Work in the authors&#39; 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.

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
<ol>
	<li>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.</li>
	<li>Confirm the sequence of the inserted cDNAs by DNA sequencing before proceeding.</li>
	<li>To perform the transfection, grow Flp-In HEK293 cells in 10 cm tissue culture dishes in a medium containing DMEM (Dulbecco&#39;s Modified Eagle Medium), 5% glutamine, and 10% FBS (fetal bovine serum).</li>
	<li>When cells reach ~70% confluency, add to each tissue culture dish a mixture of 40 &#181;l of FuGENE6 transfection reagent, 0.5 &#181;g of the appropriate pcDNA5/FRT expression plasmid, and 9.5 &#181;g of pOG44, which encodes Flp recombinase, in a total volume of 800 &#956;l.</li>
	<li>48 hr later, trypsinize and split cells at a ratio of 1:30 into 10 cm dishes, and grow them in DMEM with 5% glutamine, 10% FBS, and 100 &#181;g/ml hygromycin B for 3 - 4 weeks. Change the culture medium whenever it begins to turn yellow (typically every 3 - 5 days).</li>
	<li>To identify positive clones that express the highest level of FLAG-tagged protein, select individual hygromycin B-resistant colonies and transfer them to a single well of a 24-well plate.
	<ol>
		<li>Once the cells reach 80% confluency, harvest cells from each well in ~1 ml PBS and pellet by centrifugation at 1,000 x g for 5 min.</li>
		<li>After removing the supernatant, resuspend the cell pellet in 60 &#181;l of SDS-PAGE sample buffer.</li>
		<li>Subject half of the resuspended cell pellet to SDS page and western blotting to monitor expression of the FLAG tagged bait protein; save the other half for future analyses.</li>
	</ol>
	</li>
	<li>If no FLAG-tagged human Ino80 protein is detected in cell lysates, expand the initial clonal cell population further by plating cells from single wells of a 24-well plate into 15 cm tissue culture dishes.
	<ol>
		<li>To harvest cells from 15 cm dishes, resuspend near confluent cells in ice cold PBS and transfer to a 50 ml conical tube.</li>
		<li>Bring to a final volume of 50 ml by adding additional PBS.</li>
		<li>Pellet cells at 1,000 x g for 5 min, and remove as much as possible of the supernatant.</li>
		<li>Resuspend the cell pellet in 1 ml of Lys450 buffer (20 mM HEPES-NaOH pH 7.9, 450 mM NaCl, 0.5% TritonX-100, 10 mM KCl, 4 mM MgCl2, 0.2 mM EDTA, 10% glycerol, 1 mM DTT, 200 &#181;M PMSF, and 1:1,000 Protease Inhibitor Cocktail). 
		NOTE: here and elsewhere, always add DTT, PMSF, and protease inhibitor cocktail to buffers immediately before beginning an experiment.</li>
		<li>Immuno-precipitate FLAG-tagged proteins from the resulting whole cell lysate using 20 &#181;l of anti-FLAG agarose gel, and analyze the FLAG eluates by western blotting. [For details of immunoprecipitation procedure, see step 5.]</li>
	</ol>
	</li>
	<li>Prepare frozen stocks of desired clonal cell lines12 and store them in liquid nitrogen until use.</li>
</ol>
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.
<ol>
	<li>To each roller bottle, add 200 ml DMEM, 5% glutamine, and 10% calf serum, without hygromycin B.</li>
	<li>Transfer all of the cells from a single near-confluent 15 cm dish into each roller bottle</li>
	<li>Place roller bottles into a 37 &#176;C roller bottle incubator and rotate at 0.2 rpm.</li>
	<li>Once cells reach ~70% confluency, pour off and discard the medium.</li>
	<li>Add ~50 ml of ice cold PBS to each bottle. Holding bottles horizontally, gently swirl to loosen the cell monolayer.</li>
