Name: a chromatin assay for human brain tissue
Uploaded: 2008-03-21T19:19:00
Description: Watch this Scientific Journal Video about A Chromatin Assay for Human Brain Tissue at JoVE.com

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 (...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<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

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

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 ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Kromatin Immunoprecipitation (chip) ved hjelp Drosophila Vev

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

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

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

Generasjon av høy kvalitet Chromatin immunoprecipitation DNA Mal for High-throughput sekvensering (ChIP-seq)

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

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

Generasjon og rensing av menneske INO80 Chromatin remodeling komplekser og Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

En metode for å velge Struktur-bytte aptamerer Anvendt til en Colorimetric Gold Nanopartikkel analysen

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin Immunutfelling analyse for identifikasjon av Arabidopsis Protein-DNA interaksjoner<em&gt; I Vivo</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...