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 equipment. Avoid contact with skin and eyes. Keep away from incompatibles, such as oxidizing agents, reducing agents, acids, alkalis, and moisture.</li>
		<li>For the glycine stop-fix solution, prepare 0.125 M glycine in 1x PBS solution and store it at RT.</li>
		<li>For the cell scraping solution, prepare a 1x PBS solution in dH2O and store it on ice. Directly before use, add proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF) to a final concentration of 0.5 mM. 
		CAUTION: PMSF is classified as corrosive and toxic; wear protective clothing and use it in a ventilated area.</li>
		<li>Directly before using the cell lysis buffer (20 mM Tris-HCl;&#160;pH 8.0, 85 mM KCl,&#160;and 0.5% NP40), add 1X Protease Inhibitor Cocktail (PIC; 100X stock solution in dimethyl sulfoxide (DMSO): 104 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), 80 &#181;M aprotinin, 4 mM bestatin, 1.4 mM E-64, 2 mM leupeptin, and 1.5 mM pepstatin A) and 0.5 mM PMSF.</li>
		<li>Directly before using the nuclei lysis buffer (50 mM Tris-Cl; pH 8.0, 10 mM ethylenediaminetetraacetic acid (EDTA), and 1% SDS), add 1X PIC and 0.5 mM PMSF.</li>
	</ol>
	</li>
</ol>
2. Cell Fixation and Shearing
<ol>
	<li>Grow the cells to 70-80% confluence on 10 cm tissue culture plates. Stimulate the cells per the experimental aims.</li>
	<li>Remove the tissue culture medium, add 5 mL of fixation solution, and incubate the cells for 10 min at RT&#160;on a shaking platform.</li>
	<li>Remove the fixation solution and wash the cells twice with 10 mL of ice-cold 1x PBS.</li>
	<li>Remove the 1x PBS, add 5 mL of glycine stop-fix solution, and incubate the cells for 5 min at RT on a shaking platform.</li>
	<li>Remove the glycine stop-fix solution and wash the cells twice with 10 mL of ice-cold 1x PBS.</li>
	<li>Remove the 1x PBS, add 2 mL of cell scraping solution, and harvest the cells using a cell scraper. Transfer the harvested cells to a conical tube on ice.</li>
	<li>Centrifuge the samples for 10 min at 600 x g and 4 &#176;C.</li>
	<li>Remove the supernatant, resuspend the pellet in 1 mL of 1X cell lysis buffer, and incubate on ice for 30 min. Alternatively, snap-freeze the pellet in liquid nitrogen and store it at -80 &#176;C.</li>
	<li>Once the pellet is resuspended 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.</li>
	<li>Transfer the cell suspension to a microcentrifuge tube and centrifuge the samples for 10 min at 2,400 x g and 4 &#176;C.</li>
	<li>Discard the supernatant, resuspend the pellet in 350 &#181;L of nuclei digestion buffer, and incubate the samples for 5 min at 37 &#176;C.</li>
	<li>Add micrococcal nuclease (1 U/&#181;L) and mix by vortexing. Incubate the samples at 37 &#176;C for 5-20 min; mix the samples every 2 min by inversion. 
	NOTE: The time and enzyme concentration differ between cell lines and might require optimization in cases of suboptimal enzymatic shearing.</li>
	<li>Alternatively, achieve chromatin shearing by sonication using commercially available sonicators. 
	NOTE: Sonication conditions will require optimization. An optimization example protocol is provided below using a sample volume of 300 &#181;L and a sample processing power of 25%.
	<ol>
		<li>Following step 2.10, discard the supernatant and resuspend the nuclear pellet in 1.0 mL of commercial shearing buffer.</li>
		<li>Aliquot 300 &#181;L of the resuspended nuclear pellet into three 1.7 mL microcentrifuge tubes. Place them on ice.</li>
		<li>Shear the 3 aliquots of fixed chromatin at 25% power using 3 different conditions to determine the optimal sonication conditions: 
		5 pulses of 20 s each, with a 30 s rest on ice between each pulse. 
		10 pulses of 20 s each, with a 30 s rest on ice between each pulse. 
