Name: native chromatin immunoprecipitation using murine brain tumor neurospheres
Uploaded: 2018-01-29T14:30:00
Description: Watch this Scientific Journal Video about Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres at JoVE.com

This work was supported by National Institutes of Health/National Institute of Neurological Disorders &#38; Stroke (NIH/NINDS) Grants R37-NS094804, R01-NS074387, R21-NS091555 to M.G.C.; NIH/NINDS Grants R01-NS076991, R01-NS082311, and R01-NS096756 to P.R.L.; NIH/NINDS R01-EB022563; the Department of Neurosurgery; Leah&#39;s Happy Hearts, and Chad Though Foundation to M.G.C. and P.R.L. RNA Biomedicine Grant F046166 to M.G.C. F.M.M. is supported by an F31 NIH/NINDS-F31NS103500. R.I.Z.-V. is supported by the NIH/ NIGMS grant 5T34GM007821-37.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Michigan.
1. Generation of brain tumor NS and culturing conditions.
<ol>
	<li>Prepare Neural Stem Cell Medium (NSC) with Dulbecco&#39;s Modified Eagle Medium/F12B27 supplement (1x), N2 supplement (1x), and an antimicrobial reagent. Supplement NSC medium on the day of use with human recombinant endothelial growth factor (EGF) and basic-fibroblast growth factor (FGF) at...

<ol>
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W., 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+and+separable+roles+for+EZH2+in+neurogenic+astroglia.">Distinct and separable roles for EZH2 in neurogenic astroglia.</a> Elife. 3, e02439 (2014).</li><li>Haring, M., 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=Chromatin+immunoprecipitation:+optimization,+quantitative+analysis+and+data+normalization.">Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization.</a> Plant Methods. 3, 11 (2007).</li></ol>

The protocol presented here will enable the user to perform native ChIP on NS derived from genetically engineered brain tumors. In contrast to cross-linking ChIP, this protocol is limited for the study of proteins that associate tightly with DNA6. The number of cells used can be modified as necessary and the protocol can be scaled up. We used 1 X 106 cells per IP, however, native ChIP may also be performed with as little as 4 x 104 cells5. In this prot...

Epigenetic events are frequent in gliomas and likely play an important role in tumor pathogenesis. Indeed, in pediatric high-grade glioma, mutations in genes encoding histone variants H3.3 and H3.1 occur frequently1. The mutations affect histone modifications and have major epigenetic consequences2,3. In the adolescent to adult spectrum, recurrent mutations in isocitrate dehydrogenase gene 1/2 (IDH1/2), a mutation that inhibits &#945;-KG dependent histone and DNA de-methylasaes, and genetic alterations in other chromatin regulators such as ATRX and DAXX occur4. Therefore, it is of critical importance to study how mutations that affect epigenetic regulators alter chromatin structure and regulatory histone modifications, which, in turn, have a dramatic impact of the tumor cells&#39; transcriptome.
Chromatin immunoprecipitation (ChIP) is a powerful tool used to evaluate the impact of epigenetic modifications in the genome5,6,7. In native ChIP, chromatin is digested with micrococcal nuclease (MNase), immunoprecipitated using an antibody raised against the protein of interest, and then DNA is purified from the immunoprecipitated chromatin complex6. Cells are not fixed during the procedure so this technique is only applicable for the study of proteins that interact tightly with DNA6. The absence of cross-linking aids antibody specificity since antibodies are usually raised against unfixed peptides or proteins7. In addition, since there is no cross-linking step, this reduces the chances of fixing transient protein-DNA interactions that are non-specific and not regulatory7,8. ChIP can be used to identify the enrichment of histone modifications in a specific genomic region. Here, we detail a protocol for performing native ChIP in neurospheres (NS) generated from genetically engineered mouse models of glioma.

