Name: chromatin immunoprecipitation assay for tissue-specific genes using early-stage mouse embryos
Uploaded: 2011-04-29T16:00:00
Description: Watch this Scientific Journal Video about Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos at JoVE.com

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 desi...

<ol>
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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+resides+in+the+nucleus+and+acquires+high+affinity+for+a+conserved+enhancer+element+on+heterodimerization.">Myogenin resides in the nucleus and acquires high affinity for a conserved enhancer element on heterodimerization.</a> Genes Dev. 4, 582-595 (1990).</li><li>Rosenthal, N., Berglund, E. B., Wentworth, B. M., Donoghue, M., Winter, B., Bober, E., Braun, T., Arnold, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+conserved+enhancer+downstream+of+the+human+MLC1/3+locus+is+a+target+for+multiple+myogenic+determination+factors.">A highly conserved enhancer downstream of the human MLC1/3 locus is a target for multiple myogenic determination factors.</a> Nucleic Acids Res. 18, 6239-6246 (1990).</li><li>Braun, T., Gearing, K., Wright, W. E., Arnold, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculovirus-expressed+myogenic+determination+factors+require+E12+complex+formation+for+binding+to+the+myosin-light-chain+enhancer.">Baculovirus-expressed myogenic determination factors require E12 complex formation for binding to the myosin-light-chain enhancer.</a> Eur J Biochem. 198, 187-193 (1991).</li><li>Chakraborty, T., Brennan, T., Olson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+trans-activation+of+a+muscle-specific+enhancer+by+myogenic+helix-loop-helix+proteins+is+separable+from+DNA+binding.">Differential trans-activation of a muscle-specific enhancer by myogenic helix-loop-helix proteins is separable from DNA binding.</a> J Biol Chem. 266, 2878-2882 (1991).</li><li>French, B. A., Chow, K. L., Olson, E. N., Schwartz, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterodimers+of+myogenic+helix-loop-helix+regulatory+factors+and+E12+bind+a+complex+element+governing+myogenic+induction+of+the+avian+cardiac+alpha-actin+promoter.">Heterodimers of myogenic helix-loop-helix regulatory factors and E12 bind a complex element governing myogenic induction of the avian cardiac alpha-actin promoter.</a> Mol Cell Biol. 11, 2439-2450 (1991).</li><li>Brennan, T. J., Chakraborty, T., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutagenesis+of+the+myogenin+basic+region+identifies+an+ancient+protein+motif+critical+for+activation+of+myogenesis.">Mutagenesis of the myogenin basic region identifies an ancient protein motif critical for activation of myogenesis.</a> Proc Natl Acad Sci U S A. 88, 5675-5679 (1991).</li><li>Lassar, A. B., Davis, R. L., Wright, W. E., Kadesch, T., Murre, C., Voronova, A., Baltimore, D., Weintraub, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+activity+of+myogenic+HLH+proteins+requires+hetero-oligomerization+with+E12/E47-like+proteins+in+vivo.">Functional activity of myogenic HLH proteins requires hetero-oligomerization with E12/E47-like proteins in vivo.</a> Cell. 66, 305-315 (1991).</li><li>Chakraborty, T., Brennan, T. J., Li, L., Edmondson, D., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inefficient+homooligomerization+contributes+to+the+dependence+of+myogenin+on+E2A+products+for+efficient+DNA+binding.">Inefficient homooligomerization contributes to the dependence of myogenin on E2A products for efficient DNA binding.</a> Mol Cell Biol. 11, 3633-3641 (1991).</li><li>Cserjesi, P., Olson, E. 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+induces+the+myocyte-specific+enhancer+binding+factor+MEF-2+independently+of+other+muscle-specific+gene+products.">Myogenin induces the myocyte-specific enhancer binding factor MEF-2 independently of other muscle-specific gene products.</a> Mol Cell Biol. 11, 4854-4862 (1991).</li><li>Braun, T., Arnold, H. 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. 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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=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. 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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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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>ChIP Assay Kit</td>
<td>&nbsp;</td>
<td>Upstate Cell Signaling Solutions, Millipore</td>
<td>17-295</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase Type II</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17101015</td>
<td>Dilution by 1 x PBS</td>
</tr>
<tr>
<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>
<tr>
<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>
</tr>
<tr>
<td>Fetal bovine serum (FBS)</td>
<td>&nbsp;</td>
<td>Mediatech, Inc.</td>
<td>35-010-CV</td>
<td>&nbsp;</td>
<tr>
<td>Gel extraction kit</td>
<td>&nbsp;</td>
<td>QIAquick</td>
<td>28704</td>
<td>50 reaction kit</td>
</tr>
<tr>
<td>Penicillin/streptomycin stock solution</td>
<td>&nbsp;</td>
<td>Gibco Labs, Invitrogen</td>
<td>&nbsp;</td>
<td>5000 &mu;g/ml concentration</td>
</tr>
<tr>
<td>Protease Inhibitor Cocktail</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P8340</td>
<td>&nbsp;</td>
<tr>
<td>Salmon sperm DNA /Protein A agarose</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>16-157</td>
<td>&nbsp;</td>
<tr>
<td>myogenin antibody</td>
<td>&nbsp;</td>
<td>Santa Cruz Biotechnology, Inc.</td>
<td>sc-576</td>
<td>&nbsp;</td>
<tr>
<td>Normal rabbit IgG</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>12-370</td>
<td>&nbsp;</td>
<tr>
<td>Platinum PCR Supermix</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11306-016</td>
<td>&nbsp;</td>
<tr>
<td>GoTaq Q-PCR master mix</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>A6001</td>
<td>&nbsp;</td>		</tbody>
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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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