Philippe Berta

Direct interaction of SRY-related protein SOX9 and steroidogenic factor 1 regulates transcription of the human anti-Müllerian hormone gene.

ABSTRACT For proper male sexual differentiation, anti-Müllerian hormone (AMH) must be tightly regulated during embryonic development to promote regression of the Müllerian duct. However, the molecular mechanisms specifying the onset of AMH in male mammals are not yet clearly defined. A DNA-binding element for the steroidogenic factor 1 (SF-1), a member of the orphan nuclear receptor family, located in the AMH proximal promoter has recently been characterized and demonstrated as being essential for AMH gene activation. However, the requirement for a specific promoter environment for SF-1 activation as well as the presence of conserved cis DNA-binding elements in the AMH promoter suggest that SF-1 is a member of a combinatorial protein-protein and protein-DNA complex. In this study, we demonstrate that the canonical SOX-binding site within the human AMH proximal promoter can bind the transcription factor SOX9, a Sertoli cell factor closely associated with Sertoli cell differentiation and AMH expression. Transfection studies with COS-7 cells revealed that SOX9 can cooperate with SF-1 in this activation process. In vitro and in vivo protein-binding studies indicate that SOX9 and SF-1 interact directly via the SOX9 DNA-binding domain and the SF-1 C-terminal region, respectively. We propose that the two transcription factors SOX9 and SF-1 could both be involved in the expression of the AMH gene, in part as a result of their respective binding to the AMH promoter and in part because of their ability to interact with each other. Our work thus identifies SOX9 as an interaction partner of SF-1 that could be involved in the Sertoli cell-specific expression of AMH during embryogenesis.

SOX9 is an intestine crypt transcription factor, is regulated by the Wnt pathway, and represses the CDX2 and MUC2 genes

TCF and SOX proteins belong to the high mobility group box transcription factor family. Whereas TCFs, the transcriptional effectors of the Wnt pathway, have been widely implicated in the development, homeostasis and disease of the intestine epithelium, little is known about the function of the SOX proteins in this tissue. Here, we identified SOX9 in a SOX expression screening in the mouse fetal intestine. We report that the SOX9 protein is expressed in the intestinal epithelium in a pattern characteristic of Wnt targets. We provide in vitro and in vivo evidence that a bipartite β-catenin/TCF4 transcription factor, the effector of the Wnt signaling pathway, is required for SOX9 expression in epithelial cells. Finally, in colon epithelium-derived cells, SOX9 transcriptionally represses the CDX2 and MUC2 genes, normally expressed in the mature villus cells of the intestinal epithelium, and may therefore contribute to the Wnt-dependent maintenance of a progenitor cell phenotype.

Prostaglandin D2 induces nuclear import of the sex-determining factor SOX9 via its cAMP-PKA phosphorylation

During mammalian gonadal development, nuclear import/export of the transcription factor SOX9 is a critical step of the Sry‐initiated testis‐determining cascade. In this study, we identify a molecular mechanism contributing to the SOX9 nuclear translocation in NT2/D1 cells, which is mediated by the prostaglandin D2 (PGD2) signalling pathway via stimulation of its adenylcyclase‐coupled DP1 receptor. We find that activation of cAMP‐dependent protein kinase A (PKA) induces phosphorylation of SOX9 on its two S64 and S181 PKA sites, and its nuclear localization by enhancing SOX9 binding to the nucleocytoplasmic transport protein importin β. Moreover, in embryonic gonads, we detect a male‐specific prostaglandin D synthase expression and an active PGD2 signal at the time and place of SOX9 expression. We thus propose a new step in the sex‐determining cascade where PGD2 acts as an autocrine factor inducing SOX9 nuclear translocation and subsequent Sertoli cell differentiation.

Male specific expression suggests role of DMRT1 in human sex determination.

Sex determination in mammals is controlled by various transcription factors. Following the identification of SRY on the Y chromosome, several other factors have been identified. They can normally be identified as being involved in sex determination by the identification of sex reversal mutations or deletions, functional studies, and also by male-specific expression patterns in embryos. Here, it is shown that DMRT1, recently demonstrated to be deleted in 9p monosomies associated with sex reversal, is specifically expressed during sex determination in the genital ridge of human male, but not female, embryos, similar to SRY.

