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.

herin (E-cadherin) is a major determinant for the acquisition of epithelial cell polarity and for the maintenance of epithelial integrity. The compartments and trafficking components required to sort and transport Ecadherin to the basolateral cell surface remain to be fully defined. On the basis of previous data, we know that E-cadherin is trafficked via the recycling endosome (RE) in nonpolarized and newly polarized cells. Here we explore the role of the RE throughout epithelial morphogenesis in MDCK monolayers and cysts. Time-lapse microscopy in live cells, altering RE function biochemically, and expressing a dominant-negative form of Rab11 (DN-Rab11), each showed that the RE is always requisite for E-cadherin sorting and trafficking. The RE remained important for E-cadherin trafficking in MDCK cells from a nonpolarized state through to fully formed, polarized epithelial monolayers. During the development of epithelial cysts, DN-Rab11 disrupted E-cadherin targeting and trafficking, the subapical localization of pERM and actin, and cyst lumen formation. This final effect demonstrated an early and critical interdependence of Rab11 and the RE for E-cadherin targeting, apical membrane formation, and cell polarity in cysts.

Structural analysis of nuclear receptor subfamily V orphan nuclear receptors suggests that ligand-independent mechanisms must regulate this subclass of receptors. Here, we report that steroidogenic factor 1 (SF-1) and liver receptor homolog 1 are repressed via posttranslational SUMO modification at conserved lysines within the hinge domain. Indeed, mutating these lysines or adding the SUMO isopeptidase SENP1 dramatically increased both native and Gal4-chimera receptor activities. The mechanism by which SUMO conjugation attenuates SF-1 activity was found to be largely histone deacetylase independent and was unaffected by the AF2 corepressor Dax1. Instead, our data suggest that SUMO-mediated repression involves direct interaction of the DEAD-box protein DP103 with sumoylated SF-1. Of potential E3-SUMO ligase candidates, PIASy and PIASx␣ strongly promoted SF-1 sumoylation, and addition of DP103 enhanced both PIAS-dependent receptor sumoylation and SF-1 relocalization to discrete nuclear bodies. Taken together, we propose that DEAD-box RNA helicases are directly coupled to transcriptional repression by protein sumoylation.Steroidogenic factor 1 (SF-1) and liver receptor homolog 1 (LRH-1) are two closely related transcription factors belonging to the nuclear receptor subfamily V (NR5A) that contain a highly conserved DNA binding domain (DBD), a large hinge domain and a ligand binding domain (LBD) (Fig. 1A). Drosophila melanogaster Ftz-F1 is the founding member of this subfamily and interacts directly with the pair-rule gene product of Ftz to control parasegmentation at early embryonic stages (25). The mammalian orthologs SF-1 and LRH-1 are also critical in tissue development and organogenesis (19,27,33). During development, SF-1 is essential for male differentiation, adrenogonadal morphogenesis, and terminal differentiation of the ventromedial hypothalamus, and in the adult, this receptor regulates genes involved in steroid biosynthesis and endocrine signaling (34,44). Although SF-1 null mice die at birth from adrenal failure, SF-1 heterozygous mice live. However, further analyses of these heterozygous mice show that despite seemingly adequate levels of SF-1, the amount of active SF-1 protein is insufficient to overcome defects in adrenal morphogenesis (2, 3). In humans, SF-1 haploinsufficiency is associated with severe adrenal disease and gonadal dysgenesis (1, 28). LRH-1 acts far earlier in development than SF-1, as evidenced by the embryonic lethality observed in LRH-1 null embryos (33). In vitro and in vivo analyses have implicated LRH-1 in bile acid homeostasis (13,26), where a heterozygous phenotype has also emerged in the intestine (4). In addition, LRH-1 controls tissue conversion of androgens to estrogen by regulating aromatase gene expression (7,17) Despite the fact that the high-resolution crystal structure of LRH-1 revealed a large hydrophobic pocket within the LBD (38), natural ligands have yet to emerge for this subclass of receptors. As such, the question of how subfamily V receptors are regulated is u...

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.In mammals, male sex determination is controlled by genetic information encoded on the Y chromosome and leads to the differentiation of embryonic gonads into testes. SRY, a Y-specific gene cloned in 1990 (1), was shown to meet all of the criteria of the testis determining factor (2, 3). SRY encodes a small nuclear protein of 204 residues comprising three distinct domains, with a central domain of about 78 amino acids called the high mobility group (HMG) 1 box. This central domain includes a nuclear localization signal (4), and in vitro studies of the human SRY protein have demonstrated its sequence-specific DNA binding through this HMG box (5). To date, no function has been ascribed to the regions of the human SRY protein outside the HMG box, whereas in mouse Sry, the Cterminal part of the protein can function as a transcriptional activator (6). This apparent lack of human SRY transactivation domain and the background-dependent sex determination capabilities of certain mouse Y chromosomes (7) along with the growing list of HMG box containing proteins involved in the assembly of multiprotein complexes (8, 9) suggest that SRY might interact with other proteins. Indeed regulation of target genes by SRY may require collaboration with other factors interacting directly with the SRY protein. Here, using the two-hybrid system we show that a PDZ domain containing nuclear protein interacts with the C-terminal portion of nonrodent SRY. Our two-hybrid results were confirmed by in vitro experiments and by immunoprecipitation that demonstrate that SRY and SIP-1 are associated also in vivo. Finally, immunofluorescence experiments reveal a nuclear expression pattern for SIP-1 in a cell line as well as in human embryo cuts at the level of the genital ridges. These results suggest a model in which SIP-1 could permit to connect SRY to another protein harboring a PDZ binding motif. The nature of this other protein partner and the functional implication of such a complex in human sex determination are now under investigation. MATERIALS AND METHODSLibrary Screenings-Yeast two-hybrid screening was carried out using the HF7c yeast strain harboring HIS3 and ␤-gal reporter genes under the control of upstream GAL4-binding sites as originally described (10, 11). The bait cons...