	<li>Transfer the resuspended cells to 250 ml plastic conical bottles and place on ice.</li>
	<li>Rinse roller bottles with additional PBS, transferring it sequentially from bottle to bottle. When rinse solution is no longer clear (typically after being used to rinse about 5 bottles), add it to the cell suspension.</li>
	<li>Pellet cells by centrifugation at 400 x g for 10 min.</li>
	<li>Gently resuspend the cells in PBS, combine them into a single 250 ml conical bottle, and keep on ice until further processing.</li>
</ol>
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 &#8220;reverse&#8221; 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.
<ol>
	<li>Grow HEK293 cells stably expressing the desired subunit of the INO80 complex to near confluency in 15 cm dishes.</li>
	<li>Add a volume of of siRNA resuspension buffer (20 mM KCl, 6 mM HEPES-pH 7.5, and 0.2 mM MgCl2) sufficient to prepare a 50 &#181;M stock solution of siRNA to a tube containing lyophilized siRNA. Pipette the solution up and down a few times and incubate on a nutator for 30 min at RT to ensure the siRNA is fully dissolved.</li>
	<li>Prepare a transfection cocktail containing siRNAs and transfection reagent. Mix 10 &#956;l of the 50 &#181;M siRNA stock solution with 32 &#181;l Lipofectamine RNAiMAX and add it to 4 ml of Opti-MEM Reduced Serum Medium with very gentle mixing; allow all reagents to equilibrate to RT before use.</li>
	<li>Incubate the mixture at RT for 30 min.</li>
	<li>Prepare Flp-In HEK293 cells stably expressing the desired INO80 subunit for transfection.
	<ol>
		<li>During the incubation of Step 3.4, wash cells in the 15 cm dishes once with RT PBS.</li>
		<li>After removing PBS, treat cells with 1 ml trypsin just until they begin to lift off the plate.</li>
		<li>Immediately add 10 ml of complete medium (DMEM + 5% glutamine + 10% FBS) to trypsinized cells and mix gently. Collect cells by centrifugation at 1,000 x g for 5 min at RT.</li>
		<li>Resuspend the cell pellet in ~4 ml complete medium, and count the cell resuspension using a haemocytometer.</li>
		<li>Dilute cells with complete medium to a concentration of ~5.4 x 106/ml.</li>
	</ol>
	</li>
	<li>To each 15 cm dish, add 15 ml complete culture medium followed by 4 ml transfection cocktail. Swirl gently to ensure the medium and transfection cocktail are thoroughly mixed. Finally, add one ml of cell suspension and again swirl gently to disperse cells uniformly.</li>
	<li>After 60 hr of culture in a 37 &#176;C, 5% CO2 incubator, gently remove medium, resuspend cells in ice cold PBS, and proceed immediately to prepare nuclear extracts.</li>
</ol>
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.
<ol>
	<li>Isolation of nuclei:
	<ol>
		<li>Gently transfer cells to a suitably sized (15 or 50 ml) graduated conical tube and spin at 1,000 x g for 10 min at 4 &#176;C.</li>
		<li>Remove the supernatant, and measure the volume of the packed cell pellet. 1 ml of packed cells corresponds to ~3 x 108 HEK293 cells.</li>
		<li>Add 5 packed cell volumes of Buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl and freshly added 1 mM DTT, 200 &#181;M PMSF, and 1:1,000 Protease Inhibitor Cocktail). Resuspend the cell pellet by gentle pipetting.</li>
		<li>Incubate on ice for exactly 10 min.</li>
		<li>Pellet the cells at 1,000 x g for 10 min at 4 &#176;C, and remove the supernatant.</li>
		<li>The size of the cell pellet should increase up to 2-fold during incubation in Buffer A.</li>