<table>
	<tbody>
		<tr>
			<td>Agilent 2100 Bioanalyzer</td>
			<td>Agilent</td>
			<td>G2946-90004</td>
			<td>bioanalyzer</td>
		</tr>
		<tr>
			<td>AccuSpin Micro 17R, refrigerated</td>
			<td>Fisher Scientific</td>
			<td>13-100-676</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6</td>
			<td>Taconic</td>
			<td>B6-f</td>
			<td>C57BL/6 mouse</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Aldrich</td>
			<td>22350-6</td>
			<td>buffer reagent</td>
		</tr>
		<tr>
			<td>D-Luciferin, Potassium Salt</td>
			<td>Goldbio</td>
			<td>LuckK-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>DiaMag1.5 magnetic rack</td>
			<td>Diagenode</td>
			<td>B04000003</td>
			<td>for magnetic bead washes</td>
		</tr>
		<tr>
			<td>DiaMag Rotator EU</td>
			<td>Diagenode</td>
			<td>B05000001</td>
			<td>rotator</td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Gibco</td>
			<td>11330-057</td>
			<td>NSC component</td>
		</tr>
		<tr>
			<td>Dynabeads Protein A</td>
			<td>Thermo Fisher Scientific</td>
			<td>10001D</td>
			<td>protein A magnetic beads</td>
		</tr>
		<tr>
			<td>Dynabeads Protein G</td>
			<td>Thermo Fisher Scientific</td>
			<td>10003D</td>
			<td>protein G magnetic beads</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>PeproTech</td>
			<td>AF-100-15</td>
			<td>prepare 20 &#956;g/mL stock in 0.1% BSA and aliquot.</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma</td>
			<td>E-4884</td>
			<td>buffer reagent</td>
		</tr>
		<tr>
			<td>ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid) (EGTA)</td>
			<td>Sigma</td>
			<td>E-4378</td>
			<td>buffer reagent</td>
		</tr>
		<tr>
			<td>Fast SYBR Green Master Mix</td>
			<td>Applied Biosystems</td>
			<td>4385612</td>
			<td>qPCR reagent</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10437028</td>
			<td>for freezing cells</td>
		</tr>
		<tr>
			<td>FGF</td>
			<td>PeproTech</td>
			<td>100-18b</td>
			<td>Prepare 20 &#956;g/mL stock in 0.1% BSA and aliquot.</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11008-13</td>
			<td>for dissection of tumor</td>
		</tr>
		<tr>
			<td>Glycerol, MB Grade</td>
			<td>EMD- Millipore</td>
			<td>356352</td>
			<td></td>
		</tr>
		<tr>
			<td>H3K4me3</td>
			<td>Abcam</td>
			<td>Ab8580</td>
			<td></td>
		</tr>
		<tr>
			<td>H3K27me3</td>
			<td>Millipore</td>
			<td>07-449</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14175-103</td>
			<td>balanced salt solution</td>
		</tr>
		<tr>
			<td>Fluriso</td>
			<td>VETone</td>
			<td>501017</td>
			<td>inhalation anesthetic</td>
		</tr>
		<tr>
			<td>Ivis Spectrum</td>
			<td>Perkin-Elmer</td>
			<td>124262</td>
			<td>in vivo optical imaging system</td>
		</tr>
		<tr>
			<td>Hyqtase</td>
			<td>HyClon</td>
			<td>SV3003001</td>
			<td>cell detachment media</td>
		</tr>
		<tr>
			<td>Lithium Chloride</td>
			<td>Sigma</td>
			<td>L8895</td>
			<td>buffer reagent</td>
		</tr>
		<tr>
			<td>Low binding microtubes</td>
			<td>Corning Costar</td>
			<td>CLS3207</td>
			<td>low protein binding microcentrifuge tube</td>
		</tr>
		<tr>
			<td>Microcentrifuge tube</td>
			<td>Fisher</td>
			<td>21-402-903</td>
			<td>regular microcentrifuge tube</td>
		</tr>
		<tr>
			<td>Micrococcal Nuclease</td>
			<td>Thermo Fisher Scientific, Affymetrix</td>
			<td>70196Y</td>
			<td>each batch may differ; purchase sufficient amount for experiments and aliquot.</td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td>NSC component</td>
		</tr>
		<tr>
			<td>Normal Rabbit IgG</td>
			<td>Millipore</td>
			<td>12-372</td>
			<td></td>
		</tr>
		<tr>
			<td>Normocin</td>
			<td>Invivogen</td>
			<td>NOL-36-063</td>
			<td>anti-microbial agent, use at 0.1 mg/mL.</td>
		</tr>
		<tr>
			<td>NP-40 (Igepal CA-630)</td>
			<td>Sigma</td>
			<td>18896-50ML</td>
			<td>buffer reagent</td>
		</tr>
		<tr>
			<td>Kimble Kontes Pellet Pestle</td>
			<td>Fisher Scientific</td>
			<td>K749515-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>P8340</td>
			<td>aliquot and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Protinase K</td>
			<td>Sigma-Aldrich</td>
			<td>P2308</td>
			<td>make 10 mg/mL stock in water; aliquot and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td>DNA purification kit</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Fine Science Tools</td>
			<td>10007-16</td>
			<td>for dissection of tumor</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>VWR</td>
			<td>0241-5KG</td>
			<td>buffer reagent</td>
		</tr>
		<tr>
			<td>Sodium Deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>D670-25G</td>
			<td>buffer reagent</td>
		</tr>
		<tr>
			<td>Sodium Dodecyl sulfate (SDS)</td>
			<td>Sigma</td>
			<td>L-4390</td>
			<td>buffer reagent</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Thermo Fisher Scientific</td>
			<td>Bp152-1</td>
			<td>buffer reagent</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP 151-500</td>
			<td>polyethylene glycol octylphenyl ether</td>
		</tr>
		<tr>
			<td>Standard Mini Centrifuge</td>
			<td>Fisherbrand</td>
			<td>12-006-901</td>
			<td>standard mini centrifuge</td>
		</tr>
		<tr>
			<td>SZX16 microscope</td>
			<td>Olympus</td>
			<td>SZX16</td>
			<td>flourescent dissecting microscope</td>
		</tr>
		<tr>
			<td>ViiA 7 Real-Time PCR System with Fast 96-Well Block</td>
			<td>Applied Biosystems</td>
			<td>4453535</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop One</td>
			<td>Thermo-Fisher Scientific</td>
			<td>ND-ONEC-W</td>
			<td></td>
		</tr>
	</tbody>
</table>

