Two Independent Nuclear Localization Signals Are Present in the DNA-binding High-mobility Group Domains of SRY and SOX9

Abstract

Mammalian sex determination and early gonadal differentiation is a developmental process involving a cascade of regulatory gene interactions. Only a few of these genes, all encoding transcription factors, are known (reviewed in Ref. 1), among them the related genes SRY and SOX9. SRY encodes the Ychromosomal testis-determining factor as shown by XY sex reversal in human individuals mutant for SRY (2, 3) and by the demonstration of testis formation in chromosomally female mice transgenic for mouse Sry (4). SOX9 on chromosome 17 is an autosomal gene essential for testis development as mutations in and around this gene cause XY sex reversal in association with the skeletal malformation syndrome campomelic dysplasia (5, 6).Both SRY and SOX9 contain an 80-amino acid DNA-binding motif known as the high-mobility group (HMG) 1 domain that characterizes a whole class of transcription factors (reviewed in Ref. 7). SRY binds to the sequence AACAAT and variants thereof (8) and induces a sharp bend in the DNA (9). The three-dimensional solution structure of the SRY HMG domain complexed with its target sequence has been solved (10), as has a similar complex of the related factor LEF-1 (11). In cell transfection studies, some evidence for transcriptional activation of testis-specific genes by SRY has been presented (12). We have shown in similar transfection assays that SOX9 also functions as a transcription factor, contains a C-terminal transactivation domain (13) and can bind via its HMG domain to the motif AACAAT (14). Recently, mouse Sox9 was found to be expressed in the gonadal anlage of both sexes, with expression increasing in the developing testis and decreasing in the developing ovary, consistent with a role for SOX9/Sox9 in Sertoli cell differentiation (15, 16).As transcription factors, SRY and SOX9 must gain access to the nucleus. Studies on nuclear localization indicate that transport across the nuclear envelope is an active process mediated by one or more nuclear localization signal sequences (NLSs), usually present in the protein itself or in a cofactor (for review, see Refs. 17 and 18). With some exceptions, two main types of NLS motifs exist. One is a short cluster of mainly basic amino acids (arginine and/or lysine), its prototype found in the simian virus 40 large tumor antigen (19). The other is a bipartite NLS motif that comprises two basic amino acids, a spacer of about 10 -15 residues consisting of any amino acid, followed by generally three basic residues, as first described for nucleoplasmin (17). Specialized NLS-binding transporter proteins that carry NLS-containing proteins through the nuclear pore complex into the nucleus have been identified recently (20).Karyophilic NLS sequences are generally identified by their ability to direct an otherwise cytoplasmic protein to the nucleus when fused to it genetically or biochemically and by the effects of deletion or point mutations on nuclear entry (18). Using these approaches with ␤-galactosidase as a reporter protein, we have identified two independent NL...

A nuclear export signal within the high mobility group domain regulates the nucleocytoplasmic translocation of SOX9 during sexual determination

“…These results, together with the previous identification of two nuclear localization signal (NLS) sequences in the HMG box (12), let us to hypothesize that SOX9 is able to shuttle between the nucleus and the cytoplasm and thus may contain a nuclear export signal (NES). Prototypic NESs are short hydrophobic sequences rich in leucine residues (13)(14)(15)(16) regulating the subcellular localization of several proteins through interaction with the CRM1 nuclear export receptor (17,18).…”

Hypoxia-Independent Gene Expression Mediated by SOX9 Promotes Aggressive Pancreatic Tumor Biology

“…SOX9 acts as a transcription factor, binding to a defined consensus sequence (3,4). Phosphorylation of Sox9 on 2 S64 and S181 protein kinase A sites is cAMPdependent and associated with activation of the transcription factor (5).…”

RAR agonists stimulate SOX9 gene expression in breast cancer cell lines: evidence for a role in retinoid-mediated growth inhibition

“…This group of transcription factors interacts with DNA through a high mobility group (HMG) box, a 79-amino-acid motif originally found in the HMG group of non-histone chromatin components. Human SOX9, a 509-amino-acid protein, contains a transactivation domain at its C-terminus (Sudbeck and Scherer, 1997) that is rich in serine, proline and glutamic acid residues.…”