This work was supported by NIH R01 GM56244 to ANI, which includes funds awarded through the American Recovery and Reinvestment Act of 2009, and by NIH R01 GM87130 to JARP

1. Isolation of Embryos
Note: All operations involving mice should be performed in accordance with the appropriate animal care and usage policies and protocols
<ol>
	<li>Check for the presence of a mating plug in the female mouse the morning after mating and separate the mated females from the stud males by placing them in a different cage. Noon of the day that the mating plug is observed is considered embryonic day 0.5 (E0.5) of development.</li>
	<li>At E8.5, (or the desired stage, if different), sacrifice the mouse using an approved protocol.</li>
	<li>Wet the belly of the euthanized animal with 70% ethanol and open the abdominal cavity. The implantation sites indicating the presence of developing embryos are seen as a "beads on a string" arrangement along the length of each uterine horn (Figure 1A). Using scissors, remove both uterine horns, cut each implantation site individually to separate (Figure 1B) and place them in a 100 mm petri dish containing room temperature (RT) dissection medium (DMEM, 10% fetal bovine serum, 20 mM HEPES pH 7.4, 60 &mu;g/ml penicillin, 2 mM streptomycin).</li>
	<li>Using forceps, remove each embryo and the surrounding membranes from the uterine tissue (Figure 1C) and place them in fresh dissection medium.</li>
	<li>Isolate individual embryos under a dissecting microscope using forceps. At this stage in development, the embryos are surrounded by the parietal yolk sac, which contacts the deciduum tissue, the visceral yolk sac and the amnion. The amnion is a transparent membrane that is in direct contact with the embryo. The visceral yolk sac is located between the amnion and the parietal yolk sac and is readily distinguished in E8.5-E9.5 embryos by the presence of prominent blood vessels that nurture the embryo. Remove each embryo from its surrounding membranes and transfer individually into a new plate containing dissection media to remove excess blood. The developmental stage can vary from embryo to embryo and between litters; representative E8.5 embryos and a representative E9.5 embryo are shown in Figure 1D.</li>
	<li>Transfer each embryo to a 1.5 ml eppendorf tube containing 200 &mu;l of dissection media (described in step 4 above).</li>
</ol>
2. Homogenization of the Isolated Embryo
<ol>
	<li>Add 20 units of Collagenase type II in a volume of 200 ul (diluted in 1X Dubbecco's Phosphate Buffered Saline; DPBS) to each eppendorf tube.</li>
	<li>Gently shake samples in a 37&deg;C shaker at 100 rpm for 20 min.</li>
	<li>Resuspend well by pipetting to disrupt clumps of cells.</li>
	<li>Apply the cell suspension on the top of sterile cell strainer (40 &mu;m mesh size) placed over a 1.5 ml eppendorf tube.</li>
	<li>Immediately apply 600 &mu;l of room temperature 1X DPBS on top of the cell strainer to complete the separation. Discard the strainer.</li>
	<li>Centrifuge samples at 4&deg;C at 4000 x g for 5 min.</li>
	<li>Discard the supernatant.</li>
	<li>Resuspend the pellet in 1 ml of RT 1X DPBS.</li>
	<li>Centrifuge the samples at 4000 x g for 1 min at RT.</li>
	<li>Discard the supernatant.</li>
	<li>Resuspend the pellet in 200 &mu;l of RT dissection media.</li>
	<li>Count the embryonic cells. There is an average 3-5 x 106 cells/E8.5 embryo.</li>
</ol>
3. Cross-link Chromatin
<ol>
	<li>Add 5.6 &mu;l of 37% formaldehyde to the 200 &mu;l samples for a final concentration of 1% formaldehyde.</li>
	<li>Incubate samples for 10 min at RT.</li>
	<li>Centrifuge the samples at 4&deg;C at 4000 x g for 3 min.</li>
	<li>Discard the supernatant.</li>
