A role for SOX9 in post-transcriptional processes: insights from the amphibian oocyte.

Sox9 is a member of the gene family of SOX transcription factors, which is highly conserved among vertebrates. It is involved in different developmental processes including gonadogenesis. In all amniote species examined thus far, Sox9 is expressed in the Sertoli cells of the male gonad, suggesting an evolutionarily conserved role in testis development. However, in the anamniotes, fishes and amphibians, it is also expressed in the oocyte but the significance of such an expression remains to be elucidated. Here, we have investigated the nuclear localization of the SOX9 protein in the oocyte of three amphibian species, the urodelan Pleurodeles waltl, and two anurans, Xenopus laevis and Xenopus tropicalis. We demonstrate that SOX9 is associated with ribonucleoprotein (RNP) transcripts of lampbrush chromosomes in an RNA-dependent manner. This association can be visualized by Super-resolution Structured Illumination Microscopy (SIM). Our results suggest that SOX9, known to bind DNA, also carries an additional function in the posttranscriptional processes. We also discuss the significance of the acquisition or loss of Sox9 expression in the oocyte during evolution at the transition between anamniotes and amniotes.

MiniSOX9, a dominant-negative variant in colon cancer cells.

Inherited and acquired changes in pre-mRNA processing have significant roles in human diseases, especially cancer. Characterization of aberrantly spliced mRNAs may thus contribute to understand malignant transformation. We recently reported an anti-oncogenic potential for the SOX9 transcription factor in the colon. For instance, the Sox9 gene knock out in the mouse intestine results in an excess of proliferation with appearance of hyperplasia. SOX9 is expressed in colon cancer cells but its endogenous activity is weak. We looked for SOX9 variants that may impair SOX9 activity in colon cancer cells and we discovered MiniSOX9, a truncated version of SOX9 devoid of transactivation domain as a result of retention of the second intron. A significant overexpression of MiniSOX9 mRNA in human tumor samples compared with their matched normal tissues was observed by real-time reverse transcriptase-PCR. Immunohistochemistry revealed that MiniSOX9 is expressed at high levels in human colon cancer samples whereas it is undetectable in the surrounding healthy tissues. Finally, we discovered that MiniSOX9 behaves as a SOX9 inhibitor, inhibits protein kinase Cα promoter activity and stimulates the canonical Wnt pathway. This potential oncogenic activity of the SOX9 locus gives new insights on its role in colon cancer.

Characterization of new mutations in the 5'-flanking region of the familial Mediterranean fever gene.

Familial Mediterranean fever (FMF) is a recessive autoinflammatory disease commonly found in the Mediterranean populations. Genetic diagnosis has developed since the discovery of the causative gene MEFV in 1997. As many patients could not be confirmed genetically by routine exon screening, we searched for mutations in the 5'-flanking region of this gene. Using denaturing gradient gel electrophoresis, we screened DNA from 108 patients with clinical FMF and 91 asymptomatic individuals. We found six novel sequence variants in a region extending -825 bp upstream of the first translated codon. To investigate the potential role of these variants in altering MEFV gene expression, we first characterized the MEFV promoter. Promoter mapping assays revealed that the region located between nucleotides -949 and -152 of the initiation codon was important for regulating expression of the gene. We identified a putative enhancer element between -571 and -414. Investigation of the sequence variants found in two patients demonstrated that c.-614C>G resulted in a 70% decrease in promoter activity, whereas c.-382C>T induced a 100% increase in activity, when compared to the wild type. We observed specific DNA-protein binding to both wild-type sites, suggesting that transcription factors may bind to these sequences to modulate MEFV expression.

[Juvenile granulosa-cell tumor: clinical and molecular expression]. / Tumeurs juvéniles de la granulosa: expression clinique et moléculaire.

