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Transcriptional networks driving enhancer function in the CFTR gene.
- Source :
-
The Biochemical journal [Biochem J] 2012 Sep 01; Vol. 446 (2), pp. 203-12. - Publication Year :
- 2012
-
Abstract
- 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.
- Subjects :
- CDX2 Transcription Factor
Cell Line
Cystic Fibrosis Transmembrane Conductance Regulator chemistry
Cystic Fibrosis Transmembrane Conductance Regulator genetics
Forkhead Transcription Factors antagonists & inhibitors
Forkhead Transcription Factors chemistry
Forkhead Transcription Factors genetics
Gene Expression Regulation
Genes, Reporter
Hepatocyte Nuclear Factor 1-alpha antagonists & inhibitors
Hepatocyte Nuclear Factor 1-alpha chemistry
Hepatocyte Nuclear Factor 1-alpha genetics
Homeodomain Proteins antagonists & inhibitors
Homeodomain Proteins chemistry
Homeodomain Proteins genetics
Humans
Introns
Mutant Proteins antagonists & inhibitors
Mutant Proteins chemistry
Mutant Proteins metabolism
Peptide Fragments antagonists & inhibitors
Peptide Fragments chemistry
Peptide Fragments genetics
Peptide Fragments metabolism
Protein Interaction Domains and Motifs
Protein Isoforms antagonists & inhibitors
Protein Isoforms chemistry
Protein Isoforms metabolism
RNA Interference
RNA, Messenger metabolism
RNA, Small Interfering
Recombinant Fusion Proteins antagonists & inhibitors
Recombinant Fusion Proteins chemistry
Recombinant Fusion Proteins metabolism
Trans-Activators antagonists & inhibitors
Trans-Activators chemistry
Trans-Activators genetics
Cystic Fibrosis Transmembrane Conductance Regulator metabolism
Enhancer Elements, Genetic
Enterocytes metabolism
Forkhead Transcription Factors metabolism
Hepatocyte Nuclear Factor 1-alpha metabolism
Homeodomain Proteins metabolism
Trans-Activators metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1470-8728
- Volume :
- 446
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- The Biochemical journal
- Publication Type :
- Academic Journal
- Accession number :
- 22671145
- Full Text :
- https://doi.org/10.1042/BJ20120693