Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

Summary

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

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.

Introduction

Evolutionarily conserved SNF2 family chromatin remodeling complexes are key regulators of chromatin organization and DNA accessibility¹. These remodeling complexes always include a central SNF2-like ATPase subunit, which, in some cases, assembles with various accessory proteins and forms multi-subunit macro-molecular assemblies. To study the molecular details of the ATP-dependent chromatin remodeling process, it is important to understand the contributions of given subsets of subunits and/or domain structures to activities of the complexes. Such analyses require the ability to generate highly purified mutant complexes that lack particular protein subunits o....

Protocol

1. Generation and Culture of HEK293 Stable Cell Lines Expressing Full Length or Mutant Versions of FLAG Epitope-tagged Ino80 or Other INO80 Complex Subunits

1. Clone cDNA encoding full length or mutant human Ino80 ATPase or another INO80 subunit into the mammalian expression vector pcDNA5/FRT with an in-frame, N terminal FLAG epitope tag.
2. Confirm the sequence of the inserted cDNAs by DNA sequencing before proceeding.
3. To perform the transfection, grow Flp-In HEK293 cells in 10 cm tissue culture dishes in a medium containing DMEM (Dulbecco's Modified Eagle Medium), 5% glutamine, and 10% FBS (fetal bovine serum).
4. When cells reac....

Results

Figure 1 shows a flow chart summarizing the procedures used to generate, purify, and characterize human INO80 ATP-dependent chromatin remodeling complexes.

As illustrated in Figures 2 and 3, these procedures enable the generation of both wild type INO80 and INO80 subcomplexes that lack various subunits, thereby enabling subsequent biochemical analyses of the contribution of these missing subunits to INO80's enzymatic activities. Fi.......

Discussion

Structural and functional studies of multi-subunit mammalian chromatin remodeling complexes from higher eukaryotes have been hampered by the difficulty of preparing biochemically useful amounts of such complexes containing mutant subunits or lacking certain subunits altogether. There are a number of technical hurdles: First, genetic manipulation in mammalian cells has been technically challenging and time-consuming. Unlike yeast cells, whose genome can be readily edited and targeted using recombineering techniques, the m.......

Materials

Name	Company	Catalog Number	Comments
Name of Reagent/Material	Company	Catalog Number	Comments
Dulbecco's Modified Eagle Medium	Cellgro	10-013-CV
Glutamax-I (stablized glutamine)	Life Technologies	35050-079
Fetal Bovine Serum	SAFC	12176C
FuGENE6 transfection reagent	Promega	E2312
Hygromycin B, sterile in PBS	AG Scientific	H-1012-PBS
pcDNA5/FRT vector	Life Technologies	V6010-20
Flp-In HEK293 cells	Life Technologies	R780-07
pOG44 Flp-Recombinase Expression Vector	Life Technologies	V600520
EZview  Red ANTI-FLAG  M2 Affinity Gel	Sigma	F2426
calf serum	SAFC	12138C
TARGETplus SMARTsiRNA pool	Dharmacon / Thermo Scientific	various
5x siRNA resuspension buffer	Dharmacon / Thermo Scientific	#B-002000-UB-100
Lipofectamine RNAiMAX	Life Technologies	13778
Opti-MEM Reduced Serum Medium	Life Technologies	51985-091
PBS	Cellgro	45000	VWR
TrypLE (trypsin)	Life Technologies	12604
1x FLAG Peptide	Sigma	F3290
Micro Bio-Spin Chromatography Column	Bio-Rad	737-5021
Amicon Ultra Centrifugal Filter Device (50k MWCO)	Amicon	UFC805024	Fisher Scientific
Zeba Desalting Columns	Thermo Scientific	89882
Anti-FLAG M2 antibody, mouse	Sigma	F3165
Anti-FLAG M2 antibody, rabbit	Sigma	F7425
Protease Inhibitor Cocktail	Sigma	P8340
benzonase	Novagen	70664
Equipment	Company
Wheaton Dounce Tissue Grinders	Wheaton	06-435C
JS-4.2 rotor in a J6 centrifuge	Beckman-Coulter	339080
JA-17 rotor	Beckman-Coulter	369691
10 ml polycarbonate tubes	Beckman-Coulter	355630
70 ml polycarbonate bottles	Beckman-Coulter	355655
Type 45 Ti rotor	Beckman-Coulter	339160
Type 70.1 Ti rotor	Beckman-Coulter	342184
BD Clay Adams Nutator Mixer	BD Diagnostics	15172-203	VWR
Glas-Col Tube/Vial Rotator	Glas-Col	099A RD4512
PCR thermal cycler PTC 200	MJ Research	PTC 200
roller bottle incubator	Bellco biotechnology	353348
Immobilon-FL Transfer Membrane 7 x 8.4	Millipore	IPFL07810
lubricated 1.5ml microcentrifuge tubes	Costar	3207