		20 pulses of 20 s each, with a 30 s rest on ice between each pulse.</li>
		<li>Continue with Step 2.15, as outlined below.</li>
	</ol>
	</li>
	<li>Following the incubation with the micrococcal nuclease, add 7 &#181;L of ice-cold 0.5 M EDTA to stop the reaction.</li>
	<li>Centrifuge the samples with the sheared DNA for 10 min at 16,200 x g and 4 &#176;C.</li>
	<li>Transfer the sheared DNA-containing supernatant to a fresh microcentrifuge tube. Aliquot 50 &#181;L into a separate microcentrifuge tube to confirm the successful shearing of the DNA (section 3). Use the remaining volume immediately for immunoprecipitation (section 4) or store it at -80 &#176;C.</li>
</ol>
3. Confirmation of the Shearing Efficiency
<ol>
	<li>Add 150 &#181;L of nuclease-free dH2O and 10 &#181;L of 5 M NaCl to the 50 &#181;L aliquot of the sheared chromatin from step 2.16.</li>
	<li>Incubate the samples at 65 &#176;C for 4 h to reverse the crosslinks.</li>
	<li>Add 2 &#181;L of RNase A (10 &#181;g/&#181;L) and incubate the samples at 37 &#176;C for 15 min.</li>
	<li>Add 10 &#181;L of proteinase K (0.5 &#181;g/&#181;L) and incubate the samples at 42 &#176;C for 90 min.</li>
	<li>For purification of the DNA, perform a phenol/chloroform extraction12.
	<ol>
		<li>Add nuclease-free water to a final volume of 200 &#181;L. Add 200 &#181;L of phenol:chloroform:isoamyl alcohol (25:24:1) to the sample and vortex for 20 s.</li>
		<li>Centrifuge at room temperature for 5 min at 16,000 x g. Carefully remove the upper aqueous phase and transfer it to a fresh tube. Be sure not to carry over any phenol during the pipetting.</li>
		<li>Add 0.1 volumes of 3 M sodium acetate (NH4OAc), pH 5.2, and mix by flicking the tube several times with a finger. Add 2.5 volumes of ice-cold 100% ethanol and mix by vortexing for 5 s. Incubate the sample O/N at -20 &#176;C, or for at least 1 h at -80 &#176;C.</li>
		<li>Centrifuge the sample for 30 min at 4 &#176;C and 16,000 x g to pellet the cDNA.</li>
		<li>Carefully remove the supernatant without disrupting the pellet. Add 1 mL of RT, 70% ethanol and invert the tube several times.</li>
		<li>Centrifuge the sample for 2 min at 4 &#176;C and 16,000 x g. Carefully remove the supernatant.</li>
		<li>Dry the pellet for 5-10 min at RT. Resuspend the pellet in 30 &#181;L of dH2O.</li>
	</ol>
	</li>
	<li>Use a 5 and 10 &#181;L aliquot of the purified DNA for gel electrophoresis with a 1% TAE agarose gel. 
	NOTE: Successful chromatin shearing results in a 200-1,500 bp DNA pattern. If the shearing is unsuccessful, determine the optimal shearing conditions for the specific cells by modifying the incubation time of the shearing step (step 2.12) to 5, 10, and 15 min.</li>
	<li>Use the remaining sample to analyze the DNA concentration of the sample. Reverse-calculate the DNA concentration in the sheared chromatin sample (step 2.16); use the same DNA amounts for each treatment group for the immunoprecipitation (section 4).</li>
</ol>
4. Immunoprecipitation
<ol>
	<li>Combine 10-25 &#181;g of the sheared, crosslinked chromatin (step 2.16) with 25 &#181;L of ChIP-grade protein G magnetic beads, 10 &#181;L of immunoprecipitation dilution buffer (stock solution: 0.01% SDS, 1.1% Triton X-100, 16.7 mM Tris-HCl; pH 8.0, 1.2 mM EDTA, and 167 mM NaCl), and 1 &#181;L of PIC.</li>
	<li>After 4 h of incubation, add 1-10 &#181;g of antibody (the antibody concentration will vary based upon the affinity of the antibody; it should initially be used per manufacturer recommendations and experimentally optimized if necessary) to a 1.5 mL microcentrifuge tube and add dH2O to a final volume of 100 &#181;L. 
