Name: sequential salt extractions for the analysis of bulk chromatin binding properties of chromatin modifying complexes
Uploaded: 2017-10-02T13:00:00.000+00:00
Duration: 7 min 41 s
Description: Watch this Scientific Journal Video about Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes at JoVE.com

This work was supported by a V scholar award (V2014-004) and a V scholar plus award (D2016-030) from the V Foundation for Cancer Research, and an American Cancer Society Institutional Research Grant (ACS IRG Grant 58-006-53) to the Purdue University Center for Cancer Research. E. G. P. was supported by the Borch Graduate Endowment Award to the Purdue University Medicinal Chemistry and Molecular Pharmacology Department.

1. Preparations
<ol>
	<li>Prepare 100 mL of hypotonic solution Buffer A: 0.3 M sucrose, 60 mM KCl, 60 mM Tris pH 8.0, 2 mM EDTA, and 0.5% NP-40. Store at 4 &#186;C. 
	NOTE: Some cell lines, such as HEK293T, require less stringent lysing conditions. If nuclei lyse easily, use Modified Buffer A: 25 mM HEPES pH 7.6, 25 mM KCl, 5 mM MgCl2, 0.05 mM EDTA, 0.1% NP-40, and 10% glycerol.</li>
	<li>Prepare a 250 mL stock solution of 2x mRIPA solution: 100 mM Tris pH 8.0, 2% NP-40, and 0.5% sodium deoxycholate.</li>
	<li>Prepare a 100 mL stock solution of 5 M NaCl.</li>
	<li>Using the 2x mRIPA and 5 M NaCl solutions, prepare 50 mL of 1x mRIPA solution for each of the six salt concentrations: 0 mM, 100 mM, 200 mM, 300 mM, 400 mM, and 500 mM NaCl. Prior to starting the experiment, cool all solutions on ice.</li>
	<li>Grow cell lines under desired conditions. 
	NOTE: When determining the effects of knocking down a protein, SSE for the knockdown line must be compared to wildtype cells. These SSE must be performed side by side. For examples using chemical inhibitors or stimuli, refer to sections 3 - 5.</li>
</ol>
2. Basic Sequential Salt Extraction
<ol>
	<li>Harvest 8 million cells of each condition and wash twice with 5 mL of ice cold PBS. 
	NOTE: It is critical to have equal number of cells to have equivalent protein concentrations. The exact number of cells may need to be optimized for individual cell lines or particular proteins. It is important not to have too many, as the increase in protein concentrations will prevent observation of the elution curve, which is dependent on equilibrium binding. However, with too few&#160;cells there may not be enough protein to detect the curve, and the chromatin pellet is harder to isolate and can be lost between fractions.</li>
	<li>Re-suspend cells in 1 mL of Buffer A + protease inhibitors, transfer into 1.5 mL micro centrifuge tubes, and rotate top over bottom at 4 &#186;C for 10 min.</li>
	<li>Isolate the nuclei by centrifugation at 6000 x g for 5 min at 4 &#186;C . 
	NOTE: After this step the nuclear envelope should still be intact. If the nuclear envelope is intact, the pellet will re-suspend fully; however, when the envelope is lysed and the chromatin is released, the pellet will not re-suspend. If the nuclear envelope is not intact after Buffer A incubation, use Modified Buffer A.</li>
	<li>Add 200 &#181;L of mRIPA 0 mM NaCl + protease inhibitors to each nuclei pellet before re-suspending pellet. Homogenize each sample by pipetting 15 times with a 1 mL pipette. The pellet should re-suspend easily, however, the nuclei will lyse as it is pipetted and the sample will become thick and sticky and difficult to pipette.
	<ol>
		<li>In order to draw up the pellet into the tip, tap the end of the pipette tip against the bottom of the centrifuge tube. The pellet will not fully dissolve once the DNA is released from the nuclei, but pipetting will break it up.</li>
	</ol>
	</li>
	<li>When all the samples have been homogenized, incubate all the samples on ice for 3 min. 
	NOTE: The incubation time may need to be optimized depending on the protein of interest.</li>
	<li>Isolate chromatin pellet by centrifuging the samples for 3 min at 6500 x g at 4 &#186;C.</li>
