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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	<li>Clapier, C. R., Cairns, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+chromatin+remodeling+complexes,+Annual+Review+of+Biochemistry.">The biology of chromatin remodeling complexes, Annual Review of Biochemistry.</a> 78, 273-304 (2009).</li><li>Szerlong, H., Hinada, K., Viswanathan, R., Erdjument-Bromage, H., Tempst, P., Cairns, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+HSA+domain+binds+nuclear+actin-related+proteins+to+regulate+chromatin-remodeling+ATPases.">The HSA domain binds nuclear actin-related proteins to regulate chromatin-remodeling ATPases.</a> Nature Struct. Mol. Biol. 15 (5), 469-476 (2008).</li><li>Shen, X., Mizuguchi, G., Hamiche, A., Wu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chromatin+remodelling+complex+involved+in+transcription+and+DNA+processing.">A chromatin remodelling complex involved in transcription and DNA processing.</a> Nature. 406 (6795), 541-544 (2000).</li><li>Shen, X., Ranallo, R., Choi, E., Wu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+actin-related+proteins+in+ATP-dependent+chromatin+remodelling.">Involvement of actin-related proteins in ATP-dependent chromatin remodelling.</a> Mol. Cell. 12, 147-155 (2003).</li><li>Chen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subunit+organization+of+the+human+INO80+chromatin+remodeling+complex:+an+evolutionarily+conserved+core+complex+catalyzes+ATP-dependent+nucleosome+remodeling.">Subunit organization of the human INO80 chromatin remodeling complex: an evolutionarily conserved core complex catalyzes ATP-dependent nucleosome remodeling.</a> Journal of Biological Chemistry. 286 (13), 11283-11289 (2011).</li><li>Sauer, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+recombination:+developments+and+applications.">Site-specific recombination: developments and applications.</a> 5 (5), 521-527 (1994).</li><li>Gorman, S., Fox, D. T., Wahl, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinase-mediated+gene+activation+and+site-specific+integration+in+mammalian+cells.">Recombinase-mediated gene activation and site-specific integration in mammalian cells.</a> Science. 251 (4999), 1351-1355 (1991).</li><li>Jin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mammalian+chromatin+remodeling+complex+with+similarities+to+the+yeast+INO80+complex.">A mammalian chromatin remodeling complex with similarities to the yeast INO80 complex.</a> Journal of Biological Chemistry. 280 (50), 41207-41212 (2005).</li><li>Cai, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=YY1+functions+with+INO80+to+activate+transcription.">YY1 functions with INO80 to activate transcription.</a> Nature Structural and Molecular Biology. 14 (9), 872-874 (2007).</li><li>Yao, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+Modes+of+Regulation+of+the+Uch37+Deubiquitinating+Enzyme+in+the+Proteasome+and+in+the+Ino80+Chromatin-Remodeling+Complex.">Distinct Modes of Regulation of the Uch37 Deubiquitinating Enzyme in the Proteasome and in the Ino80 Chromatin-Remodeling Complex.</a> Mol.Cell. 31 (6), 909-917 (2008).</li><li>Brizzard, B. L., Chubet, R. G., Vizard, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoaffinity+purification+of+FLAG+epitope-tagged+bacterial+alkaline+phosphatase+using+a+novel+monoclonal+antibody+and+peptide+elution,+Biotechniques.">Immunoaffinity purification of FLAG epitope-tagged bacterial alkaline phosphatase using a novel monoclonal antibody and peptide elution, Biotechniques.</a> 16 (4), 730-735 (1994).</li><li>Barker, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> At the Bench: A Laboratory Navigator. 10, 207-245 (2005).</li><li>Dignam, J. D., Lebovitz, R. M., Roeder, R. 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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 border="0" cellpadding="0" cellspacing="0" width="890">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Name of Reagent/Material</td>
			<td style="width:212px;">Company</td>
			<td style="width:147px;">Catalog Number</td>
			<td style="width:173px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:212px;">Cellgro</td>
			<td style="width:147px;">10-013-CV</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glutamax-I (stablized glutamine)</td>
			<td>Life Technologies</td>
			<td style="width:147px;">35050-079</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>SAFC</td>
			<td style="width:147px;">12176C</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FuGENE6 transfection reagent&#160;&#160;</td>
			<td>Promega</td>
			<td style="width:147px;">E2312</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hygromycin B, sterile in PBS</td>
			<td>AG Scientific</td>
			<td style="width:147px;">H-1012-PBS</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pcDNA5/FRT vector</td>
			<td>Life Technologies</td>
			<td style="width:147px;">V6010-20</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Flp-In HEK293 cells&#160;</td>
			<td>Life Technologies</td>
			<td style="width:147px;">R780-07</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">pOG44 Flp-Recombinase Expression Vector</td>
			<td>Life Technologies</td>