Ovarian sex cord-stromal tumors are rare tumors that originate from the nongerminal cells of ovary. Two decades ago, the identification of juvenile granulosa-cell tumors (GCT), as a specific entity inside this group, allowed a better treatment of these tumors in children. However, little data have been reported on the natural course of the disease and reliable prognostic factors have not been yet defined. We here review the clinical and genetics aspects of granulosa tumors, based on a series of 40 children. This national collaborative study involved the French Society of Children Cancer and eight clinical departments of pediatric endocrinology. We found that early diagnosis of a tumor, revealed by clinical signs of hyperoestrogeny, is an important prognostic factor. The pathophysiology of these tumors is still debatable and several cellular- and molecular-abnormal signals could be implicated in their development. The role of growth factors and oncogenes through the signaling pathway of MAP kinase is still discussed. According to our data, FSH signaling-transduction pathway, such as a constitutionally activated Galphas, could also be implicated in the induction of granulosa cell proliferation and seems to modulate the invasiveness of the tumor. Last, we have described a low-expression pattern or an extinction of an ovarian-determination gene, FOXL2, which is related to a worse prognosis of this tumor.

Male specific expression of lipocalin-type prostaglandin D synthase (cPTGDS) during chicken gonadal differentiation: relationship with cSOX9.

As in mammals, the SOX9 gene (cSOX9) is specifically expressed in male differentiating Sertoli cells during chicken gonadal development. Recent studies in mouse have shown that the prostaglandin D(2) (PGD(2))/lipocalin-type prostaglandin D synthase (PTGDS) pathway is functionally associated with the regulation of SOX9 and is specifically expressed in male developing gonads. In this study, we have shown that, as in mammals, cPTGDS is a male specific gene during chicken testicular development and is temporally expressed in the same window as cSOX9 in Sertoli cells. Using a culture of gonadal explants, we have shown that exogenous PGD(2) enhances cSOX9 expression in male, and activates its ectopic expression in female gonads without up-regulating cAMH. These data indicate a conserved PGD(2) modulation of SOX9 expression during testicular differentiation between birds and mammals.

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

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.

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.

Characterization of genetic elements required for site-specific integration of Lactobacillus delbrueckii subsp. bulgaricus bacteriophage mv4 and construction of an integration-proficient vector for Lactobacillus plantarum.

Temperate phage mv4 integrates its DNA into the chromosome of Lactobacillus delbrueckii subsp. bulgaricus strains via site-specific recombination. Nucleotide sequencing of a 2.2-kb attP-containing phage fragment revealed the presence of four open reading frames. The larger open reading frame, close to the attP site, encoded a 427-amino-acid polypeptide with similarity in its C-terminal domain to site-specific recombinases of the integrase family. Comparison of the sequences of attP, bacterial attachment site attB, and host-phage junctions attL and attR identified a 17-bp common core sequence, where strand exchange occurs during recombination. Analysis of the attB sequence indicated that the core region overlaps the 3' end of a tRNA(Ser) gene. Phage mv4 DNA integration into the tRNA(Ser) gene preserved an intact tRNA(Ser) gene at the attL site. An integration vector based on the mv4 attP site and int gene was constructed. This vector transforms a heterologous host, L. plantarum, through site-specific integration into the tRNA(Ser) gene of the genome and will be useful for development of an efficient integration system for a number of additional bacterial species in which an identical tRNA gene is present.

The human testis determining factor SRY: a new member of the HMG box protein family.

The product of the sex-determining gene SRY is a member of the HMG box containing protein superfamily. The HMG box is a DNA-binding domain of about 80 amino acids shared by many proteins with diverse functions. It seems that the functions of the full length protein are restricted to the HMG box but their molecular basis remains to be determined. We have summarized here the properties of this binding domain described so far in the literature and, using a synthetic peptide mimicking the DNA binding domain (SRY80), we have confirmed the existence of DNA minor groove contacts with this domain. Using intrinsic fluorescence of the tryptophane, the interaction between SRY80 and the putative target sequence AACAAAT was also quantified. In conclusion, we also consider the possible putative action of SRY to fulfill its role in sex determination.