	<li>Resuspend the cells with cold 1X DPBS containing 80 &mu;l/ml protease inhibitor cocktail (PIC).</li>
	<li>Centrifuge samples at 4&deg;C at 4000 x g for 3 min.</li>
	<li>Discard the supernatant. At this step, the samples can be frozen at -80&deg;C. The frozen, crosslinked samples are stable for several months.</li>
</ol>
4. Sonication
<ol>
	<li>Resuspend the cell pellet in 100 &mu;l of RT SDS lysis buffer (50 mM Tris-HCl (pH 8.1), 10 mM EDTA and 1% SDS with freshly added PIC at 1 &mu;l PIC/800 &mu;l total volume). When resuspended, add an additional 100 ul of RT SDS lysis buffer to promote resuspension. Mix well by pipetting and inversion.</li>
	<li>Incubate the cell suspension on ice for 10 min.</li>
	<li>Sonicate lysed cells to shear DNA to between 200 and 500 basepairs using a Diagenode Bioruptor&trade; UCD200 (5 min x 8 times: amplitude setting of 30 sec on/30 sec off on high (320 watts) power). The samples should be surrounded by an ice slurry. Do not allow the samples to warm above 4&deg;C or to freeze. There are many types of sonicators available. Sonication conditions should be optimized for each sonicator to result in shearing of the cross-linked DNA to a length of approximately 500 bp.</li>
	<li>Centrifuge sonicated samples at 4&deg;C at 14000 x g for 10 min.</li>
	<li>Transfer the supernatant into a fresh eppendorf tube pre-cooled on ice.</li>
	<li>Divide the sonicated sample into a maximum of 5 aliquots. We have empirically determined that one E8.5 embryo can be divided into 5 aliquots. Smaller or larger sample sizes can be scaled accordingly. At this step, the sample can be stored at -80&deg;C until further use.</li>
	<li>Reserve one aliquot for use as input and store at -20&deg;C until step 6 when the cross-links will be reversed. Dilute the other aliquots 10-fold in ChIP dilution buffer (16.7 mM Tris-HCl (pH 8.1), 1.2 mM EDTA, 1.1% Triton X-100, 167 mM NaCl and 0.01% SDS) containing freshly added PIC at 1 &mu;l/800 &mu;l buffer.</li>
</ol>
5. Pre-clearing and Immunoprecipitation
<ol>
	<li>Pre-clear the diluted supernatant with 75 &mu;l of Salmon-Sperm DNA/Protein A or G Agarose-50% slurry at 4&deg;C, with rotation, for 1 hr. Use of either Protein A or Protein G beads should be determined by the antibody to be used for the ChIP. For example, protein A beads are recommended for rabbit antibodies, whereas protein G beads are recommended for goat antibodies or mouse IgG1 antibodies. For more detailed information, see the recommendations made by the bead manufacturer.</li>
	<li>Centrifuge at 4&deg;C at 2500 x g for 1 min.</li>
	<li>Transfer the supernatant to a new, pre-cooled eppendorf tube.</li>
	<li>Add antibody (4 &mu;g/sample) to the pre-cleared aliquot and incubate overnight at 4&deg;C with rotation.</li>
</ol>
6. Washing the Chromatin-protein-bead Complex
<ol>
	<li>Add 60 &mu;l of Salmon Sperm DNA/Protein A or G agarose slurry into each vial and incubate at 4&deg;C with rotation for 1 hr. Use the same bead type used for pre-clearing in step 3 above.</li>
	<li>Centrifuge at 4&deg;C at 1000 x g for 1 min.</li>
	<li>Carefully remove the supernatant and discard. Keep the DNA-protein-bead complex on ice.</li>
	<li>Resuspend and wash the pellet as directed below with wash buffers 1, 2, 3, and 4. Wash buffers 1-3 should be chilled to 4&deg;C and washes with these buffers should be performed at 4&deg;C. Wash buffer 4 should be at room temperature and washes with this buffer should be performed at room temperature. Each wash is for 5 minutes with rotation at the appropriate temperature. Following each wash, centrifuge at 4&deg;C (room temperature for wash buffer 4 washes) at 1000 x g for 1 min, then carefully aspirate or remove the supernatant.</li>
</ol>
