Roles of binding elements, FOXL2 domains, and interactions with cJUN and SMADs in regulation of FSHβ.

We previously identified FOXL2 as a critical component in FSHβ gene transcription. Here, we show that mice deficient in FOXL2 have lower levels of gonadotropin gene expression and fewer LH- and FSH-containing cells, but the same level of other pituitary hormones compared to wild-type littermates, highlighting a role of FOXL2 in the pituitary gonadotrope. Further, we investigate the function of FOXL2 in the gonadotrope cell and determine which domains of the FOXL2 protein are necessary for induction of FSHβ transcription. There is a stronger induction of FSHβ reporter transcription by truncated FOXL2 proteins, but no induction with the mutant lacking the forkhead domain. Specifically, FOXL2 plays a role in activin induction of FSHβ, functioning in concert with activin-induced SMAD proteins. Activin acts through multiple promoter elements to induce FSHβ expression, some of which bind FOXL2. Each of these FOXL2-binding sites is either juxtaposed or overlapping with a SMAD-binding element. We determined that FOXL2 and SMAD4 proteins form a higher order complex on the most proximal FOXL2 site. Surprisingly, two other sites important for activin induction bind neither SMADs nor FOXL2, suggesting additional factors at work. Furthermore, we show that FOXL2 plays a role in synergistic induction of FSHβ by GnRH and activin through interactions with the cJUN component of the AP1 complex that is necessary for GnRH responsiveness. Collectively, our results demonstrate the necessity of FOXL2 for proper FSH production in mice and implicate FOXL2 in integration of transcription factors at the level of the FSHβ promoter

Hormonal regulation of FSH beta-subunit gene expression

In the anterior pituitary, GnRH and activin are key regulators of reproduction. Furthermore, GnRH and activin synergize to induce FSH[beta] gene expression. We have identified a novel region of the FSH[beta] promoter located at -23 from the transcriptional start site that is important for GnRH responsiveness. In addition, activin has been shown to exert its effects on FSH[beta] gene transcription through Smad transcription factors as well as through the newly identified transcription factor FoxL2. In the mouse FSH[beta] promoter, we identified the FoxL2 sites at -350 and -120 to be most critical for activin induction, FoxL2 induction as well as for the synergistic induction by activin and GnRH. There seems to be controversy regarding the necessity of Smad binding sites adjacent to FoxL2 sites to convey activin responsiveness in different species. Therefore, we sought to determine whether FoxL2 or Smad binding site is a critical player in human FSH[beta] promoter by focusing on the homologous site at -132 from the transcriptional start sit