		<li>Resuspend the cells in two packed cell volumes of buffer A, and transfer the cell suspension to an appropriately sized Dounce tissue homogenizer. If starting with less than 2 ml of packed cells, use a 7 ml homogenizer; for 2 - 4 ml packed cells, use a 15 ml homogenizer; and for 10 or more ml of packed cells, use a 40 ml homogenizer.</li>
		<li>Homogenize the cell suspension with the LOOSE glass pestle of the Dounce homogenizer until 90% of the cells stain positively with 1% trypan blue.</li>
		<li>Transfer the suspension to a 45 ml high-speed centrifuge tube and spin at 25,000 x g for 20 min at 4 &#176;C in a JA-17 or similar rotor (see Table of Materials/Equipment).</li>
		<li>From the nuclear pellet, remove the supernatant, which contains cytosolic proteins or proteins that leak out of the nucleus during fractionation.</li>
	</ol>
	</li>
	<li>Extracting nuclei with salt:
	<ol>
		<li>Add Buffer C (20mM HEPES, pH 7.9, 25% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, and freshly added 1 mM DTT, 200 &#181;M PMSF, and 1:1,000 Protease Inhibitor Cocktail) to the nuclear pellet; use 2.5 ml Buffer C for every 3 ml of starting packed cell volume (~1 x 109 cells).</li>
		<li>Using a glass rod or a pipette, dislodge the nuclear pellet from the wall of the tube and transfer the entire mixture to a Dounce homogenizer of an appropriate size.</li>
		<li>Resuspend the nuclei by homogenizing the mixture with two strokes of a LOOSE pestle.</li>
		<li>Transfer the resuspended nuclear fraction into a chilled beaker. Choose a beaker such that the suspension will fill the beaker to at least 0.5 cm deep.</li>
		<li>To extract nuclear proteins from chromatin or other insoluble structures, gradually increase the salt concentration of the suspension to 0.42 M NaCl by dropwise addition of 5 M NaCl while gently stirring the suspension with a pipette or glass rod on ice; once all of the 5 M NaCl has been added, the solution should become very viscous or gel-like. Calculate the volume of 5 M NaCl needed to bring the solution to a final concentration of 0.42 M NaCl according to the following formula: 
		<img alt="Equation 1" fo:content-width="4.5in" src="/files/ftp_upload/51720/51720equation1.jpg" width="500" /></li>
		<li>Carefully transfer the viscous suspension into 10 ml polycarbonate tubes for a Type 70.1 Ti rotor (or equivalent) or 70 ml polycarbonate bottles for a Type 45 Ti or similar rotor (see Table of Materials/Equipment). Seal tightly with parafilm if using 10 ml tubes or with cap assembly if using 70 ml bottles.</li>
		<li>Slowly rock the sealed tubes at 4 &#176;C for 30 min using a nutator.</li>
		<li>Spin the samples in a Type 45 Ti or 70.1 Ti rotor for 30 min at 120,000 x g at 4 &#176;C.</li>
		<li>Transfer the supernatant to a single plastic tube or bottle. This supernatant is the nuclear extract, and the pellet contains chromatin and other nuclear debris.</li>
		<li>Divide the nuclear extract into conveniently sized aliquots, freeze it in liquid nitrogen, and store it at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
5. Immunoaffinity Purification of the Human INO80 or INO80 Subcomplexes
<ol>
	<li>To thaw frozen nuclear extract, place tubes containing the extract on the benchtop or roll tubes between hands until the frozen material becomes a slurry. Then place the tubes on ice or in the cold room until the extract is completely thawed.</li>
	<li>Transfer the thawed nuclear extract to 10 ml polycarbonate ultracentrifuge tubes, and spin at 100,000 x g for 20 min at 4 &#176;C in a Type 70.1 Ti rotor or equivalent to remove any precipitate that may have formed during the freeze-thaw cycle.</li>
