title: Generation and purification of human INO80 chromatin remodeling complexes and subcomplexes.
abstract: INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex's chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.
authors.affiliation: Stowers Institute for Medical Research; Department of Biochemistry & Molecular Biology, Kansas University Medical Center.
authors.forename: Lu
authors.initials: L
authors.lastname: Chen
authors.affiliation: Stowers Institute for Medical Research.
authors.forename: Soon-Keat
authors.initials: SK
authors.lastname: Ooi
authors.affiliation: Stowers Institute for Medical Research; Department of Biochemistry & Molecular Biology, Kansas University Medical Center; rcc@stowers.org.
authors.forename: Ronald C
authors.initials: RC
authors.lastname: Conaway
authors.affiliation: Stowers Institute for Medical Research; Department of Biochemistry & Molecular Biology, Kansas University Medical Center.
authors.forename: Joan W
authors.initials: JW
authors.lastname: Conaway
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D002846:Chromatography, Affinity / Q000379:methods; D004265:DNA Helicases / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans
chemical_list: D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D000939:Epitopes; D000074183:ATPases Associated with Diverse Cellular Activities; D004265:DNA Helicases; C524421:INO80 protein, human