	NOTE: Instead of the above-described direct immunoprecipitation method, the indirect immunoprecipiation method can be used by first combining the chromatin with the antibodies and subsequently adding protein G magnetic beads.</li>
	<li>Incubate the samples on an end-to-end rotator for 4-12 h at 4 &#176;C.</li>
</ol>
5. Elution
<ol>
	<li>Place the tubes on a magnetic stand. Once the magnetic beads aggregate on the side of the tube, carefully remove and discard the supernatant.</li>
	<li>Wash the beads three times with 800 &#181;L of wash buffer 1 (0.1% SDS, 1% Triton X-100, 2 mM EDTA pH 8, 20 mM Tris-HCl; pH 8, and 150 mM NaCl); mix by inverting the tube several times. Place the tube on the magnetic stand and carefully remove and discard the wash buffer once the magnetic beads aggregate on the side of the tube.</li>
	<li>Wash the beads once with 800 &#181;L of wash buffer 2 (0.1% SDS, 1% Triton X-100, 2 mM EDTA; pH 8, 20 mM Tris-HCl pH 8, and 500 mM NaCl); mix by inverting the tube several times. Place the tube on the magnetic stand and carefully remove and discard the wash buffer once the magnetic beads aggregate on the side of the tube.</li>
	<li>Resuspend the beads in 50 &#181;L of elution buffer (1% SDS and 100 mM NaHCO3) and incubate the samples on an end-to-end rotator for 15 min at RT.</li>
	<li>Place the tubes on a magnetic stand, and once the magnetic beads collect at the side of the tube, transfer the supernatant to a fresh tube.</li>
	<li>Add 6 &#181;L of 5 M NaCl, to reverse the crosslinking, and 2 &#181;L of proteinase K (0.5 &#181;g/&#181;L). After mixing the samples, incubate the samples for 2 h at 65 &#176;C.</li>
	<li>For purification of the DNA, perform a phenol/chloroform extraction12 (step 3.5) and subsequently resuspend the pellet in 30 &#181;L of dH2O. 
	NOTE: The samples can be directly analyzed for protein binding sites (section 5) or can be stored at -20 &#176;C.</li>
</ol>
6. Binding-site Analysis
<ol>
	<li>Perform an analysis for specific binding sites within a known nucleic acid sequence using PCR or qRT-PCR. For primer design, follow conventional PCR or qRT-PCR protocols. Design the primers to synthesize a 150-400 bp amplicon that bridges the protein binding site of interest (Figure 1).</li>
	<li>Set up the PCR using four different DNA templates to assure specificity: i) DNA from ChIP immunoprecipitated with the antibody of interest, ii) DNA from ChIP immunoprecipitated with an unspecific IgG antibody, iii) input DNA, and iv) H2O as a negative control for the PCR to rule out contamination. In the case of pathway stimulation, choose a fifth template using a different set of primers specific to a known binding site of the immunoprecipitated protein. 