	<li>Transfer the supernatant to a 15 ml conical tube. Add fresh DTT, PMSF, and Protease Inhibitor Cocktail to final concentrations of 1 mM DTT, 200 &#181;M PMSF, and 1:1,000 Protease Inhibitor Cocktail.</li>
	<li>To prepare anti-FLAG agarose for the immunopurification, transfer 200 &#181;l of 50% slurry of anti-FLAG agarose beads to a 1.5 ml microcentrifuge tube using a P200 or similar pipette with a tip from which the end has been cut off with a clean scalpel or razor blade.</li>
	<li>Pellet the beads by centrifugation in a benchtop microcentrifuge at 8,000 x g for 30 sec. Remove the supernatant, and wash the beads by resuspending the beads in 1 ml of Lys450 buffer, and pellet the beads at 8,000 x g for 30 sec. Wash the beads two more times.</li>
	<li>Resuspend the washed anti-FLAG agarose beads in about 100 &#181;l of the nuclear extract using a P200 or similar adjustable volume pipette with a tip from which the end has been cut off with a clean scalpel or razor blade and, using the same tip, transfer the resuspended beads to the 15 ml conical tube containing the extract. Repeat a few times until all of the beads have been transferred to the 15 ml tube.</li>
	<li>Incubate the extract/bead mixture for 4 hr at 4 &#176;C with slow rotation on a laboratory rotator. OPTIONAL: Include benzonase at a concentration of 25 Units/ml to remove contaminating DNA.</li>
	<li>Collect the FLAG agarose by centrifugation at 1,000 x g for 5 min at 4 &#176;C.</li>
	<li>Resuspend in 10 ml Lys450 to wash, incubate 5 min at 4 &#176;C with gentle rocking on a nutator. Pellet the beads at 1,000 x g for 5 min at 4 &#176;C.</li>
	<li>Resuspend in 100 - 150 &#181;l of Lys450 and transfer beads to a 1.5 ml microcentrifuge tube. Continue to rinse the 15 ml conical tube with 100 - 150 &#181;l of Lys450 until all the beads have been transferred to the microcentrifuge tube.</li>
	<li>Spin down the beads at 8,000 x g for 30 sec at 4 &#176;C in a microcentrifuge. Wash three times more with 1 ml Lys450 and once with 1ml EB100 buffer (10 mM HEPES pH 7.9, 10% glycerol, 100 mM NaCl, 1.5 mM MgCl2, 0.05% TritonX-100, and freshly added 1 mM DTT, 200 &#181;M PMSF, and 1:1,000 Protease Inhibitor Cocktail).</li>
	<li>To elute bound proteins, add 200 &#181;l EB100 buffer containing 0.25 mg/ml 1x FLAG peptide. Incubate 30 min at 4 &#176;C each on a nutator.</li>
	<li>Pellet the beads at 8,000 x g for 30 sec at 4 &#176;C in a microcentrifuge. Transfer the supernatant, which contains the eluted INO80 complex, to a fresh microcentrifuge tube.</li>
	<li>Repeat the elution four more times, and pool all the supernatants into a single tube.</li>
	<li>To remove any residual FLAG-agarose beads from the eluted protein fraction, pass the eluate through an empty spin column.</li>
	<li>Concentrate the eluted protein fraction ~10-fold using an ultra centrifugal filter device (50,000 molecular weight cutoff).</li>
	<li>To remove the FLAG peptide, pass the concentrated protein fraction sequentially through two desalting columns.</li>
	<li>Divide the purified, desalted protein fraction into 20 &#181;l aliquots, frozen in liquid nitrogen, and stored at -80 &#176;C.</li>
	<li>Analyze the subunit composition of INO80 or INO80 subcomplexes on silver-stained gels or by western blotting, and estimate their concentrations by semi-quantitative western blotting using preparations of recombinant INO80 subunits of known concentration as standards.</li>
</ol>

