Limits...
The transcription factor FOXL2 mobilizes estrogen signaling to maintain the identity of ovarian granulosa cells.

Georges A, L'Hôte D, Todeschini AL, Auguste A, Legois B, Zider A, Veitia RA - Elife (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Institut Jacques Monod, Paris, France.

ABSTRACT
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.


Related in: MedlinePlus

FOXL2 is required for ESR2 expression.(A) Relative amounts of Esr2 cDNA, determined by qPCR, in mouse follicular cells transfected with an anti-FOXL2 or control siRNAs for 24 hr and treated by 2 nM Activin A or a vehicle for 18 hr. The average expression level of Sdha and Actb was used as a control for normalization. The values are the mean of three independent experiments performed in duplicate. (B) FOXL2 peaks in the Esr2 TU. Three main peaks can be observed in the fourth and eighth introns. (C) Sequence around the summit of the FOXL2 peak in the eighth intron. FOXL2 and SMAD motifs are present near the peak summit. (D–F) Luciferase assays showing FOXL2/SMAD3/ESR2 transcriptional cooperation/synergy on the Esr2 regulatory elements. COV434 cells were transfected with constructs driving the expression of FOXL2, SMAD3, ESR2, and/or a constitutive mutant of TGFBR1 (T204D), as well as with constructs containing the sequences of Peaks 1–3 (D), mutated versions of Peak 3 (E) or Peak 3 alone (F) cloned upstream of a minimal CMV promoter driving the expression of the Firefly luciferase gene, and a construct for the expression of the Renilla luciferase. In the indicated conditions, the cells were treated with 2 nM Activin for 18 hr before lysis. Transcriptional activity was measured as the ratio of Firefly/Renilla luciferases. p-values according to a Student's t test between the indicated conditions and the control condition: *p < 0.05; **p < 0.01; ***p < 0.001; n.s.: non-significant. Asterisks above the histogram in (E) represent the maximum p-value of a Student's t test between full Peak 3 transcriptional activity and all three mutants. Experiments were performed in six replicates.DOI:http://dx.doi.org/10.7554/eLife.04207.013
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4356143&req=5

fig3: FOXL2 is required for ESR2 expression.(A) Relative amounts of Esr2 cDNA, determined by qPCR, in mouse follicular cells transfected with an anti-FOXL2 or control siRNAs for 24 hr and treated by 2 nM Activin A or a vehicle for 18 hr. The average expression level of Sdha and Actb was used as a control for normalization. The values are the mean of three independent experiments performed in duplicate. (B) FOXL2 peaks in the Esr2 TU. Three main peaks can be observed in the fourth and eighth introns. (C) Sequence around the summit of the FOXL2 peak in the eighth intron. FOXL2 and SMAD motifs are present near the peak summit. (D–F) Luciferase assays showing FOXL2/SMAD3/ESR2 transcriptional cooperation/synergy on the Esr2 regulatory elements. COV434 cells were transfected with constructs driving the expression of FOXL2, SMAD3, ESR2, and/or a constitutive mutant of TGFBR1 (T204D), as well as with constructs containing the sequences of Peaks 1–3 (D), mutated versions of Peak 3 (E) or Peak 3 alone (F) cloned upstream of a minimal CMV promoter driving the expression of the Firefly luciferase gene, and a construct for the expression of the Renilla luciferase. In the indicated conditions, the cells were treated with 2 nM Activin for 18 hr before lysis. Transcriptional activity was measured as the ratio of Firefly/Renilla luciferases. p-values according to a Student's t test between the indicated conditions and the control condition: *p < 0.05; **p < 0.01; ***p < 0.001; n.s.: non-significant. Asterisks above the histogram in (E) represent the maximum p-value of a Student's t test between full Peak 3 transcriptional activity and all three mutants. Experiments were performed in six replicates.DOI:http://dx.doi.org/10.7554/eLife.04207.013

Mentions: As previously mentioned, we observed that Esr2 expression dropped following Foxl2 knockdown, showing that the latter is required for the expression of ESR2 in follicular cells. Previous studies have shown that Esr2 expression is dependent on Activin/SMAD3 in granulosa cells (Kipp et al., 2007). Since cooperation between FOXL2 and Activin/SMAD3 has already been identified on some targets, we tested whether FOXL2 was required for Esr2 induction by Activin. For this, we treated primary follicular cells with siRNAs targeting FOXL2 or control siRNAs for 24 hr, and then with recombinant Activin A for 18 hr (Figure 3A). As expected, we observed an increase of Esr2 expression in the presence of Activin A and a decrease following the Foxl2 knockdown. Interestingly, no increase in Esr2 expression could be observed in cells treated with Activin A when Foxl2 was knocked-down. This clearly shows that FOXL2 is required for efficient Activin induction of Esr2 expression.10.7554/eLife.04207.013Figure 3.FOXL2 is required for ESR2 expression.


