This work was supported by the University of Texas MD Anderson Cancer Center, Houston, TX (startup funds, B.B.).

1. Preparation of Solutions
<ol>
	<li>Prepare the following solutions fresh for each experiment.
	<ol>
		<li>For the fixation solution, prepare 37% formaldehyde and store it at RT. Dilute it in 1x Phosphate-Buffered Saline (PBS) to a final concentration of 1.42%. 
		CAUTION: Formaldehyde is classified as irritant, corrosive, mutagenic, teratogenic, and carcinogenic. Do not ingest. Do not breathe gas/fumes/vapor/spray. In case of insufficient ventilation, wear suitable respiratory ...

<ol>
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M., Revzin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gel+electrophoresis+method+for+quantifying+the+binding+of+proteins+to+specific+DNA+regions:+application+to+components+of+the+Escherichia+coli+lactose+operon+regulatory+system.">A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system.</a> Nucleic Acids Res. 9 (13), 3047-3060 (1981).</li><li>Gilmour, D. S., Lis, J. 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J. <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+unknown+target+genes+of+human+transcription+factors+using+chromatin+immunoprecipitation.">Identification of unknown target genes of human transcription factors using chromatin immunoprecipitation.</a> Methods. 26 (1), 37-47 (2002).</li><li>Weinmann, A. S., Yan, P. S., Oberley, M. J., Huang, T. H., Farnham, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolating+human+transcription+factor+targets+by+coupling+chromatin+immunoprecipitation+and+CpG+island+microarray+analysis.">Isolating human transcription factor targets by coupling chromatin immunoprecipitation and CpG island microarray analysis.</a> GenesDev. 16 (2), 235-244 (2002).</li><li>Massague, J., Seoane, J., Wotton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smad+transcription+factors.">Smad transcription factors.</a> Genes Dev. 19 (23), 2783-2810 (2005).</li><li>Rojas, A., 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=A+Positive+TGF-beta/c-KIT+Feedback+Loop+Drives+Tumor+Progression+in+Advanced+Primary+Liver+Cancer.">A Positive TGF-beta/c-KIT Feedback Loop Drives Tumor Progression in Advanced Primary Liver Cancer.</a> Neoplasia. 18 (6), 371-386 (2016).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+nucleic+acids+by+extraction+with+phenol:chloroform.">Purification of nucleic acids by extraction with phenol:chloroform.</a> CSH Protoc. 2006 (1), (2006).</li><li>Dennler, S., 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=Direct+binding+of+Smad3+and+Smad4+to+critical+TGF+beta-inducible+elements+in+the+promoter+of+human+plasminogen+activator+inhibitor-type+1+gene.">Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene.</a> EMBO J. 17 (11), 3091-3100 (1998).</li><li>Shi, Y., 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=Crystal+structure+of+a+Smad+MH1+domain+bound+to+DNA:+insights+on+DNA+binding+in+TGF-beta+signaling.">Crystal structure of a Smad MH1 domain bound to DNA: insights on DNA binding in TGF-beta signaling.</a> Cell. 94 (5), 585-594 (1998).</li><li>Seidel, H. M., 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=Spacing+of+palindromic+half+sites+as+a+determinant+of+selective+STAT+(signal+transducers+and+activators+of+transcription)+DNA+binding+and+transcriptional+activity.">Spacing of palindromic half sites as a determinant of selective STAT (signal transducers and activators of transcription) DNA binding and transcriptional activity.</a> Proc Natl Acad Sci USA. 92 (7), 3041-3045 (1995).</li><li>Nelson, J. D., Denisenko, O., Bomsztyk, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+the+fast+chromatin+immunoprecipitation+(ChIP)+method.">Protocol for the fast chromatin immunoprecipitation (ChIP) method.</a> Nat Protoc. 1 (1), 179-185 (2006).</li><li>Tian, B., Yang, J., Brasier, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-step+cross-linking+for+analysis+of+protein-chromatin+interactions.">Two-step cross-linking for analysis of protein-chromatin interactions.</a> Methods Mol Biol. 809, 105-120 (2012).</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 (1), 76-82 (2003).</li><li>Horz, W., Altenburger, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+specific+cleavage+of+DNA+by+micrococcal+nuclease.">Sequence specific cleavage of DNA by micrococcal nuclease.</a> Nucleic Acids Res. 9 (12), 2643-2658 (1981).</li><li>Chung, H. 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In this report, we demonstrate the TGF-&#946;1-induced binding of SMAD2 to an SBE within the c-KIT ligand promoter and TGF-&#946;1-induced binding of STAT3 to its recognition sequence within the TGF-&#946;1 ligand gene. We demonstrate cytokine-induced binding of both transcription factors using chromatin immunoprecipitation.
Chromatin immunoprecipitation is a powerful tool to demonstrate the direct binding of a protein of interest to DNA, to characterize the stimuli that induce protein binding...

