The transcription factor FOXL2 mobilizes estrogen signaling to maintain the identity of ovarian granulosa cells.
Bottom Line: We found that FOXL2 is required for normal gene regulation by steroid receptors, and we show that estrogen receptor beta (ESR2) is the main vector of estradiol signaling in these cells.Moreover, we found that FOXL2 directly modulates Esr2 expression through a newly identified intronic element.Interestingly, we found that FOXL2 repressed the testis-determining gene Sox9 both independently of estrogen signaling and through the activation of ESR2 expression.
Affiliation: Institut Jacques Monod, Paris, France.
FOXL2 is a lineage determining transcription factor in the ovary, but its direct targets and modes of action are not fully characterized. In this study, we explore the targets of FOXL2 and five nuclear receptors in murine primary follicular cells. We found that FOXL2 is required for normal gene regulation by steroid receptors, and we show that estrogen receptor beta (ESR2) is the main vector of estradiol signaling in these cells. Moreover, we found that FOXL2 directly modulates Esr2 expression through a newly identified intronic element. Interestingly, we found that FOXL2 repressed the testis-determining gene Sox9 both independently of estrogen signaling and through the activation of ESR2 expression. Altogether, we show that FOXL2 mobilizes estrogen signaling to establish a coherent feed-forward loop repressing Sox9. This sheds a new light on the role of FOXL2 in ovarian maintenance and function.
No MeSH data available.
Mentions: We observed that a canonical estrogen receptor-binding site was present nearby FOXL2 and SMAD3 sites in the peak 3 sequence. We therefore tested whether ESR2 could activate its own expression, alone or in combination with FOXL2 and TGFβ/SMAD3 (Figure 3F). Interestingly, we observed that the overexpression of ESR2 alone or in combination with SMAD3 and TGFBR1 (T204D) had little effect on luciferase activity. However, ESR2 was able to slightly induce luciferase in the presence of FOXL2 and more strongly in the presence of FOXL2, SMAD3, and TGFBR1 (T204D). Therefore, this experiment suggests that the induction of Esr2 expression due to FOXL2 and TGFβ signals may be amplified by ESR2 action on its own enhancer, although ESR2 itself may not be able to stabilize alone its own expression. This further supports the idea that FOXL2 is required to establish a robust ESR2 expression in the ovary. Several observations corroborate this finding. Indeed, the transcriptional effect of Foxl2 knockdown is well correlated with the one of ESR2 on its target genes (Figure 4—figure supplement 1). Thirteen out of the 20 ESR2 target genes detected here are also (direct and indirect) targets of FOXL2. These shared targets represent slightly more than 10% of FOXL2 targets, confirming that FOXL2 acts upstream of ESR2. In line with this, a hierarchical clustering of FOXL2-activated targets yields two main clusters (Figure 4A). The first cluster is mainly characterized by a strong effect of Esr2 knockdown and includes ESR2 transcriptional targets, whereas no particular signature was observed for the second cluster (Figure 4B and Figure 4—figure supplement 2). These results show that gene regulation via, or along with, ESR2 may represent an important part of the transcriptional effects of FOXL2. Moreover, a third of the ESR2 targets in cultured cells, and Esr2 itself, had a decreased expression in the Foxl2 conditional-knockout ovaries, suggesting that FOXL2 regulates ESR2 (and its targets) in vivo (Figure 1—source data 1). Finally, it is interesting to note that although FOXL2 has a strong positive transcriptional effect on ESR2 targets in control conditions, it has in average no effect on the same genes when Esr2 is knocked down (Figure 4C). FOXL2 still activates a few genes, such as Fam84a, although to lower extents (Figure 4—source data 1). This suggests that the overall positive effect of FOXL2 on ESR2 targets is indeed due to the activation by FOXL2 of ESR2 expression, although FOXL2 may also have some positive or negative effects on individual ESR2 targets through independent pathways.10.7554/eLife.04207.014Figure 4.Analysis of estradiol-dependent transcription.
No MeSH data available.