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F Poulat

Publications and source records attributed to F Poulat.

At least 19 recordsLinked to original sources
PubMed2001

SOX7 transcription factor: sequence, chromosomal localisation, expression, transactivation and interference with Wnt signalling.

The Sox gene family consists of several genes related by encoding a 79 amino acid DNA-binding domain known as the HMG box. This box shares strong sequence similarity to that of the testis determining protein SRY. SOX proteins are transcription factors having critical roles in the regulation of diverse developmental processes in the animal kingdom. We have characterised the human SOX7 gene and compared it to its mouse orthologue. Chromosomal mapping analyses localised mouse Sox7 on band D of mouse chromosome 14, and assigned human SOX7 in a region of shared synteny on human chromosome 8 (8p22). A detailed expression analysis was performed in both species. Sox7 mRNA was detected during embryonic development in many tissues, most abundantly in brain, heart, lung, kidney, prostate, colon and spleen, suggesting a role in their respective differentiation and development. In addition, mouse Sox7 expression was shown to parallel mouse Sox18 mRNA localisation in diverse situations. Our studies also demonstrate the presence of a functional transactivation domain in SOX7 protein C-terminus, as well as the ability of SOX7 protein to significantly reduce Wnt/beta-catenin-stimulated transcription. In view of these and other findings, we suggest different modes of action for SOX7 inside the cell including repression of Wnt signalling.

Amino Acid Sequence↗
PubMed2001

SRYand architectural gene regulation: the kinetic stability of a bent protein-DNA complex can regulate its transcriptional potency.

Protein-directed DNA bending is proposed to regulate assembly of higher-order DNA-multiprotein complexes (enhanceosomes and repressosomes). Because transcriptional initiation is a nonequilibrium process, gene expression may be modulated by the lifetime of such complexes. The human testis-determining factor SRY contains a specific DNA-bending motif, the high-mobility group (HMG) box, and is thus proposed to function as an architectural factor. Here, we test the hypothesis that the kinetic stability of a bent HMG box-DNA complex can in itself modulate transcriptional potency. Our studies employ a cotransfection assay in a mammalian gonadal cell line as a model for SRY-dependent transcriptional activation. Whereas sex-reversal mutations impair SRY-dependent gene expression, an activating substitution is identified that enhances SRY's potency by 4-fold. The substitution (I13F in the HMG box; fortuitously occurring in chimpanzees) affects the motif's cantilever side chain, which inserts between base pairs to disrupt base pairing. An aromatic F13 cantilever prolongs the lifetime of the DNA complex to an extent similar to its enhanced function. By contrast, equilibrium properties (specific DNA affinity, specificity, and bending; thermodynamic stability and cellular expression) are essentially unchanged. This correlation between potency and lifetime suggests a mechanism of kinetic control. We propose that a locked DNA bend enables multiple additional rounds of transcriptional initiation per promoter. This model predicts the occurrence of a novel class of clinical variants: bent but unlocked HMG box-DNA complexes with native affinity and decreased lifetime. Aromatic DNA-intercalating agents exhibit analogous kinetic control of transcriptional elongation whereby chemotherapeutic potencies correlate with drug-DNA dissociation rates.

Amino Acid Sequence↗
PubMed2000

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.

Female↗
PubMed2000

The SOX8 gene is located within 700 kb of the tip of chromosome 16p and is deleted in a patient with ATR-16 syndrome.

SOX proteins are transcription factors that are characterized by a common DNA-binding motif known as the HMG domain. We describe the 5. 4-kb human SOX8 gene that codes for a 446-amino-acid protein and that is expressed strongly in brain and less abundantly in other tissues. SOX8 shows an overall identity of 47% to SOX9 and SOX10. The latter two possess a C-terminal transactivation domain, whereas in SOX8, this domain is located in the central part of the protein. We have mapped SOX8 within 700 kb of the telomeric repeats of band 16p13.3. Hemizygosity for 1 Mb from this region causes the ATR-16 syndrome characterized by alpha-thalassemia and mental retardation. We show that SOX8 is deleted in an ATR-16 patient, and from its location, we deduce that it should be deleted in all previously described cases. Thus, SOX8 is a good candidate gene contributing to the mental retardation phenotype seen in ATR-16 patients.

Amino Acid Sequence↗
PubMed2000

Expression and subcellular localization of SF-1, SOX9, WT1, and AMH proteins during early human testicular development.