	<li>Transfer supernatant to a clean 1.5 mL centrifuge tube. This will be the 0 mM fraction. This 0 mM mRIPA solution can act as a wash step to lysis the nuclei and remove any loose proteins not associated with chromatin.</li>
	<li>Add 200 &#181;L of mRIPA 100 mM NaCl + protease inhibitors to each chromatin pellet before re-suspending pellet. Break up the chromatin pellet by pipetting the pellet up and down 15 times. 
	NOTE: It is critical to be consistent with the number of times the pellet in pipetted.</li>
	<li>Incubate on ice for 3 min. This incubation step allows all samples to reach an equilibrium state, which is particularly important when there are multiple samples.</li>
	<li>Centrifuge at 6500 x g for 3 min at 4 &#186;C and transfer supernatant to a clean 1.5 mL tube.</li>
	<li>Repeat 2.6 - 2.10 for the remaining salt concentrations. 
	NOTE: After 400 mM NaCl, mRIPA the pellet should be clear and gloopy and will not stay at the bottom of the tube. The pellet can be placed in the lid of the centrifuge tube while the supernatant isolated.</li>
	<li>Add 70 &#181;L of 4x protein loading dye to each fraction and load 30 &#181;L of each fraction on to an SDS acrylamide gel for western blot analysis. 
	NOTE: To determine the binding profile of the protein, it is critical to load equivalent volumes of lysate, rather than loading equal protein concentrations.</li>
	<li>Perform a standard western blot by transferring onto a membrane and use primary antibodies for proteins of interest.</li>
	<li>To quantitate the protein eluted from the chromatin, incubate the blot in infrared fluorescence IRDye secondary antibodies and image blot with an imager. While other methods of development can be used, we recommend fluorescence or infrared imaging, as it is more quantitative in nature.</li>
	<li>Use ImageJ or similar software to calculate the intensity for the protein eluted at each salt concentration. By graphing the band intensity against the salt concentration, the elution pattern of your protein of interest can be determined.</li>
</ol>
3. Sequential Salt Extraction in the Presence of a &#34;Reader&#34; Inhibitor
<ol>
	<li>Harvest two sets of cells (4 million) and isolate the nuclei as in a standard SSE.</li>
	<li>Re-suspend both sets in 200 &#181;L of mRIPA 0 mM NaCl and incubate for 3 min. This will allow for the removal of any free protein in the nuclei.</li>
	<li>Add 200 &#181;L mRIPA 0 mM NaCl to each pellet. Add the inhibitor (2 &#181;L of 1 mM (+)JQ1) to one sample and DMSO to the control set.</li>
	<li>Agitate the pellet by pipetting 15 times and incubate on ice for 5 min.</li>
	<li>Centrifuge at 6500 x g for 3 min at 4 &#186;C and transfer supernatant to a clean 1.5 mL tube.</li>
	<li>Repeat 3.3 - 4 for all the salt concentrations and perform a standard western blot.</li>
</ol>
4. Sequential Salt Extraction in the Presence of a &#34;Writer&#34; Inhibitor
<ol>
	<li>Treat cells with the inhibitor (10 &#181;M SAHA) or DMSO for 3 h.</li>
	<li>Harvest 4 million cells for each treatment.</li>
	<li>Perform a standard SSE for the samples with the inhibitor added to all the buffers.</li>
</ol>
5. Sequential Salt Extraction Following DNA Damage
<ol>
	<li>Treat cells with 1 &#181;M doxorubicin for 1 h.</li>
	<li>Change the media on the cells and allow them to recover for 3 h.</li>
	<li>Harvest 8 million cells and perform the basic SSE.</li>
</ol>
6. Non-Sequential Salt Extraction
<ol>
	<li>Harvest 12 million cells and wash with PBS.</li>
	<li>Divide cells so that there are 2 million cells per micro centrifuge tube.</li>
	<li>Re-suspend in 500 &#181;L of Buffer A and incubate for 10 min.</li>
	<li>Isolate the nuclei by centrifugation at 6000 x g.</li>
	<li>Re-suspend pellets in a 200 &#181;L of each of the mRIPA buffers + NaCl.</li>
	<li>Homogenize each sample by pipetting 15 times with a 1 mL pipette tip.</li>
	<li>Incubate on ice for 3 min.</li>
	<li>Isolate the chromatin by centrifugation at 6500 x g and transfer the supernatant to clean tubes. Add 70 &#181;L of loading dye and run 30 &#181;L on a SDS-page gel for western blot analysis.</li>
</ol>