Buffer 1:Low salt wash buffer (20 mM Tris-HCl (pH 8.1), 2 mM EDTA, 1% Triton X-100, 
150 mM NaCl and 0.1% SDS), 1 ml x 2 washes. 
Buffer 2:High salt wash buffer (20 mM Tris-HCl (pH 8.1), 2 mM EDTA, 1% Triton X-100, 
500 mM NaCl and 0.1% SDS), 1 ml x 2 washes. 
Buffer 3:LiCl salt wash buffer (10 mM Tris-HCl (pH 8.1), 1 mM EDTA, 1% IGEPAL-CA630, 
0.25 M LiCl and 1% deoxycholic acid (sodium salt)), 1 ml x 1 wash. 
Buffer 4:TE buffer (10 mM Tris-HCl (pH 8.1), 1 mM EDTA), 1 ml x 2 washes.
7. Elution of the Chromatin-antibody Complex
<ol>
	<li>Resuspend the washed chromatin-protein-bead complex in 250 &mu;l of freshly prepared elution buffer (1% SDS, 0.1 M NaHCO3). Vigorously vortex the sample for 5 sec, then incubate at RT for 15 min with rotation.</li>
	<li>Centrifuge at 2500 x g for 1 min at RT and transfer the eluate into a new eppendorf tube.</li>
	<li>Repeat steps 1 and 2 and combine the eluates in the same eppendorf tube. The eluates should be kept at room temperature. Upon completion of this step, the eluates can be stored at -20&deg;C until further use.</li>
</ol>
8. Reverse Cross-link and Recover DNA
<ol>
	<li>Add 20 &mu;l 5 M NaCl for 500 &mu;l eluate (40 &mu;l/ml for input control).</li>
	<li>Heat the eluates at 65&deg;C in a water bath for 4 hr to overnight.</li>
	<li>10 &mu;l of each sample (including the input control) can be run on a 2% agarose gel next to size markers spanning 0.1 to 1 kbp to check the DNA fragment size. The DNA will appear as a smear and not a sharp band. The rest of the samples can be left at 65&deg;C while the gel is running.</li>
	<li>Recover the DNA from each sample using a QIAquick Gel Extraction Kit (Qiagen). Add 600 &mu;l QG buffer (supplied in the kit) to optimize DNA adsorption with the silica membrane in a QIAquick spin column and 200 &mu;l isopropanol into a 500 &mu;l sample. Note that QG buffer contains a pH indicator allowing easy determination of the pH of the sample. If the color of the sample turns from yellow to purple (pH &gt; 7.5) after mixing, add 10 &mu;l of 3 M NaAcetate (pH 5.2) to the sample and mix. This reduces the pH of the mixture to maximize DNA binding to the beads in the column.</li>
	<li>Prior to loading the column, check the mixture to determine whether any SDS precipitate is present. If SDS precipitation has occurred, the sample should be warmed in a 42&deg;C water bath until the precipitate disappears.</li>
	<li>Load 700 &mu;l of the mixture into the QIAquick spin column (purple column in the kit) and centrifuge at RT at 14000 x g for 1 min. Discard the flow through from the column before proceeding and repeat this step with the remainder of the sample.</li>
	<li>Add 500 &mu;l QG buffer to wash the column. Centrifuge at RT at 14000 x g for 2 min. Discard the flow through from the column before proceeding.</li>
	<li>Wash the column with 700 &mu;l of PE wash buffer (supplied in the kit) to which ethanol has been added as instructed by the manufacturer. Centrifuge at RT at 14000 x g for 1 min. Discard the flow through from the column before proceeding.</li>
	<li>Repeat the centrifugation step to completely remove the remainder of the PE buffer from the column. Discard the flow through and the collection tube.</li>
	<li>Elute DNA from the column into a fresh eppendorf tube by adding 60 &mu;l EB buffer (elution buffer supplied in the kit).</li>
	<li>Centrifuge at room temperature at 14000 x g for 1 min. The eluted DNA can be stored at -20&deg;C until detection. A260 readings of the recovered material typically indicate concentrations of 90-120 ng/ul, for a total recovery of ~6-7 ug per aliquot. However, the A260/280 ratio of these samples is typically &gt;1.8, suggesting that RNA is present in the sample. Thus the exact amount of recovered DNA may be lower.</li>