Many transcription factors have been identified and implicated in male sex determination pathway. Specifically involved in Sertoli cell differentiation and subsequent anti-Müllerian hormone (AMH) secretion in eutherian mammals, they include steroidogenic factor-1 (SF-1), SOX9 (SRY HMG box related gene 9), WT1 (Wilms' tumor 1), and GATA-4 (a zinc finger transcription factor). These factors have been described to execute their function in the male sex determination pathway by controlling AMH transcriptional expression. To understand the hierarchies of these factors and their involvement in the developing testis, for the first time we show the expression and subcellular localization of these factors by immunohistochemistry in the early human testis during embryogenesis compared with AMH expression. If these studies do not refute their possible synergistic interaction to control AMH expression in human embryo, they also reveal a new sexual dimorphism in SOX9 expression during the sex determination process. We show that SOX9 sex specifically shifts from the cytoplasmic to the nuclear compartment at the time of testis differentiation and AMH expression. Putative models for this subcellular distribution are discussed.

Anti-Mullerian Hormone↗
PubMed1999

Diversification pattern of the HMG and SOX family members during evolution.

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.

Animals↗
PubMed1999

[Sex determination in mammals: state of the art].

Sex determination relies on the translation of chromosomal sex established at fertilisation into gonadal sex (testis or ovary), and later into somatic sex (male or female) under the control of gonadal hormone secretions. The aim of the current review will be to highlight our knowledge of the key events which, in the presence of a Y chromosome, induce the organisation of the developing epithelial cells located inside the genital ridges into testicular cords. Many groups have tried to define the molecules relevant to this process, with a double goal: unravelling a molecular pathway which leads to cell fate decision (Sertoli cell in this particular case) during development; improving the establishment of a diagnosis and subsequent medical management in cases where chromosomal, gonadal and then somatic sexes are discordant. Recent progress made in this area will be depicted, with the introduction of several pieces to this developmental jigsaw puzzle.

Animals↗
PubMed1998

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 beta 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.

Animals↗
PubMed1998

Characterization of two Sp1 binding sites of the human sex determining SRY promoter.

To investigate the molecular basis of the human SRY gene regulation, we have examined the significance of two potential binding sites for the transcription factor Sp1 (Sp1A: -124 to -131 and Sp1B: -147 to -154) by DNase I footprinting and gel mobility shift assays. Cotransfection experiments in Drosophila SL2 cells implicated Sp1 protein in the transcriptional activation of the SRY promoter.

Animals↗
PubMed1998

Phosphorylation of an N-terminal motif enhances DNA-binding activity of the human SRY protein.

Of the several strategies that eukaryotes have evolved to modulate transcription factor activity, phosphorylation is regarded as one of the major mechanisms in signal-dependent transcriptional control. To conclusively demonstrate that the human sex-determining gene SRY is affected by such a post-translational control mechanism, we have analyzed its phosphorylation status in living cells. In the present study, we show that the cyclic AMP-dependent protein kinase (PKA) phosphorylates the human SRY protein in vitro as well as in vivo on serine residues located in the N-terminal part of the protein. This phosphorylation event was shown to positively regulate SRY DNA-binding activity and to enhance the ability of SRY to inhibit a basal promoter activity located downstream of an SRY DNA-binding site concatamer. Together these results strongly support the hypothesis that human SRY is a natural substrate for PKA in vivo and that this phosphorylation significantly modulates its major activity, DNA-binding, thereby possibly altering its biological function.

Amino Acid Sequence↗
PubMed1998

Frasier syndrome is caused by defective alternative splicing of WT1 leading to an altered ratio of WT1 +/-KTS splice isoforms.

The Wilms' tumor gene WT1 plays a key role in genitourinary development and subsequent normal function. Homozygous mutations of WT1 can be found in approximately 15% of Wilms' tumors. Furthermore, somatic heterozygous loss of WT1 is known to lead to cryptorchidism and hypospadias in males. A much more severe phenotype is seen in patients with Denys-Drash syndrome which results from heterozygous dominant-negative mutations of the gene. Characteristic features are mesangial sclerosis with early kidney failure, varying degrees of gonadal dysgenesis and high risk of Wilms' tumors. Here we show that a related disease, Frasier syndrome, characterized by focal glomerular sclerosis, delayed kidney failure and complete gonadal dysgenesis, is probably caused by specific intronic point mutations of WT1 that preferentially affect a CpG dinucleotide. Disruption of alternative splicing at the exon 9 splice donor site prevents synthesis of the usually more abundant WT1 +KTS isoform from the mutant allele. In contrast to Denys-Drash syndrome, no mutant protein is produced. The splice mutation leads to an imbalance of WT1 isoforms in vivo , as detected by RT-PCR on streak gonadal tissue. Thus, WT1 isoforms must have quite different functions, and the pathology of Frasier syndrome suggests that especially gonadal development may be particularly sensitive to imbalance or relative underrepresentation of the WT1 +KTS isoform.

