FGFR4 overexpression in pancreatic cancer is mediated by an intronic enhancer activated by HNF1alpha.
Fibroblast growth factor receptor 4 (FGFR4) is expressed in 50-70% of pancreatic carcinomas (PC) and a similar proportion of derived cell lines. Here we determine the sites of FGFR4 transcriptional initiation which show a pattern characteristic of genes with GC-rich, TATA-less promoters. We have examined the chromatin structure around the FGFR4 gene in a panel of expressing and non-expressing PC lines using the DNase I hypersensitive site assay. One region of hypersensitivity, located largely within intron 1, was found to be greatly extended in expressing cells. Subsequent functional analyses using reporter assays demonstrated that this region was able to act as a cell-specific enhancer, only showing significant activity in PC lines expressing endogenous FGFR4. Transcription factors able to bind to the enhancer were investigated using footprinting and mobility shift assays and two binding sites for Sp1 proteins and two sites able to bind hepatic nuclear factor 1 (HNF1) proteins were identified. Further reporter assays using constructs mutated in each binding site demonstrated that HNF1 binding was essential for enhancer activity in expressing cells, an observation that correlated with the increased abundance of HNF1alpha in these same cells as measured by Western blotting. Finally we show that exogenous expression of HNF1 factors in an FGFR4 non-expressing line led to an induction of enhancer activity in reporter assays and also activated expression of the endogenous gene. We conclude that HNF1alpha is a major determinant of FGFR4 expression in PC.
Shah
Riyaz N H
RN
Cancer Research UK Molecular Oncology Unit, ICSM at Hammersmith Hospital, London W12 0NN, UK.
Ibbitt
J Claire
JC
Alitalo
Kari
K
Hurst
Helen C
HC
D001483:Base Sequence; D018983:DNA Footprinting; D017931:DNA Primers; D004268:DNA-Binding Proteins; D004742:Enhancer Elements, Genetic; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology*; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta; D007438:Introns; D009687:Nuclear Proteins; D010190:Pancreatic Neoplasms / Q000235:genetics* / Q000473:pathology; D011485:Protein Binding; D051499:Receptor, Fibroblast Growth Factor, Type 4; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic
D017931:DNA Primers; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D017468:Receptors, Fibroblast Growth Factor; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta; D051499:Receptor, Fibroblast Growth Factor, Type 4