Diversification Pattern of the HMG and SOX Family Members During Evolution

Abstract. From a database containing the published HMG protein sequences, we constructed an alignment of the HMG box functional domain based on sequence identity. Due to the large number of sequences (more than 250) and the short size of this domain, several data sets were used. This analysis reveals that the HMG box superfamily can be separated into two clearly defined subfamilies: (i) the SOX/MATA/TCF family, which clusters proteins able to bind to specific DNA sequences; and (ii) the HMG/UBF family, which clusters members which bind non specifically to DNA. The appearance and diversification of these subfamilies largely predate the split between the yeast and the metazoan lineages. Particular emphasis was placed on the analysis of the SOX subfamily. For the first time our analysis clearly identified the SOX subfamily as structured in six groups of genes named SOX5/6, SRY, SOX2/3, SOX14, SOX4/22, and SOX9/18. The validity of these gene clusters is confirmed by their functional characteristics and their sequences outside the HMG box. In sharp contrast, there are only a few robust branching patterns inside the UBF/HMG family, probably because of the much more ancient diversification of this family than the diversification of the SOX family. The only consistent groups that can be detected by our analysis are HMG box 1, vertebrate HMG box 2, insect SSRP, and plant HMG. The various UBF boxes cannot be clustered together and their diversification appears to be extremely ancient, probably before the appearance of metazoans.

Male sex determination in the spiny rat Tokudaia osimensis (Rodentia: Muridae) is not Sry dependent

In mammals, gender follows from the development of the embryonic gonads as testis or ovaries and is under genetic control. In males, testis development is dependent on the inheritance of the Y Chromosome-encoded Sry gene. It is believed, as a result of transgenic experiments, that Sry is the only gene encoded on the Y Chr that is necessary to switch the genital ridges towards the testicular developmental pathway (Koopman et al. 1990). An exception to this rule is described in a recent report describing the absence ofSry in two species of an arvicoline rodent, Ellobius, which have an unconventional gonosome complement (Just et al. 1995). In this context,Tokudaia osimensis osimensis, a small murine rodent from the Amami Island (part of the Ryukyu Archipelago in the south of Japan), is an intriguing mammal. Indeed, in this particular species, the Ryukyu Spiny Rat, males and females have an identical karyotype (2n4 25,XO; Honda et al. 1977). This small (head and body length from 12 to 18 cm) spiny rat is a rare mammal (Nowak 1991), believed by IUCN (1978) to be seriously endangered by the general destruction of natural arcas on the Ryukyu Islands.Tokudaia osimensisis fully protected in Japan. The presence or the absence of Sryon either the X Chr or on one of the autosomes of this particular mammal was, therefore, investigated. Using degenerate oligonucleotides designed from an alignment of all Sry HMG box sequences reported so far from rodent only or from mammal and a nested touch-down PCR strategy (Don et al. 1991), we were unable to amplify any Srysequence from genomic DNA of both male and female T. osimensis osimensis (Fig. 1). To ensure that our primers were able to amplify Sry sequences from either a closely related rodent or from an unrelated one, we also amplified DNA of other species such as two murines Apodemus agrarius, Apodemus flavicollis, and one cricetineMesocricetus auratus.In these cases we obtained PCR products of the expected sizes (Fig. 1), which we confirmed as being Sryby sequencing (not shown). This result suggests that the lack of Sryamplification inT. osimensiswas not simply the result of strong sequence divergence owing to the high evolutionary rate of the Sry gene observed in rodents (Tucker and Lundrigan 1993; Whitfield et al. 1993). The positive result observed in the two Apodemusspecies is particularly important because of their close relationship with Tokudaia, as revealed by morphological studies (Kawamura 1989) and also from phylogenetic inferences derived from albumin immunology (Watts and Baverstock 1994) and mitochondrial DNA sequences (S. Soullier and F. Catzeflis, unpublished data). Tokudaia osimensis DNA integrity was next checked by amplification of other genes, such as the androgen receptor AR, and two transcription factorsSox3andZfx (Fig. 1 and data not shown). These results, obtained with autosomal and X Chr encoded genes, clearly demonstrate that our conditions allow the detection of single-copy genes. Taken together, these data indicate that the Sry gene is absent from the T. osimensis osimensis genome. Nevertheless, our results should be tested by additional biological material (from testis tissues) and additional experiments such as Southern blotting. This species is thus the second rodent genus described so far as lacking the mammalian sex-determining factor Sry,since two species of voles in the genus Ellobius (E. lutescensand E. tancrei ) display the same unusual feature (Just et al. 1995). Phylogenetically, the arvicolineEllobius is not closely related to the murine Tokudaia(Fig. 2), as these genera belong to different subfamilies of Muridae, thought to have diverged from each other ca. 17 to 25 My ago (Catzeflis et al. 1993). As all other rodents so far analyzed possess aSry gene (Just et al. 1995; Tucker and Lundrigan 1993; Suzuki et al. 1997), we therefore conclude that the loss of Sry as a switch mechanism in sex determination among Tokudaiaand Ellobius rodents is the result of two independent events. So, even between mammals, a high plasticity of the sex determination mechanism is observed. Such a variety of genetic strategies in sex determination within the same taxon (in this case: the Muridae family) has been previously reported in the case of dipteran insects. In this case, eithe r a Y Chr located factor, an autosomal male dominant factor, or the X:A ratio can be used as the primary signal (Dübendorfer et al. 1992). What may be the genetic switch controlling sex determination in TokudaiaandEllobius?Although several hypotheses are possible at this stage, there is no indication that the same mechanism occurs in these two rodent genera. As a hypothesis, a gene under the control ofSry in the usual genetic pathway may be the master control gene. Recently, the discovery of an X Chr inactivation event occurring during embryonic development in the male mouse urogenital ridges coincident withSry gene expression raises the intriguing possibility of a connection between these two events (Jamieson et al. 1997). To decipher the mechanism(s) of sex determination in these particular species, it is important to know whether there is a genetic difference between males and females. If not, sex determination could be the result of an imprinting mechanism involving the X Chr, since a nonrandom X inactivation has already been reported in mouse extraembryonic tissues (Marahrens et al. 1997) and in somatic cells of X*XY wood lemmings (genusMyopus,another arvicoline rodent) leading to different sexual phenotypes (Schempp et al. 1985). The description of these mechanisms will now require a more precise genetic analysis of these species and will bring us new information on sex determination mechanisms, evolution, and plasticity. Correspondence to: P. Berta Mammalian Genome 9, 590–592 (1998).

