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

Abstract

In mammals, male sex determination starts when the Y chromosome Sry gene is expressed within the undetermined male gonad. One of the earliest effect of Sry expression is to induce up-regulation of Sox9 gene expression in the developing gonad. SOX9, like SRY, contains a high mobility group domain and is sufficient to induce testis differentiation in transgenic XX mice. Before sexual differentiation, SOX9 protein is initially found in the cytoplasm of undifferentiated gonads from both sexes. At the time of testis differentiation and anti-Müllerian hormone expression, it becomes localized to the nuclear compartment in males whereas it is down-regulated in females. In this report, we used NIH 3T3 cells as a model to examine the regulation of SOX9 nucleo-cytoplasmic shuttling. SOX9-transfected cells expressed nuclear and cytoplasmic SOX9 whereas transfected cells treated with the nuclear export inhibitor leptomycin B, displayed an exclusive nuclear localization of SOX9. By using SOX9 deletion constructs in green fluorescent protein fusion proteins, we identified a functional nuclear export signal sequence between amino acids 134 and 147 of SOX9 high mobility group box. More strikingly, we show that inhibiting nuclear export with leptomycin B in mouse XX gonads cultured in vitro induced a sex reversal phenotype characterized by nuclear SOX9 and anti-Müllerian hormone expression. These results indicate that SOX9 nuclear export signal is essential for SOX9 sex-specific subcellular localization and could be part of a regulatory switch repressing (in females) or triggering (in males) male-specific sexual differentiation.

Fig 1.

SOX proteins contain conserved NES sequences. (A) Alignment of SOX9 NES sequence (amino acids 134–147) with conserved leucine-rich functional NES sequences (16). (B) Alignment of the HMG box NES sequences of SOX9 with human SOX proteins representative of each SOX family group (for the classes, see ref. 27). Group A: SRY (SwissProt accession no. Q 05066); group B: SOX2 (P 48431); group C: SOX4 (O 15370); group D: SOX5 (P 35711); group E: SOX9 (P 48436); group F: SOX7 (AAH 04299 and ref. 46), and group G: SOX20 (O 60248). Conserved hydrophobic residues are boxed. SOX9 leucine-142 (L142) is specific to group E SOX proteins and is indicated by * in A.

Fig 2.

SOX9 contains a CRM1-dependent NES. COS-7 (A) and NIH 3T3 (B) cells were transiently transfected with a SOX9 expression vector and treated with (2.5 ng/ml) LMB when indicated (+LMB). Cells were costained with SOX9 Ab (Left) and Hoechst dye (HST, Right). (A) In COS-7 cells, SOX9 was strictly nuclear and was not affected by LMB (not shown). (B) Conversely in NIH 3T3 cells, SOX9 is also cytoplasmic (−LMB). This localization is the result of nuclear export because after inhibition by LMB all SOX9 remains in the nucleus (+LMB).

Fig 3.

Mapping of the SOX9 NES. Different HMG box constructs and NES oligos were fused to GFP reporter and transfected into NIH 3T3 cells. The GFP and Hoechst dye (HST) were analyzed in control cells (−LMB) or in cells treated with 2.5 ng/ml LMB (+LMB). Schematic drawings of the different reporter constructs and their names are indicated below the complete SOX9-HMG box and to the left of the corresponding transfection experiments. HMG-GFP, ΔNLS₁-GFP (lines HMG and ΔNLS₁) and ΔNLS₂-GFP (not shown) are nuclear and cytoplasmic but are mainly nuclear in the presence of LMB. ΔNLS_1–2-GFP is cytoplasmic because of a lack of NLS peptide; when LMB is applied, the fusion protein is able to remain in the nucleus (line ΔNLS_1–2). Synthetic double-stranded oligos NES-GFP is predominantly cytoplasmic but accumulates in the nucleus when nuclear export is inhibited (line NES, with LMB). Conversely, the two mutant NES oligos 5L-5A-GFP and L142A-GFP are essentially nuclear regardless of LMB inhibition (lines 5L–5A and L142A). Black boxes are NLS sequences; white boxes are the NES oligo sequence within the HMG box.

Fig 4.

Substitution of leucine-142 is sufficient to block nuclear export. COS-7 cells were transiently transfected with wild-type (HMG) and L142 mutant SOX9 HMG (HMG-L142A) expression vectors. After 36 h of expression, cells were treated with the nuclear import inhibitor AcD 3 h before fixing (+AcD) or used as controls (−AcD). Cells were stained with Hoechst dye (HST), and GFP subcellular localization (GFP) was analyzed. When nuclear import is impaired (+AcD), some HMG-GFP fusion protein is able to accumulate in the cytoplasm and becomes visible. In the absence of AcD, the balance is in the favor of nuclear HMG-GFP, whereas inhibition by AcD reveals the nuclear export activity driven by the NES. This export activity is completely abolished by the L142A mutation, and only nuclear L142A-GFP could be detected.

Fig 5.

SOX9 and AMH expression in gonad cultures. Dissected pairs of genital ridge and mesonephros (mesogonads) from 10.5 dpc male and female mouse embryos were cultured in vitro for 4 days and sectioned. The subcellular localization of SOX9 (green in A–C) and AMH (red in D and E) were revealed with specific Abs. Germ cells were concomitantly detected by endogenous alkaline-phosphatase activity on the same sections (dark staining in a, b, d, and e and not detected in c). In male gonads, (A) nuclear SOX9- and (D) cytoplasmic AMH-expressing cells were found in areas positive for germ cell (a and d, respectively). In female gonads, germ cell-positive regions were devoid of any SOX9 (B and b) or AMH (E and e) expression. When cytoplasmic SOX9 could be observed (not colocalized with blue nuclear Hoechst staining in C), it was not in the direct vicinity of germ cells (c). (Bar = 50 μm.)

Fig 6.

LMB induces sex reversal in female gonad cultures. Dissected pairs of mesogonads from 10.5 dpc embryos were cultured in vitro with LMB for 4 days and sectioned. SOX9 (green in B, C, G, and H) and AMH (red in E, F, G, H) were revealed by specific Abs. Germ cells were concomitantly detected by endogenous alkaline-phosphatase activity on the same sections (dark staining in A and D and white in C, F, G, and H). Female gonads treated with LMB displayed a sex-reversal phenotype resembling male-expression pattern: (i) cells expressing nuclear SOX9 (B) surrounded germ cells (black in A and white in C); (ii) in an adjacent section, cytoplasmic AMH was expressed in the same area (E) and surrounded germ cells (D and F). Subcellular localization was confirmed by blue nuclear Hoechst staining (frame in B and E); (iii) nuclear SOX9-positive cells are organized in sex-cord-like circles surrounding germ cells (B and C). Expression of AMH in SOX9-positive cells was recorded on a confocal microscope after double immuno-staining in a male (G) and in a treated female mesogonad culture (H, germ cells marked in white). [Bars = 100 μm (A–F) or 50 μm (G–H).]