title: Differential contribution of cis-regulatory elements to higher order chromatin structure and expression of the CFTR locus.
abstract: Higher order chromatin structure establishes domains that organize the genome and coordinate gene expression. However, the molecular mechanisms controlling transcription of individual loci within a topological domain (TAD) are not fully understood. The cystic fibrosis transmembrane conductance regulator (CFTR) gene provides a paradigm for investigating these mechanisms.CFTR occupies a TAD bordered by CTCF/cohesin binding sites within which are cell-type-selective cis-regulatory elements for the locus. We showed previously that intronic and extragenic enhancers, when occupied by specific transcription factors, are recruited to the CFTR promoter by a looping mechanism to drive gene expression. Here we use a combination of CRISPR/Cas9 editing of cis-regulatory elements and siRNA-mediated depletion of architectural proteins to determine the relative contribution of structural elements and enhancers to the higher order structure and expression of the CFTR locus. We found the boundaries of the CFTRTAD are conserved among diverse cell types and are dependent on CTCF and cohesin complex. Removal of an upstream CTCF-binding insulator alters the interaction profile, but has little effect on CFTR expression. Within the TAD, intronic enhancers recruit cell-type selective transcription factors and deletion of a pivotal enhancer element dramatically decreases CFTR expression, but has minor effect on its 3D structure.
authors.affiliation: Human Molecular Genetics Program, Lurie Children's Research Center, Chicago, IL 60614, USA Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
authors.forename: Rui
authors.initials: R
authors.lastname: Yang
authors.affiliation: Human Molecular Genetics Program, Lurie Children's Research Center, Chicago, IL 60614, USA Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
authors.forename: Jenny L
authors.initials: JL
authors.lastname: Kerschner
authors.affiliation: Human Molecular Genetics Program, Lurie Children's Research Center, Chicago, IL 60614, USA Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
authors.forename: Nehal
authors.initials: N
authors.lastname: Gosalia
authors.affiliation: Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Neems
authors.affiliation: Human Molecular Genetics Program, Lurie Children's Research Center, Chicago, IL 60614, USA Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
authors.forename: Lidija K
authors.initials: LK
authors.lastname: Gorsic
authors.affiliation: Division of Medical Genetics, Department of Pediatrics and Center for Genomic and Computational Biology, Duke University Medical School, Durham, NC 27708, USA.
authors.forename: Alexias
authors.initials: A
authors.lastname: Safi
authors.affiliation: Division of Medical Genetics, Department of Pediatrics and Center for Genomic and Computational Biology, Duke University Medical School, Durham, NC 27708, USA.
authors.forename: Gregory E
authors.initials: GE
authors.lastname: Crawford
authors.affiliation: Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
authors.forename: Steven T
authors.initials: ST
authors.lastname: Kosak
authors.affiliation: Human Molecular Genetics Program, Lurie Children's Research Center, Chicago, IL 60614, USA Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
authors.forename: Shih-Hsing
authors.initials: SH
authors.lastname: Leir
authors.affiliation: Human Molecular Genetics Program, Lurie Children's Research Center, Chicago, IL 60614, USA Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA ann-harris@northwestern.edu.
authors.forename: Ann
authors.initials: A
authors.lastname: Harris
mesh_terms: D000076246:CCCTC-Binding Factor; D018938:Caco-2 Cells; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D002843:Chromatin / Q000737:chemistry*; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D056426:Genetic Loci; D006801:Humans; D038101:Insulator Elements; D012097:Repressor Proteins / Q000378:metabolism; D000097722:Cohesins
chemical_list: D000076246:CCCTC-Binding Factor; D018797:Cell Cycle Proteins; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D012097:Repressor Proteins; D000097722:Cohesins; C000619697:CTCF protein, human