Sox neuro, a new Drosophila Sox gene expressed in the developing central nervous system.

We describe the identification and detailed expression pattern of a second Drosophila Sox gene, SoxNeuro (SoxN), highly related to mammalian group B Sox1, 2, 3 genes. SoxN is expressed in a highly dynamic pattern during embyogenesis, being associated with the development of the central nervous system (CNS), from the early steps onwards. We present strong evidence that the early SoxN neuroectoderm expression is controlled by the zygotic dorso-ventral patterning genes (dpp, sog, brk, twi).

Steroidogenic factor-1 contributes to the cyclic adenosine monophosphate down-regulation of human SRY gene expression

Abstract In mammals, male sex determination is initiated by SRY (sex-determining region of the Y chromosome) gene expression and followed by testicular development. This study describes specific down-regulation of the human SRY gene transcription by cAMP stimulation using reverse transcription-polymerase chain reaction experiments. Using transfection experiments, conserved nuclear hormone receptor (NHR1) and Sp1 consensus binding sites were identified as essential for this cAMP transcriptional response. Steroidogenic factor-1 (SF-1), a component of the sex-determination cascade, binds specifically to the NHR1 site and activates the SRY promoter. Activation of SF-1 was abolished by cAMP pretreatment of the cells, suggesting a possible effect of cAMP on the SF-1 protein itself. Indeed, human SF-1 protein contains at least two in vitro cAMP-dependent protein kinase (PKA) phosphorylation sites, leading after phosphorylation to a modification of both DNA-binding activity and interaction with general transcription factors such as Sp1. Taken together, these data suggest that cAMP responsiveness of human SRY promoter involves both SF-1 and Sp1 sites and could act via PKA phosphorylation of the SF-1 protein itself.

Steroidogenic Factor-1 Regulates Transcription of the Human Anti-müllerian Hormone Receptor

Anti-müllerian hormone type II receptor (AMHRII) is a serine/threonine receptor and a member of type II receptors of the transforming growth factor β superfamily. AMHRII has been recently identified in humans, mice, rats, and rabbits. In the male embryo, the AMHRII gene has been shown to be expressed in Sertoli’s cells, in Leydig’s cells and in the mesenchymal cells surrounding the müllerian duct. To determine the functional region of the AMHRII promoter as well as the factors controlling AMHRII gene expression, we used a 1.1-kilobase DNA fragment from the 5′-flanking region of the human AMHRII gene to generate a series of deletion or mutation and analyzed the resulting transcriptional activities after transfection of the NT2/D1 teratocarcinoma cell line. Our results indicate that maximal expression of the AMHRII promoter in this particular cell line, a cell line positive for endogenous AMHRII expression, requires a conserved estrogen receptor half-site element (AGGTCA) identical to the binding element for steroidogenic factor-1 (SF-1). Studies of this SF-1 binding element using gel mobility shift, antibody supershift assays, and transient transfections of reporter constructs indicate that SF-1 can bind and transactivate the AMHRII promoter. Finally, SF-1 protein expression in human male embryos was shown to display a good coincidence with the previously reported AMHRII expression profile. We then propose that SF-1 may be a key transcriptional regulator of AMHRII gene expression during early human development.

Genome-wide analysis of Sox genes in Drosophila melanogaster

Genes of the Sox family encode evolutionarily conserved HMG box containing transcription factors, which play key roles in various events of cell determination/differentiation during development. The total number of Sox genes in Drosophila melanogaster was estimated to be eight, after classical molecular cloning approaches and exhaustive screening of the complete Drosophila genome. Here we report the embryonic and larval expression pattern of four previously uncharacterized Sox genes, through antibody staining and in situ hybridization experiments.