FOXL2 transcriptionally represses Sf1 expression by antagonizing WT1 during ovarian development in mice

Abstract

Steroidogenic factor 1 (SF1; Ad4BP/NR5A1) plays key roles in gonadal development. Initially, the Sf1 gene is expressed in mouse fetal gonads of both sexes, but later is up-regulated in testes and down-regulated in ovaries. While Sf1 expression is activated and maintained by Wilms tumor 1 (WT1) and LIM homeobox 9 (LHX9), the mechanism of sex-specific regulation remains unclear. We hypothesized that Sf1 is repressed by the transcription factor Forkhead box L2 (FOXL2) during ovarian development. In an in vitro system (TM3 cells), up-regulation of Sf1 by the WT1 splice variant WT1-KTS was antagonized by FOXL2, as determined by quantitative RT-PCR. Using reporter assays, we localized the Sf1 proximal promoter region involved in this antagonism to a 674-bp interval. A conserved FOXL2 binding site was identified in this interval by in vitro chromatin immunoprecipitation. Introducing mutations into this site abolished negative regulation by FOXL2 in reporter assays. Finally, in Foxl2-null mice, Sf1 expression was increased 2-fold relative to wild-type XX fetal gonads. Our results support the hypothesis that FOXL2 negatively regulates Sf1 expression by antagonizing WT1-KTS during early ovarian development in mice.

Keywords: LHX9; reproduction; sex determination.

Figure 1.

Sf1 expression decreased from 11.5 dpc and coincided with a dramatic up-regulation of Foxl2 expression. A) Time course of Sf1, Foxl2, Wt1, and Lhx9 expression during mouse gonadal development. Data sets represent mRNA expression relative to Rps29, means ± sd of 3 biologically independent experiments performed in triplicate. Bold and dotted traces indicate ovary and testis, respectively. Statistically significant differences between 11.5 dpc and other time points of Sf1 expression levels in 46,XX gonads were analyzed by using Student's t test. ***P < 0.001. B) Time course of Sf1 and Foxl2 expression during mouse ovarian development, with maximum expression levels adjusted to 100%.

Figure 2.

FOXL2 suppresses up-regulation of Sf1 expression by WT1-KTS in TM3 cells. Sf1 mRNA levels in TM3 cells transfected with constructs expressing Wt1+KTS (A; 0 and 1.5 μg), Lhx9 (A, C; 0 and 1.5 μg), Wt1-KTS (A, B; 0 and 1.5 μg) and Foxl2 (A, C; 0 and 1.5 μg; B; 0, 1.5, and 3.0 μg). Data sets represent mRNA expression relative to Gapdh (means±sd of 3 biologically independent experiments performed in triplicate). Statistical significance was determined by using 1-way ANOVA and a Tukey-Kramer post hoc test. NS, not significant. *P < 0.05.

Figure 3.

FOXL2 represses Sf1 expression by directly binding to the proximal Sf1 promoter in vitro. A) Luciferase activity driven by the Sf1 proximal promoter in TM3 cells transfected with expression plasmids for WT1-KTS (200 ng) and FOXL2 (0–400 ng) (means±sd of 3 biologically independent experiments performed in triplicate). B) Schematic representation of the structure of the proximal Sf1 promoter. FLB1, W, and L with the shade box indicate the putative FOXL2, WT1-KTS, and LHX9 binding sequence, respectively. Arrows indicate primers designed for ChIP assay. C) ChIP analysis using anti-MYC or control IgG antibodies. Samples were analyzed by qPCR using a primer set encompassing the putative FOXL2 biding site or a primer set for negative control. Shaded and open bars indicate samples treated with anti-Myc or anti-IgG antibodies, respectively. D) Schematic representation of the luciferase reporter with mutations in the FOXL2 binding site. E) Luciferase activity of the Sf1 proximal reporter vector with mutations in the FOXL2 binding site in TM3 cells transfected with WT1-KTS (200 ng) and FOXL2 (0–400 ng) (means±sd of 3 biologically independent experiments performed in triplicate). F) Schematic representation of the structure of the proximal Sf1 promoter and the primers for ChIP assay. G) Samples were extracted from TM3 cells transfected with HA-Wt1-KTS solely or along with Foxl2. ChIP analysis using anti-HA or control IgG antibodies. Samples were analyzed by qPCR using a primer set encompassing the WT1-KTS biding site or a primer set for negative control (NC2). Shaded bars and open bars indicate samples treated with anti-HA or anti-IgG antibodies, respectively. Statistical significance was determined using 1-way ANOVA followed by a Tukey-Kramer post hoc test. NS, not significant. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4.

WT1, LHX9, and FOXL2 binding motif are well conserved in eutherian mammals. Phylogenetic analysis was performed on the Sf1 proximal promoter sequence of rats, humans, cows, dogs, and mice. Each sequence was obtained from the Ensembl database and aligned using BLAST. Numbering is based on the mouse sequences, and known binding sites are labeled and shaded in gray. Putative FOXL2-binding domains (FLB1) are labeled, shaded, and boxed.

Figure 5.

Sf1 expression was increased in Foxl2-null mice during fetal ovarian development. qRT-PCR analysis of Sf1, Wt1, and Lhx9 expression at 13.5 dpc, 16.5 dpc, and P0 in XX gonads of Foxl2-null mice. Data represent mRNA expression relative to XX wild-type (WT) gonads (means±sd of 3 biologically independent experiments performed in triplicate). *P < 0.05, **P < 0.01.

Figure 6.

Model of regulation for the sexual dimorphic expression pattern of Sf1. In 46,XY gonads, SOX9 is involved in additional expression of Sf1, resulting in remarkably increased Sf1 expression. In 46,XX gonads, FOXL2 inhibits transactivation by WT1-KTS, leading to decreased Sf1 expression (present study).