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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.
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.
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 or domain structures.
Previous structure-function studies of ATP-dependent chromatin remodeling complexes have widely focused on the yeast model system due to the superior manipulability of the yeast genome (see, for example, refs1-4). Given the conservation of subunit composition and functionality among orthologous remodeling complexes, studies of the structure and function of yeast remodeling complexes have provided important insights into their counterparts in higher eukaryotes. Nonetheless, appreciable species-specific differences among remodeling complexes do exist, resulting from gain or loss of species-specific subunits, gain or loss of species-specific domains of conserved subunits, and sequence variability within conserved domains of conserved subunits. Such differences can in principle be driven by the need for higher eukaryotic cells to adapt to new molecular and cellular environments. Thus, understanding how subunits of higher eukaryotic remodeling complexes contribute to the nucleosome remodeling process is valuable, because it not only sheds light on basic mechanisms of the ATP-dependent chromatin remodeling process, but also can provide valuable insight into the mechanisms by which chromatin structure and gene expression in higher eukaryotes are regulated.
Thus far, there have been only limited structural and functional studies of multi-subunit mammalian chromatin remodeling complexes, due in part to the difficulties in obtaining biochemically defined chromatin remodeling complexes and subcomplexes. We have partially circumvented these difficulties with the procedures described below, in which immunoaffinity purification is used to prepare intact INO80 or INO80 subcomplexes from human cells stably expressing N-terminally FLAG epitope tagged wild type or mutant versions of Ino805-7 (Figure 1). To obtain intact INO80 complexes from human cells, Flp-mediated recombination is used to generate transgenic HEK293 cell lines stably expressing FLAG epitope tagged cDNAs encoding subunits of the INO80 complex8-10. Because over-expression of INO80 subunits can be somewhat toxic, it is necessary to isolate and maintain clonal cell lines under selective conditions to ensure stable transgene expression during the many passages needed for expansion of large-scale cell cultures. To obtain smaller INO80 subcomplexes that contain only a subset of subunits, we have successfully used two approaches (Figure 2A,B). In the first, we generate HEK293 Flp-In cell lines stably expressing mutant versions of Ino80 that lack domains required for interaction with specific subunits5. Alternatively, siRNA-mediated knockdown is used to deplete the desired subunit from cells expressing an appropriate FLAG-tagged INO80 subunit (unpublished data). Finally, to purify the human INO80 complexes, FLAG agarose based chromatography11 is used to enrich an INO80-containing fraction from nuclear extracts, thereby effectively reducing the presence of contaminating cytosolic proteins in the final fraction containing purified INO80 or INO80 subcomplexes.
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
2. Growing HEK293 Cell Lines in Roller Bottles
For large scale preparation of INO80 complexes, culture cells in 10 - 20 roller bottles; a typical yield from each roller bottle is ~1 ml of packed cells.
3. siRNA-mediated Knockdown of INO80 Subunits in Cells Expressing Another FLAG-tagged INO80 Subunit
To obtain INO80 subcomplexes lacking a single subunit, use FLAG-immunopurification to purify INO80 complexes from siRNA treated cells or cells stably expressing shRNA. The “reverse” siRNA (small interfering RNA) transfection protocol described here is optimized for HEK293 cells growing in 15 cm dishes. The protocol is for a single 15 cm dish of cells and should be scaled up accordingly depending on the number of cells needed. To prepare biochemically useful amounts of INO80 complex from siRNA-treated cells, one should scale up to cultures grown in 40 15 cm dishes; these will yield approximately 2 - 4 ml of packed cell pellet.
4. Preparation of Nuclear Extracts
This procedure has been modified from the protocol of Dignam13 and can be scaled up or down depending on the size of starting cell pellets. Typically, 1 ml of packed cell pellet yields 1 ml of final nuclear extract. All buffers should be ice cold, and all steps should be performed in a cold room or on ice if a suitable cold room is not available.
5. Immunoaffinity Purification of the Human INO80 or INO80 Subcomplexes
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...
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...
The authors declare that they have no competing financial interests.
Work in the authors' laboratory is supported by a grant from the National Institute of General Medical Sciences (GM41628) and by a grant to the Stowers Institute for Medical Research from the Helen Nelson Medical Research Fund at the Greater Kansas City Community Foundation.
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 |
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