The transcription factor FOXL2 mobilizes estrogen signaling to maintain the identity of ovarian granulosa cells.

Georges A, L'Hôte D, Todeschini AL, Auguste A, Legois B, Zider A, Veitia RA - Elife (2014)

FOXL2 is required for ESR2 expression.(A) Relative amounts of Esr2 cDNA, determined by qPCR, in mouse follicular cells transfected with an anti-FOXL2 or control siRNAs for 24 hr and treated by 2 nM Activin A or a vehicle for 18 hr. The average expression level of Sdha and Actb was used as a control for normalization. The values are the mean of three independent experiments performed in duplicate. (B) FOXL2 peaks in the Esr2 TU. Three main peaks can be observed in the fourth and eighth introns. (C) Sequence around the summit of the FOXL2 peak in the eighth intron. FOXL2 and SMAD motifs are present near the peak summit. (D–F) Luciferase assays showing FOXL2/SMAD3/ESR2 transcriptional cooperation/synergy on the Esr2 regulatory elements. COV434 cells were transfected with constructs driving the expression of FOXL2, SMAD3, ESR2, and/or a constitutive mutant of TGFBR1 (T204D), as well as with constructs containing the sequences of Peaks 1–3 (D), mutated versions of Peak 3 (E) or Peak 3 alone (F) cloned upstream of a minimal CMV promoter driving the expression of the Firefly luciferase gene, and a construct for the expression of the Renilla luciferase. In the indicated conditions, the cells were treated with 2 nM Activin for 18 hr before lysis. Transcriptional activity was measured as the ratio of Firefly/Renilla luciferases. p-values according to a Student's t test between the indicated conditions and the control condition: *p < 0.05; **p < 0.01; ***p < 0.001; n.s.: non-significant. Asterisks above the histogram in (E) represent the maximum p-value of a Student's t test between full Peak 3 transcriptional activity and all three mutants. Experiments were performed in six replicates.DOI:http://dx.doi.org/10.7554/eLife.04207.013
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356143&req=5

fig3: FOXL2 is required for ESR2 expression.(A) Relative amounts of Esr2 cDNA, determined by qPCR, in mouse follicular cells transfected with an anti-FOXL2 or control siRNAs for 24 hr and treated by 2 nM Activin A or a vehicle for 18 hr. The average expression level of Sdha and Actb was used as a control for normalization. The values are the mean of three independent experiments performed in duplicate. (B) FOXL2 peaks in the Esr2 TU. Three main peaks can be observed in the fourth and eighth introns. (C) Sequence around the summit of the FOXL2 peak in the eighth intron. FOXL2 and SMAD motifs are present near the peak summit. (D–F) Luciferase assays showing FOXL2/SMAD3/ESR2 transcriptional cooperation/synergy on the Esr2 regulatory elements. COV434 cells were transfected with constructs driving the expression of FOXL2, SMAD3, ESR2, and/or a constitutive mutant of TGFBR1 (T204D), as well as with constructs containing the sequences of Peaks 1–3 (D), mutated versions of Peak 3 (E) or Peak 3 alone (F) cloned upstream of a minimal CMV promoter driving the expression of the Firefly luciferase gene, and a construct for the expression of the Renilla luciferase. In the indicated conditions, the cells were treated with 2 nM Activin for 18 hr before lysis. Transcriptional activity was measured as the ratio of Firefly/Renilla luciferases. p-values according to a Student's t test between the indicated conditions and the control condition: *p < 0.05; **p < 0.01; ***p < 0.001; n.s.: non-significant. Asterisks above the histogram in (E) represent the maximum p-value of a Student's t test between full Peak 3 transcriptional activity and all three mutants. Experiments were performed in six replicates.DOI:http://dx.doi.org/10.7554/eLife.04207.013
Mentions: As previously mentioned, we observed that Esr2 expression dropped following Foxl2 knockdown, showing that the latter is required for the expression of ESR2 in follicular cells. Previous studies have shown that Esr2 expression is dependent on Activin/SMAD3 in granulosa cells (Kipp et al., 2007). Since cooperation between FOXL2 and Activin/SMAD3 has already been identified on some targets, we tested whether FOXL2 was required for Esr2 induction by Activin. For this, we treated primary follicular cells with siRNAs targeting FOXL2 or control siRNAs for 24 hr, and then with recombinant Activin A for 18 hr (Figure 3A). As expected, we observed an increase of Esr2 expression in the presence of Activin A and a decrease following the Foxl2 knockdown. Interestingly, no increase in Esr2 expression could be observed in cells treated with Activin A when Foxl2 was knocked-down. This clearly shows that FOXL2 is required for efficient Activin induction of Esr2 expression.10.7554/eLife.04207.013Figure 3.FOXL2 is required for ESR2 expression.

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.

View Article: PubMed Central - PubMed

Affiliation: Institut Jacques Monod, Paris, France.

ABSTRACT
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.


Related in: MedlinePlus