In the nucleus of eukaryotes, DNA interacts with histone proteins and nonhistone proteins and is condensed into chromatin. In the physiological, pathophysiological, and cell biological context, cellular functions are spatially and temporarily controlled by chromatin-coordinated gene expression. DNA-protein interactions have an essential role in the regulation of cellular processes, such as DNA replication, recombination, and repair, as well as protein expression. Therefore, the analysis of DNA-protein interactions is an indispensable tool in the evaluation of gene expression and cell function.
Several techniques exist to assess DNA-protein interactions in vitro, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting1,2. However, these techniques do not analyze the protein-DNA interaction within the chromatin and cellular context. ChIP is a technique that captures proteins bound to their specific DNA binding sites and thereby facilitates the identification of DNA-protein interactions within the chromatin context. The technique was originally developed by Gimour and Lis for the assessment of RNA polymerase II binding to specific genes in Escherichia coli and Drosophila melanogaster3,4. It is done by fixation of the DNA-protein complexes, followed by performing chromatin extraction and shearing the DNA into ~200 base pair (bp) fragments. Subsequently, the DNA-bound protein of interest is isolated by immunoprecipitation. After the reversal of the DNA-protein crosslink, the DNA is purified and analyzed. Several methods can be used for the analysis of the protein binding sites and depend upon the nucleic acid sequence of the protein binding site within the target gene5. In cases where the DNA sequence is known, standard Polymerase Chain Reactions (PCR) can be applied, using specific primer pairs flanking the known binding site. Quantitative Real-Time PCR (qRT-PCR) can also be used6. In cases where the sequence is unknown, ChIP can be combined with DNA microarrays (ChIP-on-chip), DNA sequencing (ChIP-seq), or cloning techniques7,8,9.
The TGF-&#946; pathway has potent tumor suppressing functions and is a key pathway in cell differentiation. It is activated through the binding of the TGF-&#946;1 ligand to its cognate receptor complex, resulting in the serine-phosphorylation of SMAD2/3 transcription factors. Following their association with the common mediator, SMAD4, the SMAD-complex translocates to the nucleus and binds to the SBE within the promoter region of the target genes, where it regulates genes controlling the cell cycle, apoptosis, and cell differentiation. The transcriptional response to TGF-&#946; stimulation is cell type- and context-specific10. Recently, we described a positive feedback loop between TGF-&#946; and the c-KIT pathway11. In this model, TGF-&#946;1-activated SMAD2 binds to the c-KIT ligand promoter and induces its expression and secretion. Subsequently, the c-KIT ligand activates the c-KIT receptor in an auto- and para-crinic&#160;fashion. c-KIT receptor activation results in STAT3 Tyr705-phosphorylation via JAK1/2. Following STAT3-activation and nuclear translocation, STAT3 binds to the TGF-&#946;1 ligand gene and regulates its expression.
Here, we demonstrate the essential role of ChIP analysis for the identification of SMAD2 binding to the c-KIT receptor ligand promoter and for the identification of the Signal Transducer (and) Activator (of) Transcription 3 (STAT3 binding to the TGF-&#946;1-gene).

<table border="0" cellpadding="0" cellspacing="0" width="907">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">HepG2 cells</td>
			<td style="width:193px;">ATCC</td>
			<td style="width:119px;">HB-8065</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">Hep3B cells</td>
			<td style="width:193px;">ATCC</td>
			<td>HB-8064</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">TGF-&#946;1</td>
			<td>R&#38;D Systems</td>
			<td>101-B1</td>
			<td>Used at a concentration of 10 ng/ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">Anti-SMAD2 antibody</td>
			<td style="width:193px;">Cell Signalling Technology</td>
			<td style="width:119px;">5339</td>
			<td>Amount used per IP: 3 &#181;g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">Anti-STAT3 antibody</td>
			<td style="width:193px;">Cell Signalling Technology</td>
			<td style="width:119px;">4904</td>
			<td>Amount used per IP: 3 &#181;g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">ChIP-IT Protein G Magnetic Beads</td>
			<td style="width:193px;">Active Motif</td>
			<td style="width:119px;">53033</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">Protease Inhibitor Cocktail</td>
			<td style="width:193px;">Active Motif</td>
			<td style="width:119px;">37490</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">Micrococcal Nuclease</td>
			<td style="width:193px;">Cell Signalling Technology</td>
			<td style="width:119px;">10011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">PCR forward primer: PAI-1</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-GGAAGAGGATAAAGGACAAGCTG-3&#8217;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">PCR reverse primer: PAI-1</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-TGCAGCCAGCCACGTGATTGTC-3&#8217;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">PCR forward primer: SCF</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-CACTGATGTTAATGTTCAGC-3&#8217;&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">PCR reverse primer: SCF</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-GCTCTAATTTAAACCTGGAGC-3&#8217;</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:256px;">PCR forward primer: TGF-&#946;1 (STB-1)</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-GAGAGAGACGTGAGTGGCATGTT-3&#8217;&#160;</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:256px;">PCR reverse primer: TGF-&#946;1 (STB-1)</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-TAGCTTTCTCTGCCTTGGTCTCCCC-3&#8217;&#160;</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:256px;">PCR forward primer: TGF-&#946;1 (STB-2)</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-GTACTGGGGGAGGAGCGGCATC-3&#8217;&#160;&#160;&#160;&#160;</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:256px;">PCR reverse primer: TGF-&#946;1 (STB-2)</td>
			<td style="width:193px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence: 5&#8217;-TGCCACTGTCTGGAGAGAGGTGTGTC-3&#8217;&#160;&#160;</td>
		</tr>
	</tbody>
</table>