native chromatin immunoprecipitation using murine brain tumor neurospheres

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory regions of oncogenes and tumor suppressors can lead to changes in gene expression and play an important role in the pathogenesis of brain tumors. Native chromatin immunoprecipitation (ChIP) is a popular technique that allows the detection of modifications or other proteins tightly bound to DNA. In contrast to cross-linked ChIP, in native ChIP, cells are not treated with formaldehyde to covalently link protein to DNA. This is advantageous because sometimes crosslinking may fix proteins that only transiently interact with DNA and do not have functional significance in gene regulation. In addition, antibodies are generally raised against unfixed peptides. Therefore, antibody specificity is increased in native ChIP. However, it is important to keep in mind that native ChIP is only applicable to study histones or other proteins that bind tightly to DNA. This protocol describes the native chromatin immunoprecipitation on murine brain tumor neurospheres.

A schematic representation of tumor NS generated from a brain tumor where brain tumor cells are Katushka positive is presented in Figure 1. Figure 2 is a schematic representation of the entire ChIP technique. Figure 3 shows the representative results of chromatin from brain tumor NS digested with MNase for 12 min, yielding a majority of mono, di-, and tri- nucleosomes. Following ChIP, a qPCR may be p...

Watch this Scientific Journal Video about Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres at JoVE.com

Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

Epigenetic mechanisms are frequently altered in glioma. Chromatin immunoprecipitation could be used to study the consequences of genetic alterations in glioma that result from changes in histone modifications which regulate chromatin structure and gene transcription. This protocol describes native chromatin immunoprecipitation on murine brain tumor neurospheres.

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory ...

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