<ol>
	<li>Ramakrishnan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+structure+and+the+organization+of+the+nucleosome.">Histone structure and the organization of the nucleosome.</a> Annu. Rev. Biophys. Biomol. Struct. 26, 83-112 (1997).</li><li>Bannister, A. J., Kouzarides, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+chromatin+by+histone+modifications.">Regulation of chromatin by histone modifications.</a> Cell Rese. 21 (3), 381-395 (2011).</li><li>Musselman, C. A., Lalonde, M. -. E., C&#244;t&#233;, J., Kutateladze, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perceiving+the+epigenetic+landscape+through+histone+readers.">Perceiving the epigenetic landscape through histone readers.</a> Nat Struct Mol Biol. 19 (12), 1218-1227 (2012).</li><li>Furey, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIP-seq+and+beyond:+new+and+improved+methodologies+to+detect+and+characterize+protein-DNA+interactions.">ChIP-seq and beyond: new and improved methodologies to detect and characterize protein-DNA interactions.</a> Nat Rev Genet. 13 (12), 840-852 (2012).</li><li>Skene, P. J., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+targeted+nuclease+strategy+for+high-resolution+mapping+of+DNA+binding+sites.">An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites.</a> eLife. 6, (2017).</li><li>Wu, S. Y., Lee, A. Y., Lai, H. T., Zhang, H., Chiang, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospho+switch+triggers+brd4+chromatin+binding+and+activator+recruitment+for+gene-specific+targeting.">Phospho switch triggers brd4 chromatin binding and activator recruitment for gene-specific targeting.</a> Mol Cell. 49 (5), 843-857 (2013).</li><li>Porter, E. G., Dykhuizen, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individual+bromodomains+of+Polybromo-1+contribute+to+chromatin+association+and+tumor+suppression+in+clear+cell+renal+carcinoma.">Individual bromodomains of Polybromo-1 contribute to chromatin association and tumor suppression in clear cell renal carcinoma.</a> J Biol Chem. 292 (7), 2601-2610 (2017).</li><li>Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycomb+group+complexes--many+combinations,+many+functions.">Polycomb group complexes--many combinations, many functions.</a> Trends cell biol. 19 (12), 692-704 (2009).</li><li>Filippakopoulos, P., 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=Selective+inhibition+of+BET+bromodomains.">Selective inhibition of BET bromodomains.</a> Nature. 468 (7327), 1067-1073 (2010).</li><li>Filippakopoulos, P., 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=Histone+recognition+and+large-scale+structural+analysis+of+the+human+bromodomain+family.">Histone recognition and large-scale structural analysis of the human bromodomain family.</a> Cell. 149 (1), 214-231 (2012).</li><li>Sanchez, R., Zhou, M. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+human+bromodomains+in+chromatin+biology+and+gene+transcription.">The role of human bromodomains in chromatin biology and gene transcription.</a> Curr opin drug disc dev. 12 (5), 659-665 (2009).</li><li>Yang, F., Teves, S. S., Kemp, C. J., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doxorubicin,+DNA+torsion,+and+chromatin+dynamics.">Doxorubicin, DNA torsion, and chromatin dynamics.</a> Biochim. Biophys. Acta - Rev Cancer. 1845 (1), 84-89 (2014).</li><li>Kakarougkas, A., 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=Requirement+for+PBAF+in+Transcriptional+Repression+and+Repair+at+DNA+Breaks+in+Actively+Transcribed+Regions+of+Chromatin.">Requirement for PBAF in Transcriptional Repression and Repair at DNA Breaks in Actively Transcribed Regions of Chromatin.</a> Mol Cell. 55 (5), 723-732 (2014).</li><li>Lee, S. Y., Park, J. -. H., Kim, S., Park, E. -. J., Yun, Y., Kwon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+proteomics+approach+for+the+identification+of+nucleophosmin+and+heterogeneous+nuclear+ribonucleoprotein+C1/C2+as+chromatin-binding+proteins+in+response+to+DNA+double-strand+breaks.">A proteomics approach for the identification of nucleophosmin and heterogeneous nuclear ribonucleoprotein C1/C2 as chromatin-binding proteins in response to DNA double-strand breaks.</a> Biochem J. 388 (Pt 1), (2005).</li><li>Donaldson, M. M., Boner, W., Morgan, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TopBP1+Regulates+Human+Papillomavirus+Type+16+E2+Interaction+with+Chromatin.">TopBP1 Regulates Human Papillomavirus Type 16 E2 Interaction with Chromatin.</a> J Virol. 81 (8), 4338-4342 (2007).</li></ol>

