title: Pituitary tumor AP-2alpha recognizes a cryptic promoter in intron 4 of fibroblast growth factor receptor 4.
abstract: Fibroblast growth factor receptors (FGFRs) have been implicated in a multitude of proliferative functions, and FGFR4 is expressed differentially in normal and neoplastic pituitary. Human pituitary tumors express a truncated FGFR4 isoform (ptd-FGFR4) for which transcription is initiated from a downstream alternative site. Analysis of FGFR4 intronic sequences predicted a possible promoter within intron 4 (In4) including a classic TATA box with a possible transcriptional start site in intron 5. We show here that the human In4 sequence can direct luciferase reporter activity in transfected pituitary GH4 cells. Four overlapping fragments (A1, A2, B1, and B2) of this intron were examined by electromobility shift assay using nuclear extracts from rat pituitary tumors. Of these, fragment B2 formed complexes with nuclear rat pituitary GH4 extracts that were competed specifically by wild type but not mutant oligonucleotides for the neural crest cell lineage-derived activating transcription factor AP-2. Conversely, an AP-2 consensus sequence probe was competed by the In4 B2 oligonucleotide but not by other fragments of the same intron. The In4 B2 complex was competed partially by NFkappaB, supershifted by an AP-2alpha-specific antibody, and co-migrated with the same probe incubated with recombinant AP-2alpha protein. We also examined the ability of primary human pituitary tumor extracts to interact with the In4 B2 fragment. Pituitary tumor-In4 B2 complexes were competed specifically by wild type AP-2 but not mutant AP-2 oligonucleotides. Western blotting revealed higher levels of AP-2alpha expression in primary human pituitary tumors than in nontumorous tissue. Mutagenesis of the putative AP-2 binding site in In4 B2 resulted in a marked loss of promoter activity in a luciferase assay. AP-2alpha transfection in the presence of the histone deacetylase inhibitor trichostatin-A resulted in enhanced expression of endogenous ptd-FGFR4. These data indicate that a cryptic promoter within intron 4 binds AP-2alpha. AP-2alpha and chromatin changes may contribute to the utilization of an alternative transcription start site leading to the genesis of the tumorigenic ptd-FGFR4 isoform.
authors.affiliation: Department of Medicine, Mount Sinai Hospital, Toronto, Ontario M5G 2M9, Canada.
authors.forename: ShunJiang
authors.initials: S
authors.lastname: Yu
authors.forename: Sylvia L
authors.initials: SL
authors.lastname: Asa
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Weigel
authors.forename: Shereen
authors.initials: S
authors.lastname: Ezzat
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007438:Introns; D016297:Mutagenesis, Site-Directed; D010911:Pituitary Neoplasms / Q000378:metabolism* / Q000473:pathology; D011401:Promoter Regions, Genetic; D051381:Rats; D051499:Receptor, Fibroblast Growth Factor, Type 4; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics*; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D004268:DNA-Binding Proteins; D017468:Receptors, Fibroblast Growth Factor; C493673:TFAP2A protein, human; D050656:Transcription Factor AP-2; D014157:Transcription Factors; C496371:FGFR4 protein, human; C496373:Fgfr4 protein, rat; D051499:Receptor, Fibroblast Growth Factor, Type 4