analysis of the c-kit ligand promoter using chromatin immunoprecipitation

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. Therefore, DNA-protein interactions regulate multiple physiological, pathophysiological, and biological functions, such as cell differentiation, cell proliferation, cell cycle control, chromosome stability, epigenetic gene regulation, and cell transformation. In eukaryotic cells, the DNA interacts with histone and nonhistone proteins and is condensed into chromatin. Several technical tools can be used to analyze DNA-protein interactions, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting. However, these techniques analyze the protein-DNA interaction in vitro, not within the cellular context. Chromatin immunoprecipitation (ChIP) is a technique that captures proteins at their specific DNA binding sites, thereby allowing for the identification of DNA-protein interactions within their chromatin context. It is done by fixation&#160;of the DNA-protein interaction, followed by&#160;immunoprecipitation of the protein of interest. Subsequently, the genomic site that the protein was bound to is characterized. Here, we describe and discuss ChIP and demonstrate its analytical value for the identification of the Transforming Growth Factor-&#946; (TGF-&#946;)-induced binding of the transcription factor SMAD2 to SMAD Binding Elements (SBE) within the promoter region of the tyrosine-protein kinase Kit (c-KIT) receptor ligand Stem Cell Factor (SCF).

The binding of the TGF-&#946;1 ligand to its cognate receptor complex results in the serine-phosphorylation of SMAD2/3 transcription factors, followed by their association with the common mediator, SMAD4. The SMAD complex translocates to the nucleus. TGF-&#946; can regulate gene transcription, either directly via SMAD binding to SBEs within regulatory regions of the target genes, or indirectly through the SMAD-regulated expression of transcriptional activators or repressors that subsequen...

Watch this Scientific Journal Video about Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation at JoVE.com

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

DNA-protein interactions are essential for multiple biological processes. During the evaluation of cellular functions, the analysis of DNA-protein interactions is indispensable for understanding gene regulation. Chromatin immunoprecipitation (ChIP) is a powerful tool to analyze such interactions in vivo.

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

The overall goal of this chromatin immunoprecipitation procedure is to evaluate TGF beta 1-induced binding of the transcription factor SMAD2 to SMAD binding elements in the C kit promoter. This method can answer key questions in the field of cellular biology such as cytokine induced gene transcription. The main advantage of this technique is that it has the potential to demonstrate direct binding of transcription factors and other transcription regulatory proteins to their DNA binding sites in vivo.Grow the cells to between 70 and 80%confluence on ten centimeter tissue culture plates. Stimulate the cells per the experimental aims. Remove the tissue culture medium, and add five milliliters of fixation solution.Then, incubate the cells for 10 minutes at room temperature on a shaking platform. Remove the fixation solution, and wash the cells twice with 10 milliliters of ice cold one X PBS. Remove the one X PBS and add five milliliters of glycine stop fix solution.After incubating the cells for five minutes at room temperature on a shaking platform, remove the glycine stop fix solution and wash the cells twice with 10 milliliters of ice cold one X PBS. After removing the PBS, add two milliliters of cell scraping solution and harvest the cells using a cell scraper. Then, transfer the harvested cells to a conical tube on ice.Centrifuge the samples for 10 minutes at 600 times G, and four degrees celsius. Remove the supernatant, re-suspend the pellet in one milliliter of one X cell lysis buffer, and incubate on ice for 30 minutes. Once the pellet is re-suspended in the cell lysis buffer, transfer the cell suspension to an ice cold Dounce homogenizer, and Dounce for 10 strokes using a type B pestle for cell disruption.Then, transfer the cell suspension to a microcentrifuge tube, and centrifuge the sample for 10 minutes at 2, 400 times G, and four degrees celsius. Discard the supernatant and re-suspend the pellet in 350 microliters of nuclei digestion buffer. After incubating the samples for five minutes at 37 degrees celsius, add micrococcal nuclease and mix by vortexing.Incubate the samples at 37 degrees celsius for five to 20 minutes, mixing the samples every two minutes by inversion. The time in some concentration, if you're between cell lines, it might require optimization in case of suboptimal enzymatic shearing. An alternative shearing procedure using commercially available sonicare can be found in the text protocol.Following the incubation with the micrococcal nuclease, add seven microliters of ice cold 0.5 molar EDTA to stop the reaction. Centrifuge the samples with the sheared DNA for 10 minutes at 16, 200 times G and four degrees celsius. Transfer the sheared DNA containing supernatant to a fresh microcentrifuge tube.Aliquot 50 microliters into a separate microcentrifuge tube, to confirm the successful shearing of the DNA as described in the text protocol. Use the remaining volume immediately for immunoprecipitation or store it at minus 80 degrees celsius. Combine 10 to 25 micrograms of the sheared cross linked chromatin with 25 microliters of chromatin immunoprecipitation grade protein G magnetic beads.Then, add 10 microliters of immunoprecipitation dilution buffer, and one microliter of protease inhibitor cocktail. After four hours of incubation, add one to 10 micrograms of antibody to a 1.5 milliliter microcentrifuge tube. And add distilled water to a final volume of 100 microliters.Incubate the samples on an end-to-end rotator for four to 12 hours at four degrees celsius. Following incubation, place the tubes on a magnetic stand. Once the magnetic beads aggregate to the side of the tube, carefully remove and discard the supernatant.Wash the beads three times with 800 microliters of wash buffer one. For each wash, mix the beads by inverting the tube several times. Then, place the tube on the magnetic stand and carefully remove the wash buffer once the magnetic beads aggregate on the side of the tube.Next, wash the beads once with 800 microliters of wash buffer two using the same procedure. After washes, re-suspend the beads in 50 microliters of elution buffer and incubate the samples on an end-to-end rotator for 15 minutes at room temperature. Then, transfer the supernatant to a fresh tube with the aid of the magnetic stand.Add six microliters of five molar sodium chloride to reverse the cross linking followed by two microliters of proteinase K.After mixing the samples, incubate them for two hours at 65 degrees celsius. At this point, the samples can be directly analyzed for protein binding sites as described in the text protocol, or, can be stored at minus 20 degrees celsius. To confirm the TGF beta 1-induced binding of SMAD2 to the SCF promoter, the chromatin immunoprecipitation assay was performed.The TGF beta 1 treatment resulted in SMAD2 binding to the SCF promoter in human liver cancer lines, HepG2 and Hep3B. In the absence of TGF beta 1 stimulation, no SMAD binding was noted. As a positive control, for TGF beta 1-induced SMAD activation, chromatin immunoprecipitation for PAI-1 was performed.The PAI-1 promoter is a known target of TGF beta and SMAD. Specificity of the SMAD2 immunoprecipitation was confirmed through use of unspecific IgG antibodies. No SMAD2 binding to the SBE was noted after immunoprecipitation with IgG.To confirm the TGF Beta 1-induced binding of STAT3 to the TGF beta ligand gene, chromatin immunoprecipitation assays were performed using TGF beta 1 treated HepG2 and Hep3B cells. The TGF beta 1 treatment resulted in STAT3 binding to the second putative STAT3 binding site of the TGF beta gene, but not to the first one. In this example, the positive STAT3 binding to STB-2 serves as an internal positive control.Similar to the other chromatin immunoprecipitation experiment, STAT3 binding to the ST-2 was not seen after immunoprecipitation using IgG. Once mastered, this technique can be done in one or two days depending on the antibody if it is performed properly. While attempting this procedure, it's important to follow laboratory safety guidelines and be highly accurate at every step of the protocol.Precautions such as checking MSDS and wearing protective gear such as gloves, white coat and safety glasses should be taken while performing this procedure. After watching this video, you should have a good understanding of how to analyze protein binding to the specific DNA binding sequences. Following this protocol, other methods like promoter assays can be performed in order to confirm the functional relevance of protein DNA binding demonstrated by chromatin immunoprecipitation.

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7.6K 次观看.  The University of Texas MD Anderson Cancer Center. The overall goal of this chromatin immunoprecipitation procedure is to evaluate TGF beta 1-induced binding of the transcription factor SMAD2 to SMAD binding elements in the C kit promoter. This method can answer key questions in the field of cellular biology such as cytokine induced gene transcription. The main advantage of this technique is that it has the potential to demonstrate direct binding of transcription factors and other transcription regulatory proteins to their DNA binding sites i...