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The authors declare that they have no competing financial interests.

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...

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.

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Cellgro</td><td>10-013-CV</td><td></td></tr><tr><td>Glutamax-I (stablized glutamine)</td><td>Life Technologies</td><td>35050-079</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>SAFC</td><td>12176C</td><td></td></tr><tr><td>FuGENE6 transfection reagent</td><td>Promega</td><td>E2312</td><td></td></tr><tr><td>Hygromycin B, sterile in PBS</td><td>AG Scientific</td><td>H-1012-PBS</td><td></td></tr><tr><td>pcDNA5/FRT vector</td><td>Life Technologies</td><td>V6010-20</td><td></td></tr><tr><td>Flp-In HEK293 cells</td><td>Life Technologies</td><td>R780-07</td><td></td></tr><tr><td>pOG44 Flp-Recombinase Expression Vector</td><td>Life Technologies</td><td>V600520</td><td></td></tr><tr><td>EZview Red ANTI-FLAG M2 Affinity Gel</td><td>Sigma</td><td>F2426</td><td></td></tr><tr><td>calf serum</td><td>SAFC</td><td>12138C</td><td></td></tr><tr><td>TARGETplus SMARTsiRNA pool</td><td>Dharmacon / Thermo Scientific</td><td>various</td><td></td></tr><tr><td>5x siRNA resuspension buffer</td><td>Dharmacon / Thermo Scientific</td><td>#B-002000-UB-100</td><td></td></tr><tr><td>Lipofectamine RNAiMAX</td><td>Life Technologies</td><td>13778</td><td></td></tr><tr><td>Opti-MEM Reduced Serum Medium</td><td>Life Technologies</td><td>51985-091</td><td></td></tr><tr><td>PBS</td><td>Cellgro</td><td>45000</td><td>VWR</td></tr><tr><td>TrypLE (trypsin)</td><td>Life Technologies</td><td>12604</td><td></td></tr><tr><td>1x FLAG Peptide</td><td>Sigma</td><td>F3290</td><td></td></tr><tr><td>Micro Bio-Spin Chromatography Column</td><td>Bio-Rad</td><td>737-5021</td><td></td></tr><tr><td>Amicon Ultra Centrifugal Filter Device (50k MWCO)</td><td>Amicon</td><td>UFC805024</td><td>Fisher Scientific</td></tr><tr><td>Zeba Desalting Columns</td><td>Thermo Scientific</td><td>89882</td><td></td></tr><tr><td>Anti-FLAG M2 antibody, mouse</td><td>Sigma</td><td>F3165</td><td></td></tr><tr><td>Anti-FLAG M2 antibody, rabbit</td><td>Sigma</td><td>F7425</td><td></td></tr><tr><td>Protease Inhibitor Cocktail</td><td>Sigma</td><td>P8340</td><td></td></tr><tr><td>benzonase</td><td>Novagen</td><td>70664</td><td></td></tr><tr><td colspan="4">Equipment</td></tr><tr><td>JS-4.2 rotor in a J6 centrifuge</td><td>Beckman-Coulter</td><td>339080</td><td></td></tr><tr><td>JA-17 rotor</td><td>Beckman-Coulter</td><td>369691</td><td></td></tr><tr><td>10 ml polycarbonate tubes</td><td>Beckman-Coulter</td><td>355630</td><td></td></tr><tr><td>70 ml polycarbonate bottles</td><td>Beckman-Coulter</td><td>355655</td><td></td></tr><tr><td>Type 45 Ti rotor</td><td>Beckman-Coulter</td><td>339160</td><td></td></tr><tr><td>Type 70.1 Ti rotor</td><td>Beckman-Coulter</td><td>342184</td><td></td></tr><tr><td>BD Clay Adams Nutator Mixer</td><td>BD Diagnostics</td><td>15172-203</td><td>VWR</td></tr><tr><td>Glas-Col Tube/Vial Rotator</td><td>Glas-Col</td><td>099A RD4512</td><td></td></tr><tr><td>PCR thermal cycler PTC 200</td><td>MJ Research</td><td>PTC 200</td><td></td></tr><tr><td>roller bottle incubator</td><td>Bellco biotechnology</td><td>353348</td><td></td></tr><tr><td>Immobilon-FL Transfer Membrane 7 x 8.4</td><td>Millipore</td><td>IPFL07810</td><td></td></tr><tr><td>lubricated 1.5 ml microcentrifuge tubes</td><td>Costar</td><td>3207</td><td></td></tr></tbody></table>

generation and purification of human ino80 chromatin remodeling complexes and subcomplexes

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&#39;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.

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&#39;s enzymatic activities. Fi...

Watch this Scientific Journal Video about Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes at JoVE.com

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

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 ...

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9.8K Views.  Stowers Institute for Medical Research. 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. 

Video: Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous nature of most mammalian chromatin-modifying complexes. In order to overcome these hurdles, we have adapted a sequential salt extraction (SSE) assay for evaluating the relative binding affinities of chromatin-bound complexes. This easy and straightforward assay can be used by non-experts to evaluate the relative difference in binding affinity of two related complexes, the changes in affinity of a complex when a subunit is lost or an individual domain is inactivated, and the change in binding affinity after alterations to the chromatin landscape. By sequentially re-suspending bulk chromatin in increasing amounts of salt, we are able to profile the elution of a particular protein from chromatin. Using these profiles, we are able to determine how alterations in a chromatin-modifying complex or alterations to the chromatin environment affect binding interactions. Coupling SSE with other in vitro and in vivo assays, we can determine the roles of individual domains and proteins on the functionality of a complex in a variety of chromatin environments.

