The Human Testis Determining Factor SRY Binds a Nuclear Factor Containing PDZ Protein Interaction Domains

Abstract

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...

Ezrin–Radixin–Moesin-Binding Phosphoprotein 50 Is Expressed At the Apical Membrane of Rat Liver Epithelia

“…Interestingly, SIP-1 in a human embryonic cell line and fetal gonadal cells also displays a cytoplasmic/nuclear distribution. 42 Immunofluorescence of nephron segments in the renal cortex, which displays two E3KARP protein isoforms by Western blotting, also shows a differential staining pattern in different nephron segments. Collecting ducts show strong intracellular staining whereas proximal tubules, which express NHE3 at their apical membrane, show E3KARP localized to the apical domain (Fig.…”

“…42 The principal difference resides in the sequence at the COOH terminus. cDNA sequence analysis and Western blotting show that liver expresses the short form (Figs.…”

SRY gene structure and phylogeny in the cetacean species

“…Mouse Sry has a repeat sequence at the C-terminal region that codes for glutamine-rich amino acid residues (Bowles et al 1999). Human SRY has PDZ (postsynaptic density protein, disc-large, zo-1) domains at the end of the C-terminal region that codes for DNA binding protein (Poulat et al 1997). However, neither was detected for the C-terminal region of the cetacean SRY in this study.…”

Spatially dynamic expression of Sry in mouse genital ridges

“…Sry encodes a transcription factor that binds to, and bends, specific DNA sequences (Harley et al, 1992;Giese et al, 1994;Pontiggia et al, 1995). Some evidence has emerged that SRY protein may directly activate (Dubin and Ostrer, 1994) or repress (McElreavey et al, 1993) transcription of its target genes, perhaps in concert with other proteins (Poulat et al, 1997;Zhang et al, 1999;Bowles et al, 1999). Sry expression appears to have several cellular consequences in the genital ridges, including induction of cell proliferation (Schmahl et al, 2000), stimulation of ingression of mesonephric cells, and subsequent cord formation Merchant-Larios et al, 1993;Merchant-Larios and Moreno-Mendoza, 1998;Martineau et al, 1997;Capel et al, 1999;Tilmann and Capel, 1999), and induction of Sertoli cell differentiation (Burgoyne et al, 1988;Palmer and Burgoyne, 1991;Patek et al, 1991).…”