Alternative Splicing↗
PubMed1998

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

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.

Animals↗
PubMed1997

The human testis determining factor SRY binds a nuclear factor containing PDZ protein interaction domains.

The human Y-linked testis determining gene SRY encodes a protein with a DNA binding domain from the high mobility group box family. To date, no function has been assigned to amino acid sequences located outside this DNA binding motif. Here, we identify in a yeast two-hybrid screen a PDZ protein termed SIP-1, as an interacting protein with human SRY. In vitro, biochemical analysis, immunoprecipitation experiments, as well as expression of SIP-1 in human embryonic testis confirm that the two proteins can interact together. Interacting domains were mapped to the C-terminal seven amino acids of SRY and to the PDZ domains of SIP-1, respectively. We hypothesize that SIP-1 could connect SRY to other transcription factors providing SRY for its missing trans-regulation domain.

Amino Acid Sequence↗
PubMed1997

SOX22 is a new member of the SOX gene family, mainly expressed in human nervous tissue.

SOX (SRY box-containing) genes share a particular DNA-binding domain, called HMG, with the mammalian testis-determining gene SRY Several SOX genes have already been shown to be transcription factors involved in the decision of important cell fates during development. Here we report the cloning of a new human member of the SOX gene family, SOX22. The corresponding protein contains several domains that are also present in other paralogous SOX proteins. The SOX22 gene maps to chromosome 20 on band p13 and does not appear to be clustered with any other SOX gene mapped to date. SOX22 mRNA is expressed in various fetal and adult organs and tissues, suggesting that this gene plays roles in both differentiation and maintenance of several cell types.

Age Factors↗
PubMed1996

PEST motifs are not required for rapid calpain-mediated proteolysis of c-fos protein.

Cytoplasmic degradation of c-fos protein is extremely rapid. Under certain conditions, it is a multi-step process initiated by calcium-dependent and ATP-independent proteases called calpains. PEST motifs are peptide regions rich in proline, glutamic acid/aspartic acid and serine/threonine residues, commonly assumed to constitute built-in signals for rapid recognition by intracellular proteases and particularly by calpains. Using a cell-free degradation assay and site-directed mutagenesis, we report here that the three PEST motifs of c-fos are not required for rapid cleavage by calpains. Testing the susceptibility of PEST motif-bearing and non-bearing transcription factors including GATA1, GATA3, Myo D, c-erbA, Tal-1 and Sry, demonstrates that PEST sequences are neither necessary nor sufficient for specifying degradation of other proteins by calpains. This conclusion is strengthened by the observation that certain proteins, reportedly known to be cleavable by calpains, are devoid of PEST motifs.

Base Sequence↗
PubMed1995

Nuclear localization of the testis determining gene product SRY.

We have studied the expression of the human SRY protein (termed p27SRY) in two different cell lines by using specific antibodies. Confocal microscopy enabled us to localize p27SRY precisely in the nucleus in a discrete punctuate pattern. Furthermore, through microinjection experiments, we have demonstrated that the localization of the p27SRY protein into the nucleus was an event involving the NH2-terminal part of the high mobility group (HMG) domain. With the help of several synthetic peptides and various p27SRY mutants, we have characterized a bipartite basic motif in this part of the protein corresponding to a nuclear localization signal. This nuclear localization signal appears to be highly conserved in SRY box- and HMB box-containing proteins, suggesting common properties of nuclear targeting within the HMG box protein family.

Amino Acid Sequence↗
PubMed1994

Description and functional implications of a novel mutation in the sex-determining gene SRY.

The sex-determining gene SRY was screened for molecular alteration in an XY sex-reversed female by single-strand conformation polymorphism (SSCP) technique. An A-to-G transition was detected which leads to an exchange of a tyrosine by a cysteine in the SRY protein. The affected tyrosine residue located at the C terminus of the DNA binding protein is evolutionarily strongly conserved among the members of the HMG box containing proteins. Using gel shift assay and peptide synthesis such a mutation is shown to abolish the SRY protein DNA binding ability. The involvement of this particular amino acid in the binding specificity is also discussed.

Adolescent↗