Sequential salt extraction of chromatin bound proteins is a useful tool for determining the binding properties of large protein complexes. This method can be employed to evaluate the role of individual subunits or domains in the overall affinity of a protein complex to bulk chromatin.

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous ...

Genetics

Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of a wide range of transcription factors (TFs) and cofactors mediating transcriptional activation or repression via changes in chromatin structure. Chromatin immunoprecipitation (ChIP) is a useful molecular biology approach for mapping histone modifications and profiling transcription factors/cofactors binding to DNA, thus providing a snapshot of the dynamic nuclear changes occurring during different biological processes.
To study transcriptional regulation in adipose tissue, samples derived from in vitro cell cultures of immortalized or primary cell lines are often favored in ChIP assays because of the abundance of starting material and reduced biological variability. However, these models represent a limited snapshot of the actual chromatin state in living organisms. Thus, there is a critical need for optimized protocols to perform ChIP on adipose tissue samples derived from animal models.
Here we describe a protocol for efficient ChIP-seq of both histone modifications and non-histone proteins in brown adipose tissue (BAT) isolated from a mouse. The protocol is optimized for investigating genome-wide localization of proteins of interest and epigenetic markers in the BAT, which is a morphologically and physiologically distinct tissue amongst fat depots.

Here we describe a protocol for efficient chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) of brown adipose tissue (BAT) isolated from a mouse. This protocol is suitable for both mapping histone modifications and investigating genome-wide localization of non-histone proteins of interest in vivo.

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of ...

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination of chromatin function and DNA-associated processes. This modification involves a sequential action of several enzymes including E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase and is reversed by deubiquitinases (DUBs). Ubiquitination induces degradation of proteins or alteration of protein function including modulation of enzymatic activity, protein-protein interaction and subcellular localization. A critical step in demonstrating protein ubiquitination or deubiquitination is to perform in vitro reactions with purified components. Effective ubiquitination and deubiquitination reactions could be greatly impacted by the different components used, enzyme co-factors, buffer conditions, and the nature of the substrate.&#160; Here, we provide step-by-step protocols for conducting ubiquitination and deubiquitination reactions. We illustrate these reactions using minimal components of the mouse Polycomb Repressive Complex 1 (PRC1), BMI1, and RING1B, an E3 ubiquitin ligase that monoubiquitinates histone H2A on lysine 119. Deubiquitination of nucleosomal H2A is performed using a minimal Polycomb Repressive Deubiquitinase (PR-DUB) complex formed by the human deubiquitinase BAP1 and the DEUBiquitinase ADaptor (DEUBAD) domain of its co-factor ASXL2. These ubiquitination/deubiquitination assays can be conducted in the context of either recombinant nucleosomes reconstituted with bacteria-purified proteins or native nucleosomes purified from mammalian cells. We highlight the intricacies that can have a significant impact on these reactions and we propose that the general principles of these protocols can be swiftly adapted to other E3 ubiquitin ligases and deubiquitinases.

Ubiquitination is a post-translational modification that plays important roles in cellular processes and is tightly coordinated by deubiquitination. Defects in both reactions underlie human pathologies. We provide protocols for conducting ubiquitination and deubiquitination reaction in vitro using purified components.

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...

Biochemistry

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo)

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) is widely used to identify genome-wide binding locations of DNA-binding proteins. However, the ChIP-seq method is limited by its heterogeneity in length of sonicated DNA fragments and non-specific background DNA, resulting in low mapping resolution and uncertainty in DNA-binding sites. To overcome these limitations, the combination of ChIP with exonuclease digestion (ChIP-exo) utilizes 5&#8217; to 3&#8217; exonuclease digestion to trim the heterogeneously sized immunoprecipitated DNA to the protein-DNA crosslinking site. Exonuclease treatment also eliminates non-specific background DNA. The library-prepared and exonuclease-digested DNA can be sent for high-throughput sequencing. The ChIP-exo method allows for near base-pair mapping resolution with greater detection sensitivity and reduced background signal. An optimized ChIP-exo protocol for mammalian cells and next-generation sequencing is described below.

