title: Expression of microphthalmia-associated transcription factor (MITF), which is critical for melanoma progression, is inhibited by both transcription factor GLI2 and transforming growth factor-β.
abstract: The melanocyte-specific transcription factor M-MITF is involved in numerous aspects of melanoblast lineage biology including pigmentation, survival, and migration. It plays complex roles at all stages of melanoma progression and metastasis. We established previously that GLI2, a Kruppel-like transcription factor that acts downstream of Hedgehog signaling, is a direct transcriptional target of the TGF-β/SMAD pathway and contributes to melanoma progression, exerting antagonistic activities against M-MITF to control melanoma cell invasiveness. Herein, we dissected the molecular mechanisms underlying both TGF-β and GLI2-driven M-MITF gene repression. Using transient cell transfection experiments with M-MITF promoter constructs, chromatin immunoprecipitation, site-directed mutagenesis, and electrophoretic mobility shift assays, we identified a GLI2 binding site within the -334/-296 region of the M-MITF promoter, critical for GLI2-driven transcriptional repression. This region is, however, not needed for inhibition of M-MITF promoter activity by TGF-β. We determined that TGF-β rapidly repressed protein kinase A activity, thus reducing both phospho-cAMP-response element-binding protein (CREB) levels and CREB-dependent transcription of the M-MITF promoter. Increased GLI2 binding to its cognate cis-element, associated with reduced CREB-dependent transcription, allowed maximal inhibition of the M-MITF promoter via two distinct mechanisms.
authors.affiliation: Institut Curie, Centre de Recherche, INSERM U1021, CNRS UMR3347, and Université Paris XI, 91400 Orsay, France.
authors.forename: Marie-Jeanne
authors.initials: MJ
authors.lastname: Pierrat
authors.forename: Véronique
authors.initials: V
authors.lastname: Marsaud
authors.forename: Alain
authors.initials: A
authors.lastname: Mauviel
authors.forename: Delphine
authors.initials: D
authors.lastname: Javelaud
mesh_terms: D001483:Base Sequence; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000235:genetics; D018450:Disease Progression; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000502:physiology*; D008545:Melanoma / Q000473:pathology*; D051739:Microphthalmia-Associated Transcription Factor / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000502:physiology*; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000502:physiology*; D000075746:Zinc Finger Protein Gli2
chemical_list: D017931:DNA Primers; D004273:DNA, Neoplasm; C082593:GLI2 protein, human; D051741:Kruppel-Like Transcription Factors; C497234:MITF protein, human; D051739:Microphthalmia-Associated Transcription Factor; D009687:Nuclear Proteins; D016212:Transforming Growth Factor beta; D000075746:Zinc Finger Protein Gli2