The authors have no competing financial interests.

Characterization of protein and chromatin interactions through salt extractions is a common method that has been employed for decades14,15; however, it has not been systematically optimized before to reveal its full utility. We demonstrate how this sequential method provides a rapid and inexpensive way to distinguish changes in chromatin binding when the protein or the environment is altered. SSE is highly adaptable and optimizable, and importantly, it is technic...

DNA regulation in eukaryotic cells is an intricate and sophisticated system that is tightly controlled by an assortment of proteins that coordinate responses to intracellular and extracellular stimuli. DNA is wrapped around histone octamers to form nucleosomes, which can be loosely distributed along DNA or compacted into tight coils1. This structural arrangement of DNA and histones is known as chromatin, which is regulated by a network of proteins that read, write, and erase post translational modifications (PTM) on histones2. Some histone PTMs, such as acetylation, change the charge of the amino acid they are deposited on, altering interactions between histones and DNA2. Histone PTMs also serve to recruit transcriptional regulators, chromatin remodelers, DNA damage repair machinery, and DNA replication machinery to specific regions of the genome3.
Most methods for studying chromatin interactions either probe small scale interactions or involve large genome-wide analyses. In vitro binding studies often utilize individual recombinant domains with histone peptides or DNA in assays such as electrophoresis mobility shift assays (EMSA), isothermal titration calorimetry, fluorescence polarization, and peptide pulldowns. Because these assays typically focus on an individual protein domain, they facilitate the understanding of a small piece of the puzzle, but do not allow us to understand the cooperative nature of multi-domain proteins, let alone their role in multi-protein complexes. Another layer of intricacy is added by the heterogeneous composition of most mammalian chromatin-modifying complexes. This protein heterogeneity, in combination with the dynamic nature of the chromatin landscape, makes it challenging to recapitulate the in vivo binding interactions of chromatin proteins to chromatin in vitro.
In vivo methods have made significant advances; however, they are often expensive, time consuming, and technically challenging. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is very useful for determining the localization of proteins and histone modifications across the genome, however it requires substantial optimization4. Proteins are often crosslinked to chromatin to preserve interactions; however, this can produce artificial interactions and may cause epitope masking5. Furthermore, the immunoprecipitations (IP) require highly specific antibodies, and extensive optimization of DNA shearing and IP conditions by ChIP-qPCR using a known binding site, which is often not available a priori. After optimization of ChIP conditions, processing of the samples is costly and requires several weeks to months to sequence and analyze. Though this method is invaluable for identifying the localization of chromatin bound proteins across the genome, the cost and time commitment make it prohibitive to use this method to generate hypotheses about how small changes may affect global binding properties.
In this paper, we describe how a sequential salt extraction (SSE) assay can be used to examine global binding profiles of chromatin-bound proteins and distinguish how changes in a protein, complex, or global PTM profile can alter interactions. Though salt extractions are a commonly and broadly used technique, we demonstrate how this sequential method is highly reproducible and versatile. SSE allows us to characterize how a single subunit of a complex or even a single domain contributes to the complex&#39;s overall affinity for bulk chromatin. SSE can also be used to determine if the binding of a protein is influenced by changes in chromatin landscape, providing interesting hypotheses for histone mark targeting that can be confirmed using ChIP-seq and other genome wide studies.
We originally adapted this method from Wu et al., to examine of the function of Polybromo1 (PBRM1) in the binding of the PBAF chromatin remodeler6,7. Using this technique, we determined the role of PBRM1 for chromatin binding within the PBAF chromatin remodeling complex and then determined the relative contribution of the six individual bromodomains to this function7.
Here we describe how to optimize this method to explore chromatin binding in different cell types, to assess the relative binding affinity of similar chromatin modifying complexes, to examine the displacement of a protein from chromatin by a chemical inhibitor, and to determine the effects of chromatin binding after alterations to the chromatin landscape.

