Genet. 12, 1755– 1765 33 Sock, E. et al. (2003) Loss of DNA-dependent dimerization of the transcription factor SOX9 as a cause for campomelic dysplasia. Hum. Mol. Genet. 12, 1439 – 1447 34 Su¨dbeck, P. et al. (1996) Sex reversal by loss of the C-terminal transactivation domain of human SOX9. Nat. Genet. 13, 230– 232 35 Argentaro, A. et al. (2003) A SOX9 defect of calmodulin-dependent nuclear import in campomelic dysplasia/autosomal sex reversal. J. Biol. Chem. 278, 33839 – 33847 36 de Santa Barbara, P. et al. (2000) Expression and subcellular localization of SF-1, SOX9, WT1, and AMH proteins during early human testicular development. Dev. Dyn. 217, 293– 298 37 Pingault, V. et al. (1998) SOX10 mutations in patients with Waardenburg-Hirschsprung disease. Nat. Genet. 18, 171– 173 38 Bowles, J. et al. (2000) Phylogeny of the SOX family of developmental transcription factors based on sequence and structural indicators. Dev. Biol. 227, 239 – 255 39 Gasiorowski, J.Z. and Dean, D.A. (2003) Mechanisms of nuclear transport and interventions. Adv. Drug Deliv. Rev. 55, 703– 716 40 Harley, V.R. et al. (2003) The molecular action and regulation of the testis-determining factors, SRY (Sex-determining Region on the Y chromosome) and SOX9 (SRY-related high-mobility group (HMG) Box 9). Endocr. Rev. 24, 466 – 487
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