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. Matched sample and control pairs are processed simultaneously.</li>
</ol>
2. Micrococcal Nuclease (MN) Digestion
<ol>
<li>Add 5U/mL of Micrococcal nuclease to the sample and mix within the solution by pipetting up and down before placing on ice. *&nbsp;CRITICAL STEP -&nbsp;It is important to do this step quickly since MN has the ability to act at even 4&deg;C.</li>
<li>Incubate samples for 7 minutes at 37&deg;C.</li>
<li>After the 7 minute incubation, add 0.5M EDTA to a concentration of 10mM to stop the MNase activity.</li>
</ol>
3. Hypotonisation
<ol>
<li>Place samples into a 15 mL falcon tube. Add 10X the sample volume of 0.2mM EDTA, 1/2000 sample volume of 0.2M benzamidine and 1/1000 sample volume of 0.1M phenylmethanesulphonylfluoride (PMSF). The latter two compounds are used as Protease Inhibitors. *&nbsp;CRITICAL STEP -&nbsp;It is important to keep the samples on ice during all these steps.</li>
<li>Incubate sample for 1 hour, while vortexing it every 10 minutes.</li>
<li>At the end of the hour long incubation, add 1/2000 sample volume of 3M DTT, yet another Protease inhibitor.</li>
<li>Vortex sample once more and centrifuge at 3175 RCF for 10 minutes at 4&deg;C. *&nbsp;OPTIONAL STEP - Precleansing with Protein G Agarose.<ol>
<li>Take supernatant and put in new 15 mL falcon tube.</li>
<li>Add 500 &micro;L of Protein G Agarose.</li>
<li>Rotate at room temperature for 30 min.</li>
<li>Centrifuge at 4000 rpm for 10 min at 4&deg;C.</li>
</ol></li>
<li>Distribute supernatant so that 500 &micro;L are used as Input control (containing only genomic DNA), and the rest is split into two tubes containing 1600 &micro;L of sample each -- which are for chromatin immunoprecipitation (ChIP) samples.</li>
<li>The Input control is placed at -80&deg;C O/N until further use.</li>
<li>To the ChIP samples - add 1:10 volume of 10XFSB and 4&mu;g of antibody, then vortex the samples and rotate them at 4&deg;C overnight. ! CAUTION ! - Amount and dilution of antibody may require optimization.</li>
</ol>
2nd Day
! CAUTION ! -&nbsp;Begin 2nd day by washing the Protein G Agarose that will be used to isolate nucleosomal DNA. Since agarose beads are very sensitive, it is necessary to cut off the heads of the tips whenever pipetting any solution containing agarose beads.
1. Probing Protein G Agarose Beads to DNA
<ol>
<li>Add 1.6 mL 1XFSB to 245 &micro;L protein G-agarose (enough for 4 tubes) in a 2 mL microcentrifuge tube (loop).</li>
<li>Separate the solution into two 2 mL tubes and refill each up to 1.6 mL with 1X FSB.</li>
<li>Rotate at RT for 30 sec and centrifuge at 0.1 RCF for 30 sec.</li>
<li>Remove the supernatant using a vacuum.</li>
<li>Add 1.6 mL 1X FSB once again. Rotate the samples for 30 sec and then centrifuge at 0.1 RCF for 30 sec.</li>
<li>Remove the supernatant once again, and combine both tubes with 1.5 mL 1XFSB.</li>
<li>Add 15 &micro;L sonicated Salmon sperm DNA (10mg/mL). ! CAUTION ! - This step should, in principle, reduce non-specific binding of the immunoprecipitate to the beads. However, this also could lead to false positives for some of the (human) DNA sequences.</li>
<li>Rotate at R.T. for 30 min, then centrifuge at 0.1 RCF for 30 sec.</li>
<li>Remove the supernatant. Add 200 &mu;L of 1XFSB.</li>
<li>Add 90 &micro;L of agarose beads into each ChIP sample. Rotate at 4&deg;C for 1 hr.</li>
<li>Add 1mL of 1XFSB into remaining agarose beads to serve as a negative control and rotate at 4&deg;C for 1 hr.&nbsp;</li>
<li>After the hour long incubation, centrifuge the samples and negative control at 0.1 RCF for 30 sec. Discard the supernatant.</li>
</ol>
2. Washing the Beads
! CAUTION ! -&nbsp;Washing buffers must be kept at 4&deg;C until use, and are to be used only if are less than a month old.
<ol>
<li>Add 1 mL of each washing solution to the beads and rotate for 3 min at RT.</li>
<li>Centrifuge at 0.1 RCF for 30 sec.&nbsp;</li>
<li>Discard supernatant using vacuum.</li>
</ol>
* Each washing solution
-- Low salt washing buffer
-- High salt washing buffer
-- Lithium chloride solution - only rotate at RT for 1 minute!
-- TE buffer (10 mM Tris, 1 mM EDTA pH=8)
3. Elution