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease ChIP-Exo

Precise determination of protein-binding locations across the genome is important for understanding gene regulation. Here we describe a&#160;genomic mapping method that treats chromatin-immunoprecipitated DNA with exonuclease digestion (ChIP-exo) followed by high-throughput sequencing. This method detects protein-DNA interactions with near base-pair mapping resolution and high signal-to-noise ratio in mammalian neurons.

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

Locus-Specific Chromatin Isolation from Yeast Cells

Single-Copy Gene Locus Chromatin Purification in Saccharomyces cerevisiae

The basic organizational unit of eukaryotic chromatin is the nucleosome core particle (NCP), which comprises DNA wrapped ~1.7 times around a histone octamer. Chromatin is defined as the entity of NCPs and numerous other protein complexes, including transcription factors, chromatin remodeling and modifying enzymes. It is still unclear how these protein-DNA interactions are orchestrated at the level of specific genomic loci during different stages of the cell cycle. This is mainly due to the current technical limitations, which make it challenging to obtain precise measurements of such dynamic interactions. Here, we describe an improved method combining site-specific recombination with an efficient single-step affinity purification protocol to isolate a single-copy gene locus of interest in its native chromatin state. The method allows for the robust enrichment of the target locus over genomic chromatin, making this technique an effective strategy for identifying and quantifying protein interactions in an unbiased and systematic manner, for example by mass spectrometry. Further to such compositional analyses, native chromatin purified by this method likely reflects the in vivo situation regarding nucleosome positioning and histone modifications and is, therefore, amenable to further structural and biochemical analyses of chromatin derived from virtually any genomic locus in yeast.

Author Spotlight: Advancing Chromatin Research and Overcoming Limitations with a High-Enrichment Locus-Specific Chromatin Isolation Protocol 

This protocol presents a locus-specific chromatin isolation method based on site-specific recombination to purify a single-copy gene locus of interest in its native chromatin context from budding yeast, Saccharomyces cerevisiae.

The basic organizational unit of eukaryotic chromatin is the nucleosome core particle (NCP), which comprises DNA wrapped ~1.7 times around a histone ...

Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

Chromatin immunoprecipitation (ChIP) is a widely-used method for determining the interactions of different proteins with DNA in chromatin of living cells. Examples include sequence-specific DNA binding transcription factors, histones and their different modification states, enzymes such as RNA polymerases and ancillary factors, and DNA repair components. Despite its ubiquity, there is a lack of up-to-date, detailed methodologies for both bench preparation of material and for accurate analysis allowing quantitative metrics of interaction. Due to this lack of information, and also because, like any immunoprecipitation, conditions must be re-optimized for new sets of experimental conditions, the ChIP assay is susceptible to inaccurate or poorly quantitative results.
Our protocol is ultimately derived from seminal work on transcription factor:DNA interactions1,2 , but incorporates a number of improvements to sensitivity and reproducibility for difficult-to-obtain cell types. The protocol has been used successfully3,4 , both using qPCR to quantify DNA enrichment, or using a semi-quantitative variant of the below protocol.
This quantitative analysis of PCR-amplified material is performed computationally, and represents a limiting factor in the assay. Important controls and other considerations include the use of an isotype-matched antibody, as well as evaluation of a control region of genomic DNA, such as an intergenic region predicted not to be bound by the protein under study (or anticipated not to show changes under the experimental conditions). In addition, a standard curve of input material for every ChIP sample is used to derive absolute levels of enrichment in the experimental material. Use of standard curves helps to take into account differences between primer sets, regardless of how carefully they are designed, and also efficiency differences throughout the range of template concentrations for a single primer set. Our protocol is different from others that are available5-8 in that we extensively cover the later, analysis phase.

We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.

Chromatin immunoprecipitation (ChIP) is a  widely-used method for determining the interactions of different proteins with  DNA in chromatin of living ...

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.

Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.