Transcriptional networks driving enhancer function in the CFTR gene.
A critical cis-regulatory element for the CFTR (cystic fibrosis transmembrane conductance regulator) gene is located in intron 11, 100 kb distal to the promoter, with which it interacts. This sequence contains an intestine-selective enhancer and associates with enhancer signature proteins, such as p300, in addition to tissue-specific TFs (transcription factors). In the present study we identify critical TFs that are recruited to this element and demonstrate their importance in regulating CFTR expression. In vitro DNase I footprinting and EMSAs (electrophoretic mobility-shift assays) identified four cell-type-selective regions that bound TFs in vitro. ChIP (chromatin immunoprecipitation) identified FOXA1/A2 (forkhead box A1/A2), HNF1 (hepatocyte nuclear factor 1) and CDX2 (caudal-type homeobox 2) as in vivo trans-interacting factors. Mutation of their binding sites in the intron 11 core compromised its enhancer activity when measured by reporter gene assay. Moreover, siRNA (small interfering RNA)-mediated knockdown of CDX2 caused a significant reduction in endogenous CFTR transcription in intestinal cells, suggesting that this factor is critical for the maintenance of high levels of CFTR expression in these cells. The ChIP data also demonstrate that these TFs interact with multiple cis-regulatory elements across the CFTR locus, implicating a more global role in intestinal expression of the gene.
Human Molecular Genetics Program, Children's Memorial Research Center, Chicago, IL 60614, USA.
Jenny L
JL
Kerschner
Ann
A
Harris
D000071616:CDX2 Transcription Factor; D002460:Cell Line; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D020895:Enterocytes / Q000378:metabolism*; D051858:Forkhead Transcription Factors / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D051538:Hepatocyte Nuclear Factor 1-alpha / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007438:Introns; D050505:Mutant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D015534:Trans-Activators / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000071616:CDX2 Transcription Factor; C491270:CDX2 protein, human; C505032:CFTR protein, human; D051858:Forkhead Transcription Factors; C496487:HNF1A protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; D018398:Homeodomain Proteins; D050505:Mutant Proteins; D010446:Peptide Fragments; D020033:Protein Isoforms; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D019005:Cystic Fibrosis Transmembrane Conductance Regulator