*&nbsp;CRITICAL STEP - Make fresh Elution Buffer for every experiment on the day it is to be used.
<ol>
<li>Add 250 &micro;L of freshly prepared elution buffer to each sample.</li>
<li>Rotate for 15 min at RT, then centrifuge at 0.4 RCF for 1 min.</li>
<li>Save the supernatant in a 2.0 mL microcentrifuge tube (Loop).</li>
<li>Add 250 &micro;L of elution buffer to each sample and vortex by hand for a few seconds. Then, vortex samples for 15 minutes on a mulitvortexer.</li>
<li>Centrifuge at 16 RCF rpm for 4 minutes and save supernatant in same 2.0 mL tube (Loop).</li>
</ol>
4. Digest Protein
<ol>
<li>Add 10 &mu;l 0.5M EDTA, 25 &mu;l 0.8M Tris-HCl, pH 6.5, 10 mg/ml Proteinase K (1/200 sample) to each ChIP sample.</li>
<li>To each Input control, add lysis buffer for proteinase K digestion (1/10 of sample buffer) and 10mg/ml Proteinase K (1/200 of sample buffer).</li>
<li>Incubate Input and ChIP samples in 52&deg;C for at least 3 hrs.</li>
</ol>
5. Phenol/Chloroform extraction
! CAUTION ! - Experiment requiring phenol/chloroform should be performed under the hood. Use nitrile gloves when handling phenol/chloroform.
<ol>
<li>After the 3 hour incubation, we add the 500 &mu;l phenol-chloroform to each sample.</li>
<li>Vortex all samples for several seconds, then centrifuge at 13 RCF rpm for 5 min.</li>
<li>At this point, two phases will be present and we are interested in the content of the top phase.&nbsp; Take out the top phase and put it into a 2 mL microcentrifuge tube.</li>
<li>Add a mixture of 2&micro;L of Glycogen and 50&micro;L of 3M sodium acetate to each sample along with 1.375 mL of 100% Ethanol.</li>
<li>Vortex all samples vigorously. Place them in -80&deg;C overnight.</li>
</ol>
3rd Day
<ol>
<li>Remove samples form -80&deg;C and place them on ice for them to thaw.</li>
<li>Centrifuge at 15 RCF for 10 minutes at 4&deg;C.</li>
<li>Carefully remove supernatant without disturbing the pellet at the bottom of the tube.</li>
<li>Add 1 mL of cold 75% Ethanol to each sample, &nbsp;then invert them 4-6 times.</li>
<li>Centrifuge at 18 RCF for 5 min at 4&deg;C.</li>
<li>Remove supernatant once more and allow the pellets to air dry.</li>
<li>Dissolve dried pellets in 50 &micro;L of 4mM Tris-HCl, pH8 and store at -80&deg;C until further use.</li>
</ol>

<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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		<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 visualizações.  University of Massachusetts Medical School. Até recentemente, estudos de expressão no cérebro humano eram limitados a quantificação de RNA ou proteína. Com as técnicas de imunoprecipitação de cromatina descritas neste documento, será possível mapear a metilação da histona e outros reguladores epigenética da expressão gênica no cérebro pós-morte.

Vídeo: A Chromatin Assay for Human Brain Tissue

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 tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind 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 characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

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, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan.  Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays. 

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

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

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

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

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

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 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 ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

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)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

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

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

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

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

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

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

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

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...