title: An insulator element 3' to the CFTR gene binds CTCF and reveals an active chromatin hub in primary cells.
abstract: Regulation of expression of the CFTR gene is poorly understood. Elements within the basal promoter of the gene do not fully explain CFTR expression patterns, suggesting that cis-regulatory elements are located elsewhere, either within the locus or in adjacent chromatin. We previously mapped DNase I hypersensitive sites (DHS) in 400 kb spanning the CFTR locus including a cluster of sites close to the 3'-end of the gene. Here we focus on a DHS at +6.8 kb from the CFTR translation end-point to evaluate its potential role in regulating expression of the gene. This DHS, which encompasses a consensus CTCF-binding site, was evident in primary human epididymis cells that express abundant CFTR mRNA. We show by DNase I footprinting and electophoretic mobility shift assays that the cis-regulatory element within this DHS binds CTCF in vitro. We further demonstrate that the element functions as an enhancer blocker in a well-established in vivo assay, and by using chromatin immunoprecipitation that it recruits CTCF in vivo. Moreover, we reveal that in primary epididymis cells, the +6.8 kb DHS interacts closely with the CFTR promoter, suggesting that the CFTR locus exists in a looped conformation, characteristic of an active chromatin hub.
authors.affiliation: Human Molecular Genetics Program, Children's Memorial Research Center, Northwestern University Feinberg School of Medicine, Chicago, IL 60614, USA.
authors.forename: Neil P
authors.initials: NP
authors.lastname: Blackledge
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Ott
authors.forename: Austin E
authors.initials: AE
authors.lastname: Gillen
authors.forename: Ann
authors.initials: A
authors.lastname: Harris
mesh_terms: D024509:3' Flanking Region; D001665:Binding Sites; D000076246:CCCTC-Binding Factor; D002478:Cells, Cultured; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004742:Enhancer Elements, Genetic; D004822:Epididymis / Q000166:cytology / Q000378:metabolism; D006801:Humans; D038101:Insulator Elements; D008297:Male; D012097:Repressor Proteins / Q000378:metabolism*
chemical_list: D000076246:CCCTC-Binding Factor; C505032:CFTR protein, human; C000619697:CTCF protein, human; D002843:Chromatin; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D003850:Deoxyribonuclease I