This work was supported by a grant from the National Institute of Mental Health (5R01MH071476).

Procedure:
1st Day
1. Homogenize 50-500 mg of frozen post-mortem gray matter tissue with Douncing Buffer.
! CAUTION ! - Human tissue must be handled with care under strict safety conditions. It should be handled at BSL-2 or higher safety standards.
<ol>
<li>Take previously dissected, post-mortem brain from -80&deg;C, dounce them in 5X brain volume of Douncing Buffer, and place in 2.0 mL microcentrifuge tube. Match...

<ol>
	<li>Huang, H. S., Matevossian, A., Jiang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Akbarian+S.+Chromatin+immunoprecipitation+in+postmortem+brain.">Akbarian S. Chromatin immunoprecipitation in postmortem brain.</a> J Neurosci Methods. 156, 284-292 .</li><li>Huang, H. S., Matevossian, A., Whittle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prefrontal+dysfunction+in+schizophrenia+involves+mixed-lineage+leukemia+1-regulated+histone+methylation+at+GABAergic+gene+promoters.">Prefrontal dysfunction in schizophrenia involves mixed-lineage leukemia 1-regulated histone methylation at GABAergic gene promoters.</a> J Neurosci. 27, 11254-11262 .</li><li>O'Neill, L. P., Turner, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoprecipitation+of+native+chromatin:+NChIP.">Immunoprecipitation of native chromatin: NChIP.</a> Methods. 31, 76-82 .</li><li>Spiteri, E., Konopka, G., Coppola, G., 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+the+transcriptional+targets+of+FOXP2,+a+gene+linked+to+speech+and+language,+in+developing+human+brain.">Identification of the transcriptional targets of FOXP2, a gene linked to speech and language, in developing human brain.</a> Am J Hum Genet. 81, 1144-1157 .</li><li>Huang, H. u. a. n. g., Akbarian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GAD1+mRNA+expression+and+DNA+methylation+in+prefrontal+cortex+of+subjects+with+schizophrenia.">GAD1 mRNA expression and DNA methylation in prefrontal cortex of subjects with schizophrenia.</a> PLoS ONE. 2, .</li><li>Stadler, F., Kolb, G., Rubusch, L., Baker, S. P., Jones, E. G., Akbarian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+methylation+at+gene+promoters+is+associated+with+developmental+regulation+and+region-specific+expression+of+ionotropic+and+metabotropic+glutamate+receptors+in+human+brain.">Histone methylation at gene promoters is associated with developmental regulation and region-specific expression of ionotropic and metabotropic glutamate receptors in human brain.</a> J Neurochem. 94, 324-336 .</li></ol>