</ol>
9. Analysis of Recovered DNA
<ol>
	<li>SDS can interfere with the activity of PCR Taq polymerase. SDS should be removed completely before performing PCR. Incubate the DNA on ice to precipitate any residual SDS and centrifuge at 4&deg;C at 14000 x g for 1 min. Transfer the supernatant to a new eppendorf tube.This step is strongly recommended.</li>
	<li> The DNA eluates can be detected either by conventional PCR or by quantitative real-time PCR (Q-PCR) with primers specific for each sequence to be analyzed. 5-10 &mu;l from total DNA eluate may be used for one PCR or Q-PCR reaction. PCR with the input control can be performed with a 5-10 fold dilution of the DNA compared with the amount of other samples. PCR and Q-PCR conditions will vary, however, the limited amount of starting material requires additional PCR cycles. For conventional PCR detection, we recommend starting with 40 cycles and adjusting as needed.</li>
</ol>
10. Representative Results
We have used this protocol to perform ChIP from both E8.5 and E9.5 embryos (Figure 2). The results demonstrate that myogenin is present on the myogenin promoter in E8.5 and E9.5 embryos. ChIP purified DNAs were analyzed by conventional PCR (Figure 2A) and by quantitative real-time PCR (Figure 2B). In contrast, there was no indication of myogenin binding to the myogenin promoter in the yolk sac, where myogenin is not expressed. The interferon-&gamma; (IFN&gamma;) promoter, which contains sequences matching the myogenin binding site, was used as a negative sequence control. As expected, myogenin was not bound to the IFN&gamma; promoter in any of the tissue samples tested. In E8.5 and 9.5 embryos, myogenin is specifically expressed in the somites (Figure 3; 6,10), which are the precursors to skeletal muscle. Thus the results indicate that myogenin is bound to the myogenin promoter in the somites at E8.5 and E9.5.
<img src="/files/ftp_upload/2677/2677fig1.jpg" alt="Figure 1" /> 
Figure 1. E8.5 embryo dissection. (A) Isolated uterine horn (top panel; higher magnification shown in lower panel). (B) Uterine horn cut with scissors to separate individual implantation sites containing individual embryos. (C) Embryos protruding from uterine tissue during dissection. Arrows mark embryos still covered by extra-embryonic tissue. (D) Two E8.5 embryos from the same litter. The embryo on the left has not begun the process of turning. The embryo on the right is undergoing turning. Far right - representative E9.5 embryo.
<img src="/files/ftp_upload/2677/2677fig2.jpg" alt="Figure 2" /> 
Figure 2. ChIP assay demonstrating myogenin binding to the myogenin promoter in E8.5 and E9.5 embryos. (Top) Conventional PCR analysis of 5 ul of DNA purified from ChIP experiments using a myogenin antibody or non-specific IgG was performed with primers that amplify a portion of the myogenin promoter from -79 to +69 relative to the start site of transcription that contains a myogenin binding site located at -12 or primers that amplify a portion of the IFN&gamma; promoter that contains a sequence matching the myogenin binding site located ~ 1075 bp upstream of the transcription start site. The IFN&gamma; primer sequences used were 5'-GCT GAC TCA AGA CCC CGA GGC-3' and 5'-TGA GGA TGG GGC AGG AGG CC-3'. (Bottom) Quantitative Real-time PCR analysis of the same samples used in (A). The data are plotted as % of input +/- standard deviation.
<img src="/files/ftp_upload/2677/2677fig3.jpg" alt="Figure 3" /> 
Figure 3. Myogenin is specifically expressed in the somites of E8.5 and E9.5 embryos. Whole mount in situ hybridization of myogenin shows specific mRNA expression in the somites. Size bar in E8.5 image - 200 &mu;m. Size bar in E9.5 image - 500 &mu;m.

