title: Aromatase is a direct target of FOXL2: C134W in granulosa cell tumors via a single highly conserved binding site in the ovarian specific promoter.
abstract: BACKGROUND
  Granulosa cell tumors (GCT) of the ovary often express aromatase and synthesize estrogen, which in turn may influence their progression. Recently a specific point mutation (C134W) in the FOXL2 protein was identified in >94% of adult-type GCT and it is likely to contribute to their development. A number of genes are known to be regulated by FOXL2, including aromatase/CYP19A1, but it is unclear which are direct targets and whether the C134W mutation alters their regulation. Recently, it has been reported that FOXL2 forms a complex with steroidogenic factor 1 (SF-1) which is a known regulator of aromatase in granulosa cells.
  
  
  RESULTS
  In this work, the human GCT-derived cell lines KGN and COV434 were heterozygous and wildtype for the FOXL2:C134W mutation, respectively. KGN had abundant FOXL2 mRNA expression but it was not expressed in COV434. Expression of exogenous FOXL2:C134W in COV434 cells induced higher expression of a luciferase reporter for the ovarian specific aromatase promoter, promoter II (PII) (-516bp) than expression of wildtype FOXL2, but did not alter induction of a similar reporter for the steroidogenic acute regulatory protein (StAR) promoter (-1300bp). Co-immunoprecipitation confirmed that FOXL2 bound SF-1 and that it also bound its homologue, liver receptor homologue 1 (LRH-1), however, the C134W mutation did not alter these interactions or induce a selective binding of the proteins. A highly conserved putative binding site for FOXL2 was identified in PII. FOXL2 was demonstrated to bind the site by electrophoretic mobility shift assays (EMSA) and site-directed mutagenesis of this element blocked its differential induction by wildtype FOXL2 and FOXL2:C134W.
  
  
  CONCLUSIONS
  These findings suggest that aromatase is a direct target of FOXL2:C134W in adult-type GCT via a single distinctive and highly conserved binding site in PII and therefore provide insight into the pathogenic mechanism of this mutation.
authors.affiliation: Prince Henry's Institute of Medical Research, Clayton, Victoria, Australia.
authors.forename: Nicholas I
authors.initials: NI
authors.lastname: Fleming
authors.forename: Kevin C
authors.initials: KC
authors.lastname: Knower
authors.forename: Kyren A
authors.initials: KA
authors.lastname: Lazarus
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Fuller
authors.forename: Evan R
authors.initials: ER
authors.lastname: Simpson
authors.forename: Colin D
authors.initials: CD
authors.lastname: Clyne
mesh_terms: D001141:Aromatase / Q000378:metabolism*; D001665:Binding Sites; D045744:Cell Line, Tumor; D005260:Female; D000074842:Forkhead Box Protein L2; D051858:Forkhead Transcription Factors / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic; D006106:Granulosa Cell Tumor / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010051:Ovarian Neoplasms / Q000235:genetics*; D010750:Phosphoproteins / Q000235:genetics; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D000098682:Steroidogenic Acute Regulatory Protein
chemical_list: C421604:FOXL2 protein, human; D000074842:Forkhead Box Protein L2; D051858:Forkhead Transcription Factors; D010750:Phosphoproteins; D000098682:Steroidogenic Acute Regulatory Protein; D001141:Aromatase