The protocol outlined here is particularly useful for investigators interested in histone and/or DNA methylation signatures of human brain, because these chromatin markings may be less prone to postmortem artifacts as compared to other types of modifications, including (histone) acetylation and phosphorylation1, 2. The postmortem brain is amenable to the study of mono-nucleosomal preparations; the DNA remains largely attached to the core histones, at least in specimens with representative autolysis intervals (...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Tris-HCl</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>9310</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium Chloride Hexahydrate</td>
<td>&nbsp;</td>
<td>OmniPur</td>
<td>5980</td>
<td>&nbsp;</td>
<tr>
<td>Calcium Chloride</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>C614-3</td>
<td>&nbsp;</td>
<tr>
<td>EDTA, 0.5M Solution, pH8.0</td>
<td>&nbsp;</td>
<td>OmniPur</td>
<td>4055</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride</td>
<td>&nbsp;</td>
<td>Mallinckrodt Chemicals</td>
<td>7581-06</td>
<td>&nbsp;</td>
<tr>
<td>SDS Solution 10% (w/v)</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>161-0416</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>93426</td>
<td>&nbsp;</td>
<tr>
<td>Igepal CA-630</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I-3021</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Deoxycholate </td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D6750-25G</td>
<td>&nbsp;</td>
<tr>
<td>Lithium Chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L9650-100G</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S7277-250G</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Acetate (anhydrous)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S-2889</td>
<td>&nbsp;</td>
<tr>
<td>Nuclease micrococcal from Staphylococcus</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>N3755-200UN</td>
<td>&nbsp;</td>
<tr>
<td>Benzamidine</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>12072</td>
<td>&nbsp;</td>
<tr>
<td>Phenylmethanesulfonylfluoride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P7626-1G</td>
<td>&nbsp;</td>
<tr>
<td>3M DTT</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>43815</td>
<td>&nbsp;</td>
<tr>
<td>Protein G Agarose, Fast Flow</td>
<td>&nbsp;</td>
<td>Upstate</td>
<td>16-266</td>
<td>&nbsp;</td>
<tr>
<td>Sonicated Salmon Sperm DNA Kit</td>
<td>&nbsp;</td>
<td>Stratagene</td>
<td>201190</td>
<td>&nbsp;</td>
<tr>
<td>Proteinase K from Engyodontium album</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P2308</td>
<td>&nbsp;</td>
<tr>
<td>Phenol:Chloroform 1:1</td>
<td>&nbsp;</td>
<td>OmniPur</td>
<td>6810</td>
<td>&nbsp;</td>
<tr>
<td>Glycogen, From Mussels</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1767-1VL</td>
<td>&nbsp;</td>
<tr>
<td>Ethyl Alcohol (200 Proof)</td>
<td>&nbsp;</td>
<td>Pharmco-AAPER</td>
<td>111000200</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
			Solutions:
<ul>
<li>nChIP Douncing Buffer : 10 mM Tris, 4 mM MgCl2, 1 mM CaCl2 adjust pH to 7.5</li>
<li>10x FSB: 50 mM EDTA, 200 mM Tris, 500 mM NaCl adjust pH to 7.5</li>
<li>Low salt washing buffer: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris (pH 8), 150 mM NaCl</li>
<li>High salt washing Buffer: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris (Ph 8), 500 mM NaCl</li>
<li>Lithium Chloride Buffer: 1% IGEPAL-CA 630, 1% Deoxycholic acid, 1 mM EDTA (pH 8), 10 mM Tris (pH 8), 0.25 M LiCl</li>
<li>TE buffer: 10 mM Tris (pH 8), 1 mM EDTA</li>
<li>Elution Buffer: 0.1M NaHCO3, 1% SDS</li>
<li>Proteinase K digestion buffer (10X): 1M Tris, 50 mM EDTA, 2% SDS, 2M NaCl adjust pH to 8.0</li>
<li>3 M Sodium Acetate: 24.61 g anhydrous sodium acetate (M.W.=82.03) in 100 mL of autoclaved distilled water. Adjust pH to 5.2 using concentrated acetic acid.</li>
</ul>		</div>

a chromatin assay for human brain tissue

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental factors that might result in epigenetic alterations of gene expression and other molecular pathology. Traditionally, however, expression studies in postmortem brain were confined to quantification of mRNA or protein. The limitations encountered in postmortem brain research   such as variabilities in autolysis time and tissue integrities   are also likely to impact any studies of higher order chromatin structures. However, the nucleosomal organization of genomic DNA   including DNA:core histone binding - appears to be largely preserved in representative samples provided by various brain banks. Therefore, it is possible to study the methylation pattern and other covalent modifications of the core histones at defined genomic loci in postmortem brain. Here, we present a simplified native chromatin immunoprecipitation (NChIP) protocol for frozen (never-fixed) human brain specimens. Starting with micrococcal nuclease digestion of brain homogenates, NChIP followed by qPCR can be completed within three days. The methodology presented here should be useful to elucidate epigenetic mechanisms of gene expression in normal and diseased human brain.

Watch this Scientific Journal Video about A Chromatin Assay for Human Brain Tissue at JoVE.com

A Chromatin Assay for Human Brain Tissue

Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

Research

JoVE Journal

Biology

12.8K Görüntüleme.  University of Massachusetts Medical School. Yakın zamana kadar, insan beyni ifade çalışmaları RNA veya protein ölçümü ile sınırlı kalmıştır. Bu yazıda anlatılan kromatin immunoprecipitation teknikleri, postmortem beyin gen ekspresyonu histon metilasyon ve diğer epigenetik düzenleyiciler haritaya mümkün olacaktır.