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+four+human+muscle+regulatory+helix-loop-helix+proteins+Myf3-Myf6+exhibit+similar+hetero-dimerization+and+DNA+binding+properties.">The four human muscle regulatory helix-loop-helix proteins Myf3-Myf6 exhibit similar hetero-dimerization and DNA binding properties.</a> Nucleic Acids Res. 19, 5645-5651 (1991).</li><li>Serna, d. e. l. a., L, I., Ohkawa, Y., Berkes, C. A., Bergstrom, D. A., Dacwag, C. S., Tapscott, S. J., Imbalzano, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MyoD+targets+chromatin+remodeling+complexes+to+the+myogenin+locus+prior+to+forming+a+stable+DNA-bound+complex.">MyoD targets chromatin remodeling complexes to the myogenin locus prior to forming a stable DNA-bound complex.</a> Mol Cell Biol. 25, 3997-4009 (2005).</li><li>Blais, A., Tsikitis, M., Acosta-Alvear, D., Sharan, R., Kluger, Y., Dynlacht, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+initial+blueprint+for+myogenic+differentiation.">An initial blueprint for myogenic differentiation.</a> Genes Dev. 19, 553-569 (2005).</li><li>Cao, Y., Kumar, R. M., Penn, B. H., Berkes, C. A., Kooperberg, C., Boyer, L. A., Young, R. A., Tapscott, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+and+gene-specific+analyses+show+distinct+roles+for+Myod+and+Myog+at+a+common+set+of+promoters.">Global and gene-specific analyses show distinct roles for Myod and Myog at a common set of promoters.</a> EMBO J. 25, 502-511 (2006).</li><li>Ohkawa, Y., Yoshimura, S., Higashi, C., Marfella, C. G., Dacwag, C. S., Tachibana, T., Imbalzano, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myogenin+and+the+SWI/SNF+ATPase+Brg1+maintain+myogenic+gene+expression+at+different+stages+of+skeletal+myogenesis.">Myogenin and the SWI/SNF ATPase Brg1 maintain myogenic gene expression at different stages of skeletal myogenesis.</a> J Biol Chem. 282, 6564-6570 (2007).</li><li>Davie, J. K., Cho, J. H., Meadows, E., Flynn, J. M., Knapp, J. R., Klein, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+gene+selectivity+of+the+myogenic+basic+helix-loop-helix+transcription+factor+myogenin+in+embryonic+muscle.">Target gene selectivity of the myogenic basic helix-loop-helix transcription factor myogenin in embryonic muscle.</a> Dev Biol. 311, 650-664 (2007).</li><li>Metivier, R., Penot, G., Hubner, M. R., Reid, G., Brand, H., Kos, M., Gannon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+receptor-alpha+directs+ordered,+cyclical,+and+combinatorial+recruitment+of+cofactors+on+a+natural+target+promoter.">Estrogen receptor-alpha directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter.</a> Cell. 115, 751-763 (2003).</li><li>Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., 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=.">.</a> Current Protocols in Molecular Biology. , (2010).</li></ol>

