FoxL2 and Smad3 Coordinately Regulate Follistatin Gene Transcription

doi:10.1074/jbc.M806676200

FoxL2 and Smad3 Coordinately Regulate Follistatin Gene Transcription*

Clayton Foundation Laboratories for Peptide Biology, Salk Institute for Biological Studies, La Jolla, California 92037

2 To whom correspondence should be addressed: Clayton Foundation Laboratories for Peptide Biology, Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-453-4100 (Ext. 1539); Fax: 858-552-546; E-mail: bilezikjian{at}salk.edu.

Abstract

Follistatin is a transcriptional target and a modulator of activin action. Through an autocrine/paracrine loop, activin controls follistatin levels and thus regulates its own bioavailability. In gonadotropic αT3-1 cells, activin induces follistatin transcription primarily through the action of Smad3 at an intronic Smad-binding element (SBE1). Using a proteomics approach, we searched for endogenous αT3-1 proteins that participate in SBE1-mediated transcription. We identified FoxL2, a member of the forkhead family, as a candidate modulator of SBE1 function. Mutations of FoxL2 are associated with the blepharophimosis/ptosis/epicanthus inversus syndrome characterized with craniofacial defects and premature ovarian failure. FoxL2 localizes to α-glycoprotein subunit- and follicle-stimulating hormone β-positive cells of the adult mouse pituitary and is present in αT3-1 and LβT2 cells, but its pituitary actions remain largely unknown. We have determined that FoxL2 binds to a forkhead-binding element (FKHB) located just downstream of the SBE1 site of the follistatin gene and functions as a Smad3 partner to drive SBE1-mediated transcription in αT3-1 cells treated with activin. Chromatin immunoprecipitation assays confirm that endogenous FoxL2 and Smad3 are recruited to the intronic enhancer of the follistatin gene where the SBE1 and FKHB sites are located. Exogenous FoxL2 enhances SBE1-mediated transcription, and short hairpin RNA-mediated knockdown of endogenous FoxL2 protein compromises this effect in αT3-1 cells. FoxL2 directly associates with Smad3 but not Smad2 or Smad4. This association between Smad3 and FoxL2 is mediated by the MH2 domain of Smad3 and is dependent on an intact forkhead domain in FoxL2. Altogether, these observations highlight a novel role for FoxL2 and suggest that it may function as a transcriptional regulator and a coordinator of Smad3 targets.

Footnotes

↵3 The abbreviations used are: TGF-β, transforming growth factor β; SBE1, Smad-binding element; FSH, follicle-stimulating hormone; FKHB, forkhead-binding site; ChIP, chromatin immunoprecipitation; shRNA, short hairpin RNA; hSmad, human Smad; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; BPES, blepharophimosis/ptosis/epicanthus inversus syndrome; mAb, monoclonal antibody; MOPS, 4-morpholinepropanesulfonic acid; FACS, fluorescence-activated cell sorting; GFP, green fluorescent protein; LH, luteinizing hormone; ESI-LC/MS/MS, electrospray ionization-liquid chromatography/tandem mass spectrometry; αGSU, α-glycoprotein subunit; MH2, Mad homology 2; GnRH, gonadotropin-releasing hormone.
↵4 A. L. Blount, W. W. Vale, and L. M. Bilezikjian, unpublished observations, and D. J. Bernard, personal communication.
↵* This work was supported, in whole or in part, by National Institutes of Health Grant 5R01HD046941-05 (NICHD). This work was also supported in part by the Clayton Medical Research Foundation, Inc., and the Adler Foundation. The mass spectrometry facility at the Salk Institute is supported by the Vincent J. Coates Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵1 Senior Investigator of the Clayton Medical Research Foundation, Inc., and the Helen McLoraine Professor of Molecular Neurobiology.
- Received August 28, 2008.
- Revision received December 10, 2008.
The American Society for Biochemistry and Molecular Biology, Inc.