<table><tbody><tr><td>Sodium Chloride, Crystal 2.5 Kg AR (ACS)</td><td>Avantor/Macron</td><td>7581-06</td><td></td></tr><tr><td>Sucrose, Crystal 500 G AR&amp;uml; (ACS)</td><td>Avantor/Macron</td><td>8360-04</td><td></td></tr><tr><td>Potassium Chloride, Granular 500 G AR&amp;uml; (ACS)</td><td>Avantor/Macron</td><td>6858-04</td><td></td></tr><tr><td>Trizma(R) base,Primary Standard and Buffer, &gt;=99.9% (titration), crystalline</td><td>Sigma-Aldrich</td><td>T1503-1kg</td><td></td></tr><tr><td>EDTA (Ethylenedinitrilo) Tetraacetic Acid, Disodium Salt, Dihydrate 125 G AR (ACS)</td><td>Avantor/Macron</td><td>4931-02</td><td></td></tr><tr><td>NONIDET P-40 SUBSTRATE, 100mL UN3082</td><td>Amresco</td><td>E109-100ML</td><td></td></tr><tr><td>HEPES Buffer Solution (1M)</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>Magnesium Chloride, 6-Hydrate, Crystal 500 G AR&amp;uml; (ACS)</td><td>Avantor/Macron</td><td>5958-04</td><td></td></tr><tr><td>GLYCEROL, 1L</td><td>Amresco</td><td>0854-1L</td><td></td></tr><tr><td>DEOXYCHOLIC ACID SODIUM SALT, 100g</td><td>Amresco</td><td>0613-100G</td><td></td></tr><tr><td>Eppendorf Research plus pipette</td><td>Fisher Scientific</td><td>13-690-032</td><td></td></tr><tr><td>Eppendorf 5424 R refrigerated microcentrifuge</td><td>Eppendorf</td><td>5424 R</td><td></td></tr><tr><td>Secondary mouse IgG HRP-linked</td><td>Cell Signaling</td><td>7076</td><td></td></tr><tr><td>Secondary rabbit IgG HRP-linked</td><td>Cell Signaling</td><td>7074</td><td></td></tr><tr><td>BMI-1 antibody</td><td>Millipore</td><td></td><td></td></tr><tr><td>PBRM1 antibody</td><td>Bethyl</td><td>A301-591A</td><td></td></tr><tr><td>ARID1a antibody</td><td>Santa Cruz</td><td>sc-32761</td><td></td></tr><tr><td>BRD4 antibody</td><td>Bethyl</td><td>A301-9852a</td><td></td></tr><tr><td>(+) JQ1</td><td>Cayman Chemical Company</td><td>11187</td><td></td></tr><tr><td>SAHA</td><td>Cayman Chemical Company</td><td>10009929</td><td></td></tr><tr><td>Doxorubicin</td><td>AK Scientific</td><td>25316-40-9</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Macron</td><td>4948-02</td><td></td></tr><tr><td>Mini Trans-Blot Cell</td><td>BioRad</td><td>1703930</td><td></td></tr><tr><td>Mini Gel Tank</td><td>ThermoFisher</td><td>A25977</td><td></td></tr><tr><td>Albumin, Bovine (BSA)</td><td>Amresco</td><td>0332-100g</td><td>Used in as a 5% solution in PBS-T as blocking solution for Western blots</td></tr><tr><td>Bolt MOPS SDS Running Buffer (20x)</td><td>Life Technologies</td><td>B0001</td><td></td></tr><tr><td>Bolt LDS Sample Buffer (4x)</td><td>Life Technologies</td><td>B0007</td><td></td></tr><tr><td>PageRuler Plus Prestained Protein Ladder, 10 to 250 kDa</td><td>ThermoFisher</td><td>26619</td><td></td></tr><tr><td>Methyl Alcohol, Anhydrous</td><td>Macron</td><td>3041-10</td><td></td></tr><tr><td>Trypsin kEDTA, 1X</td><td>Cellgro</td><td>25-053-CI</td><td></td></tr><tr><td>SODIUM AZIDE, 250g UN1687</td><td>Amresco</td><td>0639-250G</td><td></td></tr><tr><td>SODIUM DODECYL SULFATE (SDS)500g UN1325</td><td>Amresco</td><td>0227-500G</td><td></td></tr><tr><td>Glycine,for electrophoresis, &gt;=99%</td><td>Sigma-Aldrich</td><td>G8898-1kg</td><td></td></tr><tr><td>BigTop Microcentrifuge Tubes, Polypropylene</td><td>VWR</td><td>20170-333</td><td></td></tr><tr><td>1000 &amp;micro;L Pipet Tips</td><td>VWR</td><td>83007-382</td><td></td></tr><tr><td>NuPAGE Bis-Tris Precast Gels 4-12%</td><td>Invitrogen</td><td>NW04125BOX</td><td></td></tr><tr><td>Leupeptin Hemisulfate, 5 MG</td><td>RPI Research Products</td><td>22035-0.005</td><td>Used for Protease inhibitor solution.</td></tr><tr><td>APROTININ, 10 MG</td><td>RPI Research Products</td><td>20550-0.01</td><td>Used for Protease inhibitor solution.</td></tr><tr><td>PEPSTATIN A, 5 MG</td><td>RPI Research Products</td><td>30100-0.005</td><td>Used for Protease inhibitor solution.</td></tr><tr><td>OVCA429 cells</td><td>Gift from Karen Cowden Dahl. Ph.D.</td><td></td><td></td></tr><tr><td>HEK293T</td><td>ATCC</td><td>CRL-3216</td><td></td></tr><tr><td>HeLa cells</td><td>ATCC</td><td>CCL-2</td><td></td></tr><tr><td>McCoy&#039;s 5A media</td><td>Corning Mediatech</td><td>10-050-CV</td><td></td></tr><tr><td>DMEM</td><td>Corning Mediatech</td><td>10-013-CV</td><td></td></tr><tr><td>Minimum Essential Medium (MEM), Powder</td><td>Corning Mediatech</td><td>50-011-PC</td><td></td></tr><tr><td>MEM NEAA (100X) non-essential amino acid</td><td>Gibco</td><td>11140-050</td><td></td></tr><tr><td>FB-11: Fetal Bovine Serum, U.S. Source</td><td>Omega Scientific</td><td>FB-11</td><td></td></tr><tr><td>Penicillin-Streptomycin Solution</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>Glutamax</td><td>Life Technologies</td><td>35050-061</td><td></td></tr><tr><td>sodium pyruvate</td><td>Life Technologies</td><td>11360070</td><td></td></tr><tr><td>VWR Power Source</td><td>VWR</td><td>13-690-032</td><td></td></tr></tbody></table>

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.

In this paper, we demonstrate the advantages and applications of the commonly used sequential salt extraction (SSE) method that we have adapted from the literature6. In Figure 1, we compare the reproducibility of SSE to extracting proteins non-sequentially by detecting the elution patterns of ARID1a and PBRM1. We consistently observe that ARID1a, a BAF subunit, elutes primarily at 200 mM NaCl and PBRM1, an exclusive PBAF subunit, elute...

Watch this Scientific Journal Video about Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes at JoVE.com

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

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 ...

sequential-salt-extractions-for-analysis-bulk-chromatin-binding

Research

JoVE Journal

Genetics

8.3K Views.  Purdue University. 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.

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

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

Quantitative Analysis of Chromatin Proteomes in Disease

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due to the high percentage of binucleated cells,1 the predominantly heterochromatic state of the DNA, and the non-dividing nature of the cardiomyocyte which renders adult nuclei in a permanent state of interphase.2 Transcriptional regulation during development and disease have been well studied in this organ,3-5 but what remains relatively unexplored is the role played by the nuclear proteins responsible for DNA packaging and expression, and how these proteins control changes in transcriptional programs that occur during disease.6 In the developed world, heart disease is the number one cause of mortality for both men and women.7 Insight on how nuclear proteins cooperate to regulate the progression of this disease is critical for advancing the current treatment options.
Mass spectrometry is the ideal tool for addressing these questions as it allows for an unbiased annotation of the nuclear proteome and relative quantification for how the abundance of these proteins changes with disease. While there have been several proteomic studies for mammalian nuclear protein complexes,8-13 until recently14 there has been only one study examining the cardiac nuclear proteome, and it considered the entire nucleus, rather than exploring the proteome at the level of nuclear sub compartments.15 In large part, this shortage of work is due to the difficulty of isolating cardiac nuclei. Cardiac nuclei occur within a rigid and dense actin-myosin apparatus to which they are connected via multiple extensions from the endoplasmic reticulum, to the extent that myocyte contraction alters their overall shape.16 Additionally, cardiomyocytes are 40% mitochondria by volume17 which necessitates enrichment of the nucleus apart from the other organelles. Here we describe a protocol for cardiac nuclear enrichment and further fractionation into biologically-relevant compartments. Furthermore, we detail methods for label-free quantitative mass spectrometric dissection of these fractions-techniques amenable to in vivo experimentation in various animal models and organ systems where metabolic labeling is not feasible.

Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due ...

Medicine

Assays for Validating Histone Acetyltransferase Inhibitors

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene expression. KATs, such as CBP/p300, are under intense investigation as therapeutic targets due to their critical role in tumorigenesis of diverse cancers. The development of novel small molecule inhibitors targeting the histone acetyltransferase (HAT) function of KATs is challenging and requires robust assays that can validate the specificity and potency of potential inhibitors.
This article outlines a pipeline of three methods that provide rigorous in vitro validation for novel HAT inhibitors (HATi). These methods include a test tube HAT assay, Chromatin Hyperacetylation Inhibition (ChHAI) assay, and Chromatin Immunoprecipitation-quantitative PCR (ChIP-qPCR). In the HAT assay, recombinant HATs are incubated with histones in a test tube reaction, allowing for acetylation of specific lysine residues on the histone tails. This reaction can be blocked by a HATi and the relative levels of site-specific histone acetylation can be measured via immunoblotting. Inhibitors identified in the HAT assay need to be confirmed in the cellular environment.
The ChHAI assay uses immunoblotting to screen for novel HATi that attenuate the robust hyperacetylation of histones induced by a histone deacetylase inhibitor (HDACi). The addition of an HDACi is helpful because basal levels of histone acetylation can be difficult to detect via immunoblotting.
The HAT and ChHAI assays measure global changes in histone acetylation, but do not provide information regarding acetylation at specific genomic regions. Therefore, ChIP-qPCR is used to investigate the effects of HATi on histone acetylation levels at gene regulatory elements. This is accomplished through selective immunoprecipitation of histone-DNA complexes and analysis of the purified DNA through qPCR. Together, these three assays allow for the careful validation of the specificity, potency, and mechanism of action of novel HATi.

Inhibitors of histone acetyltransferases (HATs, also known as lysine acetyltransferases), such as CBP/p300, are potential therapeutics for treating cancer. However, rigorous methods for validating these inhibitors are needed. Three in vitro methods for validation include HAT assays with recombinant acetyltransferases, immunoblotting for histone acetylation in cell culture, and ChIP-qPCR.

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene ...

Cancer Research

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 ...

Biochemistry

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Biology

Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14. 
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8. 
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.

Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation

Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control.

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such ...

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 ...

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular interactions and the genetic programs controlled by the myogenic factors is essential for the rational design of efficient therapies.

MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ...

The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision

Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA interactions. ChIP coupled to high throughput sequencing (ChIP-seq) is commonly used to determine the genomic location of proteins that interact with chromatin. However, ChIP-seq is hampered by relatively low mapping resolution of several hundred base pairs and high background signal. The ChIP-exo method is a refined version of ChIP-seq that substantially improves upon both resolution and noise. The key distinction of the ChIP-exo methodology is the incorporation of lambda exonuclease digestion in the library preparation workflow to effectively footprint the left and right 5&#39; DNA borders of the protein-DNA crosslink site. The ChIP-exo libraries are then subjected to high throughput sequencing. The resulting data can be leveraged to provide unique and ultra-high resolution insights into the functional organization of the genome. Here, we describe the ChIP-exo method that we have optimized and streamlined for mammalian systems and next-generation sequencing-by-synthesis platform.

Here, we present a protocol to achieve near base pair resolution of protein-DNA interactions. This is obtained by exonuclease treatment of DNA fragments selectively enriched by chromatin immunoprecipitation (ChIP-exo) followed by high throughput sequencing.

Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA ...

Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method

Genome duplication is orchestrated by replisome proteins, including helicases, which unwind double-stranded DNA into individual antiparallel single strands, each requiring distinct modes of replication: continuous (leading) and discontinuous (lagging). Understanding the interactions of chromatin-associated proteins with replicating DNA strands in vivo is crucial for elucidating the mechanisms of chromatin assembly and DNA repair coupled to DNA replication. This protocol presents the enrichment and sequencing of protein-associated nascent DNA (eSPAN) method, designed to quantify relative protein levels on nascent leading and lagging DNA strands at replication forks. The eSPAN procedure starts with chromatin immunoprecipitation (ChIP, in yeast) or cleavage under targets and tagmentation (CUT&amp;Tag; in mammalian cells) of a protein of interest, followed by enrichment of the protein-associated nascent DNA by bromodeoxyuridine (BrdU) immunoprecipitation. Strand-specific next-generation sequencing is applied to isolated ssDNA. This technique can be used to determine whether a protein is enriched at leading or lagging replication forks. The eSPAN provides genome-wide strand-preference of chromatin-associated proteins, including histones at replication forks.

Enrichment and sequencing of protein-associated nascent DNA (eSPAN) was developed to detect the relative abundance of a chromatin-associated protein on two replicating DNA strands, thereby revealing molecular insight into chromatin replication and its coupled processes. This protocol describes eSPAN procedures in yeast and mouse embryonic stem (ES) cells.

Genome duplication is orchestrated by replisome proteins, including helicases, which unwind double-stranded DNA into individual antiparallel single ...