Experiments on animals were performed in accordance with the guidelines and regulations set forth by the University of Massachusetts Medical School IACUC.

In the described ChIP protocol, we show that the myogenic regulator myogenin is associated with the myogenin promoter in skeletal muscle precursor tissue present in single E8.5 and E9.5 embryos. Prior studies have extensively characterized myogenin binding to E box containing sequences, beginning with the initial in vitro gel shift experiments utilizing in vitro translated or bacterially produced myogenin and radiolabeled DNA encoding the relevant portion of target gene regulatory sequences 11-20</...

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<td>ChIP Assay Kit</td>
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<td>Upstate Cell Signaling Solutions, Millipore</td>
<td>17-295</td>
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<td>Collagenase Type II</td>
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<td>Dilution by 1 x PBS</td>
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<td>Dulbecco&#x2019;s modified eagle medium (DMEM) </td>
<td>&nbsp;</td>
<td>Gibco Labs, Invitrogen</td>
<td>12100-061</td>
<td>High glucose content</td>
</tr>
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<td>Dulbecco&#x2019;s phosphate buffered saline 1X (DPBS)</td>
<td>&nbsp;</td>
<td>Gibco Labs, Invitrogen</td>
<td>14190-144</td>
<td>Calcium chloride free, Magnesium chloride free</td>
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<td>Fetal bovine serum (FBS)</td>
<td>&nbsp;</td>
<td>Mediatech, Inc.</td>
<td>35-010-CV</td>
<td>&nbsp;</td>
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<td>Gel extraction kit</td>
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<td>Penicillin/streptomycin stock solution</td>
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<td>Gibco Labs, Invitrogen</td>
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<td>Millipore</td>
<td>16-157</td>
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<td>myogenin antibody</td>
<td>&nbsp;</td>
<td>Santa Cruz Biotechnology, Inc.</td>
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<td>Normal rabbit IgG</td>
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<td>Platinum PCR Supermix</td>
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<td>&nbsp;</td>
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<td>GoTaq Q-PCR master mix</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>A6001</td>
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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.

Watch this Scientific Journal Video about Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos at JoVE.com

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse 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 ...

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18.0K Views.  University of Massachusetts Medical School. 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.

Video: Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

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.

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 ...

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 ...

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 ...

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. 
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. 
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.

A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

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 ...

Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

Chromatin immunoprecipitation (ChIP) is a widely-used method for determining the interactions of different proteins with DNA in chromatin of living cells. Examples include sequence-specific DNA binding transcription factors, histones and their different modification states, enzymes such as RNA polymerases and ancillary factors, and DNA repair components. Despite its ubiquity, there is a lack of up-to-date, detailed methodologies for both bench preparation of material and for accurate analysis allowing quantitative metrics of interaction. Due to this lack of information, and also because, like any immunoprecipitation, conditions must be re-optimized for new sets of experimental conditions, the ChIP assay is susceptible to inaccurate or poorly quantitative results.
Our protocol is ultimately derived from seminal work on transcription factor:DNA interactions1,2 , but incorporates a number of improvements to sensitivity and reproducibility for difficult-to-obtain cell types. The protocol has been used successfully3,4 , both using qPCR to quantify DNA enrichment, or using a semi-quantitative variant of the below protocol.
This quantitative analysis of PCR-amplified material is performed computationally, and represents a limiting factor in the assay. Important controls and other considerations include the use of an isotype-matched antibody, as well as evaluation of a control region of genomic DNA, such as an intergenic region predicted not to be bound by the protein under study (or anticipated not to show changes under the experimental conditions). In addition, a standard curve of input material for every ChIP sample is used to derive absolute levels of enrichment in the experimental material. Use of standard curves helps to take into account differences between primer sets, regardless of how carefully they are designed, and also efficiency differences throughout the range of template concentrations for a single primer set. Our protocol is different from others that are available5-8 in that we extensively cover the later, analysis phase.

We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.

Chromatin immunoprecipitation (ChIP) is a  widely-used method for determining the interactions of different proteins with  DNA in chromatin of living ...

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 ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...