title: A functional insulator screen identifies NURF and dREAM components to be required for enhancer-blocking.
abstract: Chromatin insulators of higher eukaryotes functionally divide the genome into active and inactive domains. Furthermore, insulators regulate enhancer/promoter communication, which is evident from the Drosophila bithorax locus in which a multitude of regulatory elements control segment specific gene activity. Centrosomal protein 190 (CP190) is targeted to insulators by CTCF or other insulator DNA-binding factors. Chromatin analyses revealed that insulators are characterized by open and nucleosome depleted regions. Here, we wanted to identify chromatin modification and remodelling factors required for an enhancer blocking function. We used the well-studied Fab-8 insulator of the bithorax locus to apply a genome-wide RNAi screen for factors that contribute to the enhancer blocking function of CTCF and CP190. Among 78 genes required for optimal Fab-8 mediated enhancer blocking, all four components of the NURF complex as well as several subunits of the dREAM complex were most evident. Mass spectrometric analyses of CTCF or CP190 bound proteins as well as immune precipitation confirmed NURF and dREAM binding. Both co-localise with most CP190 binding sites in the genome and chromatin immune precipitation showed that CP190 recruits NURF and dREAM. Nucleosome occupancy and histone H3 binding analyses revealed that CP190 mediated NURF binding results in nucleosomal depletion at CP190 binding sites. Thus, we conclude that CP190 binding to CTCF or to other DNA binding insulator factors mediates recruitment of NURF and dREAM. Furthermore, the enhancer blocking function of insulators is associated with nucleosomal depletion and requires NURF and dREAM.
authors.affiliation: Institut für Genetik, Justus-Liebig-Universität, Giessen, Giessen, Germany.
authors.forename: Dorte
authors.initials: D
authors.lastname: Bohla
authors.affiliation: Institut für Genetik, Justus-Liebig-Universität, Giessen, Giessen, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Herold
authors.affiliation: Institut für Genetik, Justus-Liebig-Universität, Giessen, Giessen, Germany.
authors.forename: Imke
authors.initials: I
authors.lastname: Panzer
authors.affiliation: Institut für Genetik, Justus-Liebig-Universität, Giessen, Giessen, Germany.
authors.forename: Melanie K
authors.initials: MK
authors.lastname: Buxa
authors.affiliation: Institut für Genetik, Justus-Liebig-Universität, Giessen, Giessen, Germany; Faculty of Science, Benha University, Benha, Egypt.
authors.forename: Tamer
authors.initials: T
authors.lastname: Ali
authors.affiliation: Proteomics Centre, Erasmus Medical Centre, Rotterdam, Netherlands.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Demmers
authors.affiliation: Biomolecular Mass Spectrometry, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany.
authors.forename: Marcus
authors.initials: M
authors.lastname: Krüger
authors.affiliation: Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Maren
authors.initials: M
authors.lastname: Scharfe
authors.affiliation: Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Jarek
authors.affiliation: Institut für Genetik, Justus-Liebig-Universität, Giessen, Giessen, Germany.
authors.forename: Marek
authors.initials: M
authors.lastname: Bartkuhn
authors.affiliation: Institut für Genetik, Justus-Liebig-Universität, Giessen, Giessen, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Renkawitz
mesh_terms: D000818:Animals; D001665:Binding Sites; D000076246:CCCTC-Binding Factor; D002843:Chromatin / Q000235:genetics* / Q000378:metabolism; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics* / Q000378:metabolism; D038101:Insulator Elements; D013058:Mass Spectrometry; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000076246:CCCTC-Binding Factor; C107964:CP190 protein, Drosophila; C524946:CTCF protein, Drosophila; D002843:Chromatin; D029721:Drosophila Proteins; D008869:Microtubule-Associated Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins