Name: chromatin immunoprecipitation assay using micrococcal nucleases in mammalian cells
Uploaded: 2019-05-10T14:30:00
Description: Watch this Scientific Journal Video about Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells at JoVE.com

This research is supported by Genentech royalties to City of Hope. This work is not supported in whole or in part by the National Institutes of Health.

1. Preparation of Reagents
<ol>
	<li>Make 18.5% paraformaldehyde (PFA) solution. Add 0.925 g of PFA, 4.8 mL of water (use ultrapurified water throughout the protocol) and 35 &#181;L of 1 M KOH in a 50 mL conical plastic tube. Close the cap tightly and heat the tube in a 400-600 mL glass beaker containing approximately 200 mL of water using a microwave. Remove the tube before the water starts boiling and vortex the tube to dissolve PFA. Allow PFA to cool to room temperature and store on ice. 
	NOTE:</strong...

<ol>
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B., Kingston, R. E., Moore, D. D., Smith, J. A., Struhl, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Genomic+DNA.">Preparation of Genomic DNA.</a> Short Protocols in Molecular Biology. , (1992).</li><li>Crouse, J., Amorese, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+Precipitation:+Ammonium+Acetate+as+an+Alternative+to+Sodium+Acetate.">Ethanol Precipitation: Ammonium Acetate as an Alternative to Sodium Acetate.</a> Focus. 9 (2), 3-5 (1987).</li><li>Zeugin, J. A., Hartley, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+Precipitation+of+DNA.">Ethanol Precipitation of DNA.</a> Focus. 7 (4), 1-2 (1985).</li><li>Barski, 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=High-resolution+profiling+of+histone+methylations+in+the+human+genome.">High-resolution profiling of histone methylations in the human genome.</a> Cell. 129 (4), 823-837 (2007).</li><li>Guenther, M. G., Levine, S. S., Boyer, L. A., Jaenisch, R., Young, R. A. <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+landmark+and+transcription+initiation+at+most+promoters+in+human+cells.">A chromatin landmark and transcription initiation at most promoters in human cells.</a> Cell. 130 (1), 77-88 (2007).</li><li>Louie, M. C., 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=Androgen-induced+recruitment+of+RNA+polymerase+II+to+a+nuclear+receptor-p160+coactivator+complex.">Androgen-induced recruitment of RNA polymerase II to a nuclear receptor-p160 coactivator complex.</a> Proceedings of the National Academy of Sciences of the United States of America. 100 (5), 2226-2230 (2003).</li><li>Liu, X., 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=Positive+feedback+loop+mediated+by+protein+phosphatase+1alpha+mobilization+of+P-TEFb+and+basal+CDK1+drives+androgen+receptor+in+prostate+cancer.">Positive feedback loop mediated by protein phosphatase 1alpha mobilization of P-TEFb and basal CDK1 drives androgen receptor in prostate cancer.</a> Nucleic Acids Research. 45 (7), 3738-3751 (2017).</li><li>Zhao, H., 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=Identification+of+valid+reference+genes+for+mRNA+and+microRNA+normalisation+in+prostate+cancer+cell+lines.">Identification of valid reference genes for mRNA and microRNA normalisation in prostate cancer cell lines.</a> Scientific reports. 8 (1), 1949 (2018).</li><li>Nelson, J. D., Denisenko, O., Bomsztyk, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+the+fast+chromatin+immunoprecipitation+(ChIP)+method.">Protocol for the fast chromatin immunoprecipitation (ChIP) method.</a> Nature. 1 (1), 179-185 (2006).</li><li>Gade, P., Kalvakolanu, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+immunoprecipitation+assay+as+a+tool+for+analyzing+transcription+factor+activity.">Chromatin immunoprecipitation assay as a tool for analyzing transcription factor activity.</a> Methods in molecular biology. 809, 85-104 (2012).</li><li>Lund, E. G., Duband-Goulet, I., Oldenburg, A., Buendia, B., Collas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+features+of+lamin+A-interacting+chromatin+domains+mapped+by+ChIP-sequencing+from+sonicated+or+micrococcal+nuclease-digested+chromatin.">Distinct features of lamin A-interacting chromatin domains mapped by ChIP-sequencing from sonicated or micrococcal nuclease-digested chromatin.</a> Nucleus. 6 (1), 30-39 (2015).</li><li>Dahl, J. A., Collas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+micro+chromatin+immunoprecipitation+assay+(microChIP).">A rapid micro chromatin immunoprecipitation assay (microChIP).</a> Nature protocols. 3 (6), 1032-1045 (2008).</li><li>Yamakawa, T., Waer, C., Itakura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AT-rich+interactive+domain+5B+regulates+androgen+receptor+transcription+in+human+prostate+cancer+cells.">AT-rich interactive domain 5B regulates androgen receptor transcription in human prostate cancer cells.</a> Prostate. 78 (16), 1238-1247 (2018).</li></ol>

Although sonication is commonly used to obtain fragmented chromatin, it is time-consuming and cumbersome to identify reproducible conditions. In this protocol, we used MNase digestion because enzyme digestion should be easier to identify reproducible conditions. A brief sonication step after MNase digestion (see step 2.2) was necessary to break the cell membrane and to release the digested chromatin. Therefore, the sonication power in our protocol should be as low as possible. We use the same sonication conditions for al...

Gene expression in mammalian cells is tightly and dynamically regulated, and transcription is one of the key steps. Gene transcription is mainly regulated by transcription factors and histones. A transcription factor is a protein that binds to specific DNA sequences and controls gene transcription. These factors either promote or inhibit the recruitment of RNA polymerase II (PolII), which initiates mRNA synthesis from genomic DNA as a template1. Histone modifications such as acetylation and methylation of histone tail residues positively and negatively affect gene transcription by changing the chromatin structure2. Since alterations in gene expression affect the cellular context, it is essential to examine the molecular mechanisms by which transcription is regulated.
To date, several methods for investigating the regulation of gene transcription are available. Electrophoretic mobility shift assay (EMSA), also called a gel shift assay, is used for analyzing a protein-DNA interaction3. A nuclear extract from cells of interest is incubated with a radioactive isotope (for example, 32P)-labeled DNA probe and electrophoresed on a polyacrylamide gel. Its autoradiogram shows that the DNA-protein complex migrates slower than the probe in a gel. In the presence of an antibody against the protein, the DNA-protein-antibody complex migrates in a gel more slowly than the DNA-protein complex. This supershifted band reveals specific binding between the DNA and protein. However, EMSA only determines a specific DNA-protein interaction in a cell-free system, and therefore it remains unknown whether the interaction controls transcription in living cells. The transient reporter assay, commonly called luciferase reporter assay, was developed to address gene expression regulation in cells. Typically, an upstream genomic region of a gene of interest is inserted into a reporter plasmid, transiently transfected into cells, and the reporter activity is measured. A variety of deletion mutants allows the identification of regions that are responsible for gene regulation. Even though a reporter assay is a useful tool for identifying transcription factors and binding DNA sequences controlling transcription, this method has a major disadvantage in that a reporter plasmid is free of chromatin structure and does not reflect &#8220;real&#8221; transcription machinery. In addition, changes in histone modifications cannot be determined by the system.
The development of the chromatin immunoprecipitation (ChIP) method was based on Jackson and Chalkley&#8217;s reports that &#8220;whole cell&#8221; fixation with formaldehyde preserved chromatin structure4,5. Since then, many related techniques have been developed and improved6. In ChIP assays, cells are fixed with formaldehyde to cross-link DNA and proteins. The chromatin is fragmented and then immunoprecipitated with antibodies of interest. The immune complex is washed, and DNA is purified. PCR amplification with primers targeted to a particular region of the genome reveals the occupancy of proteins of interest in the genome.
Although ChIP is a powerful tool to identify the interactions of proteins such as transcription factors and modified histones with DNA, the method involves some difficulties, such as a chromatin fragmentation step, in practice. Sonication has been widely used for shearing chromatin; however, it is cumbersome to identify reproducible conditions. Micrococcal nuclease (MNase) treatment is an alternative method for chromatin shearing. MNase is an endo-exonuclease that digests double-stranded, single-stranded, circular and linear DNA and RNA. It is relatively easy to determine the conditions, including the amounts of chromatin and enzyme, temperature, and incubation time, for optimum chromatin fragmentation. We modified and simplified the existing protocols, and we established a straightforward and reproducible method. This paper provides the protocol for a ChIP assay using MNase in mammalian cells.

<table border="0" cellpadding="0" cellspacing="0" style="width:1643px;" width="1642">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">0.5 M EDTA (pH 8.0)</td>
			<td style="width:173px;">Thermo Scientific</td>
			<td style="width:119px;">AM9010</td>
			<td style="width:793px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">2 M KCl</td>
			<td style="width:173px;">Thermo Scientific</td>
			<td style="width:119px;">AM9010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">2X iQ SYBR Green supermix</td>
			<td style="width:173px;">Bio-Rad</td>
			<td align="right" style="width:119px;">1706862</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">5 M NaCl</td>
			<td style="width:173px;">Thermo Scientific</td>
			<td style="width:119px;">AM9010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">50 bp DNA ladder</td>
			<td style="width:173px;">New England Biolabs</td>
			<td style="width:119px;">N3236S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:557px;">Agarose</td>
			<td style="width:173px;">Research Product International</td>
			<td style="width:119px;">A20090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Branched octylphenoxy poly(ethyleneoxy)ethanol</td>
			<td style="width:173px;">Millipore Sigma</td>
			<td style="width:119px;">I8896</td>
			<td>IGEPAL CA-630</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">ChIP-grade protein G magnetic beads</td>
			<td style="width:173px;">Cell signaling technology</td>
			<td style="width:119px;">9006S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Chromatin Immunoprecipitation (ChIP) Dilution Buffer</td>
			<td style="width:173px;">Millipore Sigma</td>
			<td style="width:119px;">20-153</td>
			<td>Buffer composition: 0.01% SDS, 1.1% Triton X- 100, 1.2mM EDTA, 16.7mM Tris-HCl, pH 8.1, 167mM NaCl.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Gel Loading Dye Purple (6X)</td>
			<td style="width:173px;">New England Biolabs</td>
			<td style="width:119px;">B7024S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Glycine</td>
			<td style="width:173px;">Bio-Rad</td>
			<td style="width:119px;">161-0724</td>
			<td>Electropheresis grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Glycogen</td>
			<td style="width:173px;">Millipore Sigma</td>
			<td style="width:119px;">G1767</td>
			<td>19-22 mg/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100x)</td>
			<td style="width:173px;">Thermo Scientific</td>
			<td style="width:119px;">78445</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">High Salt Immune Complex Wash Buffer</td>
			<td style="width:173px;">Millipore Sigma</td>
			<td style="width:119px;">20-155</td>
			<td>Buffer composition: 0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 500mM NaCl.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Histone H3K4me3 antibody (pAb)</td>
			<td style="width:173px;">Active Motif</td>
			<td style="width:119px;">39915</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">LiCl Immune Complex Wash Buffer</td>
			<td style="width:173px;">Millipore Sigma</td>
			<td style="width:119px;">20-156</td>
			<td>Buffer composition: 0.25M LiCl, 1% IGEPAL CA630, 1% deoxycholic acid (sodium salt), 1mM EDTA, 10mM Tris, pH 8.1.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Low Salt Immune Complex Wash Buffer</td>
			<td style="width:173px;">Millipore Sigma</td>
			<td style="width:119px;">20-154</td>
			<td>Buffer composition: 0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 150mM NaCl.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Magna GrIP Rack (8 well)</td>
			<td style="width:173px;">Millipore Sigma</td>
			<td style="width:119px;">20-400</td>
			<td style="width:793px;">Any kind of magnetic separation stands that are compatible with a 1.5 mL tube is fine.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Micrococcal nuclease</td>
			<td style="width:173px;">New England Biolabs</td>
			<td style="width:119px;">M0247S</td>
			<td>comes with 10 x buffer (500 mM Tris-HCl, 50 mM CaCl2, pH 7.9 @ 25 &#176;C) and 100 x BSA (10 mg/ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">NaHCO3</td>
			<td style="width:173px;">JT Baker</td>
			<td style="width:119px;">3506-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Normal rabbit IgG</td>
			<td style="width:173px;">Millipore Sigma</td>
			<td style="width:119px;">12-370</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">PIPES</td>
			<td style="width:173px;">Millipore Sigma</td>
			<td style="width:119px;">P6757</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Proteinase K</td>
			<td style="width:173px;">Millipore Sigma</td>
			<td style="width:119px;">3115887001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Real-time PCR system</td>
			<td style="width:173px;">Bio-Rad</td>
			<td style="width:119px;">CFX96, C1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">RNA pol II CTD phospho Ser5 antibody</td>
			<td style="width:173px;">Active Motif</td>
			<td style="width:119px;">39749</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">SDS</td>
			<td style="width:173px;">Boehringer Mannheim</td>
			<td style="width:119px;">100155</td>
			<td>Electropheresis grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">sodium acetate</td>
			<td style="width:173px;">Millipore Sigma</td>
			<td style="width:119px;">S5636</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">Sonicator equipped with a microtip probe</td>
			<td style="width:173px;">QSONICA</td>
			<td style="width:119px;">Q700</td>
			<td style="width:793px;">Any kind of sonicators that are compatible with a 1.5 mL tube is fine.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:557px;">UltraPure Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)</td>
			<td style="width:173px;">Thermo Scientific</td>
			<td style="width:119px;">15593031</td>
			<td>pH 8.05</td>
		</tr>
	</tbody>
</table>

chromatin immunoprecipitation assay using micrococcal nucleases in mammalian cells

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene expression. The cellular transcriptional machinery and its chromatin modification proteins coordinate to regulate transcription. To analyze transcriptional regulation at the molecular level, several experimental methods such as electrophoretic mobility shift, transient reporter and chromatin immunoprecipitation (ChIP) assays are available. We describe a modified ChIP assay in detail in this article because of its advantages in directly showing histone modifications and the interactions between proteins and DNA in cells. One of the key steps in a successful ChIP assay is chromatin shearing. Although sonication is commonly used for shearing chromatin, it is difficult to identify reproducible conditions. Instead of shearing chromatin by sonication, we utilized enzymatic digestion with micrococcal nuclease (MNase) to obtain more reproducible results. In this article, we provide a straightforward ChIP assay protocol using MNase.

Digesting chromatin is one of the important steps for a ChIP assay. We used MNase to digest chromatin to obtain a mixture of nucleosome oligomers. In the MNase digestion step, MNase can go through the nuclear membrane and digest chromatin. However, the digested chromatin cannot go through the membrane and remains in the nuclei. To release the digested chromatin from the nuclei, brief sonication is needed. Figure 1A shows microphotographs before and after sonication of VCaP cell suspension. W...

Watch this Scientific Journal Video about Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells at JoVE.com

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...

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