title: RNAi-independent role for Argonaute2 in CTCF/CP190 chromatin insulator function.
abstract: A major role of the RNAi pathway in Schizosaccharomyces pombe is to nucleate heterochromatin, but it remains unclear whether this mechanism is conserved. To address this question in Drosophila, we performed genome-wide localization of Argonaute2 (AGO2) by chromatin immunoprecipitation (ChIP)-seq in two different embryonic cell lines and found that AGO2 localizes to euchromatin but not heterochromatin. This localization pattern is further supported by immunofluorescence staining of polytene chromosomes and cell lines, and these studies also indicate that a substantial fraction of AGO2 resides in the nucleus. Intriguingly, AGO2 colocalizes extensively with CTCF/CP190 chromatin insulators but not with genomic regions corresponding to endogenous siRNA production. Moreover, AGO2, but not its catalytic activity or Dicer-2, is required for CTCF/CP190-dependent Fab-8 insulator function. AGO2 interacts physically with CTCF and CP190, and depletion of either CTCF or CP190 results in genome-wide loss of AGO2 chromatin association. Finally, mutation of CTCF, CP190, or AGO2 leads to reduction of chromosomal looping interactions, thereby altering gene expression. We propose that RNAi-independent recruitment of AGO2 to chromatin by insulator proteins promotes the definition of transcriptional domains throughout the genome.
authors.affiliation: Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Nellie
authors.initials: N
authors.lastname: Moshkovich
authors.forename: Parul
authors.initials: P
authors.lastname: Nisha
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Boyle
authors.forename: Brandi A
authors.initials: BA
authors.lastname: Thompson
authors.forename: Ryan K
authors.initials: RK
authors.lastname: Dale
authors.forename: Elissa P
authors.initials: EP
authors.lastname: Lei
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D060565:Argonaute Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D000076246:CCCTC-Binding Factor; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D016000:Cluster Analysis; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D022041:Euchromatin / Q000235:genetics / Q000378:metabolism; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D020869:Gene Expression Profiling; D049750:Genome, Insect / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: C433807:AGO2 protein, Drosophila; C062393:Abd-B proteins, Drosophila; D060565:Argonaute Proteins; D000076246:CCCTC-Binding Factor; C107964:CP190 protein, Drosophila; C524946:CTCF protein, Drosophila; D029721:Drosophila Proteins; D022041:Euchromatin; D018398:Homeodomain Proteins; D008869:Microtubule-Associated Proteins; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D012097:Repressor Proteins