			<td style="width:147px;">V600520</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EZview&#160; Red ANTI-FLAG&#160; M2 Affinity Gel&#160;</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:147px;">F2426</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">calf serum</td>
			<td>SAFC</td>
			<td style="width:147px;">12138C</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TARGETplus SMARTsiRNA pool</td>
			<td>Dharmacon / Thermo Scientific</td>
			<td style="width:147px;">various</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5x siRNA resuspension buffer&#160;</td>
			<td>Dharmacon / Thermo Scientific</td>
			<td style="width:147px;">#B-002000-UB-100</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamine RNAiMAX&#160;</td>
			<td>Life Technologies</td>
			<td style="width:147px;">13778</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Opti-MEM Reduced Serum Medium&#160;</td>
			<td>Life Technologies</td>
			<td style="width:147px;">51985-091</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS&#160;</td>
			<td style="width:212px;">Cellgro</td>
			<td style="width:147px;">45000</td>
			<td style="width:173px;">VWR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">TrypLE (trypsin)</td>
			<td>Life Technologies</td>
			<td style="width:147px;">12604</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1x FLAG Peptide</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:147px;">F3290</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro Bio-Spin Chromatography Column</td>
			<td style="width:212px;">Bio-Rad</td>
			<td style="width:147px;">737-5021</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amicon Ultra Centrifugal Filter Device (50k MWCO)</td>
			<td style="width:212px;">Amicon</td>
			<td style="width:147px;">UFC805024&#160;</td>
			<td style="width:173px;">Fisher Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zeba Desalting Columns&#160;</td>
			<td style="width:212px;">Thermo Scientific</td>
			<td style="width:147px;">89882</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Anti-FLAG M2 antibody, mouse</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:147px;">F3165</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Anti-FLAG M2 antibody, rabbit</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:147px;">F7425</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease Inhibitor Cocktail</td>
			<td style="width:212px;">Sigma</td>
			<td style="width:147px;">P8340</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">benzonase&#160;</td>
			<td>Novagen</td>
			<td style="width:147px;">70664</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;"></td>
			<td style="width:212px;"></td>
			<td style="width:147px;"></td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Equipment</td>
			<td style="width:212px;">Company</td>
			<td style="width:147px;"></td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wheaton Dounce Tissue Grinders</td>
			<td>Wheaton</td>
			<td style="width:147px;">06-435C&#160;</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">JS-4.2 rotor in a J6 centrifuge&#160;</td>
			<td>Beckman-Coulter</td>
			<td style="width:147px;">339080</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">JA-17 rotor</td>
			<td>Beckman-Coulter</td>
			<td style="width:147px;">369691</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 ml polycarbonate tubes&#160;</td>
			<td>Beckman-Coulter</td>
			<td style="width:147px;">355630</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70 ml polycarbonate bottles&#160;</td>
			<td>Beckman-Coulter</td>
			<td style="width:147px;">355655</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Type 45 Ti rotor</td>
			<td>Beckman-Coulter</td>
			<td style="width:147px;">339160</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Type 70.1 Ti rotor&#160;</td>
			<td>Beckman-Coulter</td>
			<td style="width:147px;">342184</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Clay Adams Nutator Mixer</td>
			<td style="width:212px;">BD Diagnostics</td>
			<td style="width:147px;">15172-203&#160;</td>
			<td style="width:173px;">VWR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glas-Col Tube/Vial Rotator</td>
			<td style="width:212px;">Glas-Col</td>
			<td style="width:147px;">099A RD4512</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR thermal cycler PTC 200</td>
			<td>MJ Research</td>
			<td style="width:147px;">PTC 200</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">roller bottle incubator</td>
			<td style="width:212px;">Bellco biotechnology</td>
			<td style="width:147px;">353348</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Immobilon-FL Transfer Membrane 7 x 8.4</td>
			<td style="width:212px;">Millipore</td>
			<td style="width:147px;">IPFL07810</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">lubricated 1.5ml microcentrifuge tubes&#160;</td>
			<td style="width:212px;">Costar</td>
			<td style="width:147px;">3207</td>
			<td style="width:173px;"></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.  Этот протокол описывает процедуру для генерации и очистки дикого типа и мутантных версий человеческого INO80 хроматина комплекса. Эпитопов помечены варианты субъединиц INO80 стабильно экспрессируются в клетках НЕК293, и полные комплексы и комплексы, не имеющие к?...

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

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A nucleoprotein (NP) into viral ribonulceoprotein particles (vRNPs). The influenza vRNPs are enclosed within the viral envelope. During cell entry, however, these vRNP complexes are released into the cytoplasm, where they gain access to the host nuclear transport machinery. In order to study the nuclear import of influenza vRNPs and the replication of the influenza genome, it is useful to work with isolated vRNPs so that other components of the virus do not interfere with these processes. Here, we describe a procedure to purify these vRNPs from the influenza A virus. The procedure starts with the disruption of the influenza A virion with detergents in order to release the vRNP complexes from the enveloped virion. The vRNPs are then separated from the other components of the influenza A virion on a 33-70% discontinuous glycerol gradient by velocity sedimentation. The fractions obtained from the glycerol gradient are then analyzed on via SDS-PAGE after staining with Coomassie blue. The peak fractions containing NP are then pooled together and concentrated by centrifugation. After concentration, the integrity of the vRNPs is verified by visualization of the vRNPs by transmission electron microscopy after negative staining. The glycerol gradient purification is a modification of that from Kemler et al. (1994)1, and the negative staining has been performed by Wu et al. (2007).2

The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza A vRNPs.

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A ...

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristics, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004 and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification 3. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods 9. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37&deg;C overnight culture before islets were transplanted or used for research.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves required for peristalsis and mediate neuronal input from the enteric nervous system1. Different subtypes of ICC form distinct networks in the muscularis of the GI tract 2,3. Loss or injury to these networks is associated with a number of motility disorders4. ICC cells express the KIT receptor tyrosine kinase on the plasma membrane and KIT immunostaining has been used for the past 15 years to label the ICC network5,6. Importantly, normal KIT activity is required for ICC development5,6. Neoplastic transformation of ICC cells results in gastrointestinal stromal tumor (GIST), that frequently harbor gain-of-function KIT mutations7,8. We recently showed that ETV1 is a lineage-specific survival factor expressed in the ICC/GIST lineage and is a master transcriptional regulator required for both normal ICC network formation and for of GIST tumorigenesis9. We further demonstrate that it cooperates with activating KIT mutations in tumorigenesis. Here, we describe methods for visualization of ICC networks in mice, largely based on previously published protocols10,11. More recently, the chloride channel anoctamin 1 (ANO1) has also been characterized as a specific membrane marker of ICC11,12. Because of their plasma membrane localization, immunofluorescence of both proteins can be used to visualize the ICC networks. Here, we describe visualization of the ICC networks by fixed-frozen cyrosections and whole mount preparations.

Visualization of the Interstitial Cells of Cajal ICC Network in Mice

The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves ...

Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for magnetic resonance imaging (MRI). Many commercially available ligands are especially useful because they contain 
functional groups that allow for fast, high-purity, and high-yielding conjugation to macromolecules and biomolecules via amine-reactive 
activated esters and isothiocyanate groups or thiol-reactive maleimides. While metalation of these ligands is considered common 
knowledge in the field of bioconjugation chemistry, subtle differences in metalation procedures must be taken into account when 
selecting metal starting materials. Furthermore, multiple options for purification and characterization exist, and selection of the most 
effective procedure partially depends on the selection of starting materials. These subtle differences are often neglected in published 
protocols. Here, our goal is to demonstrate common methods for metalation, purification, and characterization of lanthanide complexes 
that can be used as contrast agents for MRI (Figure 1). We expect that this publication will enable biomedical scientists to incorporate 
lanthanide complexation reactions into their repertoire of commonly used reactions by easing the selection of starting materials and 
purification methods.

We demonstrate the metalation, purification, and characterization of lanthanide complexes. The complexes described here can be conjugated to macromolecules to enable tracking of these molecules using magnetic resonance imaging.

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (A&beta;, tau), Parkinson's Disease (&alpha;-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for A&beta;6, tau7-9, &alpha;-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
 Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic &alpha;-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.

The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, 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 ...