	NOTE: In the case of the c-KIT ligand promoter analysis, use conventional PCR to demonstrate TGF-&#946;-induced SMAD binding at the SBE within the c-KIT ligand promoter region. As a positive control, perform PCR on plasminogen activator inhibitor-1 (PAI-1) as a known target of TGF-&#946;-induced SMAD binding.</li>
</ol>

<ol><li>Brenowitz, M., Senear, D. F., Shea, M. A., Ackers, G. K. <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/0076687986300119">Quantitative DNase footprint titration: a method for studying protein-DNA interactions.</a> Methods Enzymol. 130, 132-181 (1986).</li>
<li>Garner, M. M., Revzin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+gel+electrophoresis+method+for+quantifying+the+binding+of+proteins+to+specific+DNA+regions%3A+application+to+components+of+the+Escherichia+coli+lactose+operon+regulatory+system%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">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. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detecting+protein-DNA+interactions+in+vivo%3A+distribution+of+RNA+polymerase+on+specific+bacterial+genes%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+USA%22%5BJournal%5D)">Detecting protein-DNA interactions in vivo: distribution of RNA polymerase on specific bacterial genes.</a> Proc Natl Acad Sci USA. 81 (14), 4275-4279 (1984).</li>
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<li>Mukhopadhyay, A., Deplancke, B., Walhout, A. J., Tissenbaum, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromatin+immunoprecipitation+%28ChIP%29+coupled+to+detection+by+quantitative+real-time+PCR+to+study+transcription+factor+binding+to+DNA+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Chromatin immunoprecipitation (ChIP) coupled to detection by quantitative real-time PCR to study transcription factor binding to DNA in Caenorhabditis elegans.</a> Nat Protoc. 3 (4), 698-709 (2008).</li>
<li>Weinmann, A. S., Bartley, S. M., Zhang, T., Zhang, M. Q., Farnham, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+chromatin+immunoprecipitation+to+clone+novel+E2F+target+promoters%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">Use of chromatin immunoprecipitation to clone novel E2F target promoters.</a> Mol Cell Biol. 21 (20), 6820-6832 (2001).</li>
<li>Weinmann, A. S., Farnham, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+unknown+target+genes+of+human+transcription+factors+using+chromatin+immunoprecipitation%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">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/pubmed?term=((Isolating+human+transcription+factor+targets+by+coupling+chromatin+immunoprecipitation+and+CpG+island+microarray+analysis%5BTitle%5D)+AND+%22GenesDev%22%5BJournal%5D)">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/pubmed?term=((Smad+transcription+factors%5BTitle%5D)+AND+%22Genes+Dev%22%5BJournal%5D)">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/pubmed?term=((A+Positive+TGF-beta%2Fc-KIT+Feedback+Loop+Drives+Tumor+Progression+in+Advanced+Primary+Liver+Cancer%5BTitle%5D)+AND+%22Neoplasia%22%5BJournal%5D)">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/pubmed?term=((Purification+of+nucleic+acids+by+extraction+with+phenol%3Achloroform%5BTitle%5D)+AND+%22CSH+Protoc%22%5BJournal%5D)">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/pubmed?term=((Direct+binding+of+Smad3+and+Smad4+to+critical+TGF+beta-inducible+elements+in+the+promoter+of+human+plasminogen+activator+inhibitor-type+1+gene%5BTitle%5D)+AND+%22EMBO+J%22%5BJournal%5D)">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/pubmed?term=((Crystal+structure+of+a+Smad+MH1+domain+bound+to+DNA%3A+insights+on+DNA+binding+in+TGF-beta+signaling%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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>
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<li>Nelson, J. D., Denisenko, O., Bomsztyk, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protocol+for+the+fast+chromatin+immunoprecipitation+%28ChIP%29+method%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">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/pubmed?term=((Two-step+cross-linking+for+analysis+of+protein-chromatin+interactions%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">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.sciencedirect.com/science/article/pii/S1046202303000902">Immunoprecipitation of native chromatin: NChIP.</a> Methods. 31 (1), 76-82 (2003).</li>
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The authors declare that they have no competing financial interests.

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><tbody><tr><td>HepG2 cells</td><td>ATCC</td><td>HB-8065</td><td></td></tr><tr><td>Hep3B cells</td><td>ATCC</td><td>HB-8064</td><td></td></tr><tr><td>TGF-&amp;beta;1</td><td>R&amp;amp;D Systems</td><td>101-B1</td><td>Used at a concentration of 10 ng/mL</td></tr><tr><td>Anti-SMAD2 antibody</td><td>Cell Signalling Technology</td><td>5339</td><td>Amount used/IP: 3 &amp;micro;g</td></tr><tr><td>Anti-STAT3 antibody</td><td>Cell Signalling Technology</td><td>4904</td><td>Amount used/IP: 3 &amp;micro;g</td></tr><tr><td>ChIP-IT Protein G Magnetic Beads</td><td>Active Motif</td><td>53033</td><td></td></tr><tr><td>Protease Inhibitor Cocktail</td><td>Active Motif</td><td>37490</td><td></td></tr><tr><td>Micrococcal Nuclease</td><td>Cell Signalling Technology</td><td>10011</td><td></td></tr><tr><td>PCR forward primer: PAI-1</td><td></td><td></td><td>Sequence: 5&amp;rsquo;-GGAAGAGGATAAAGGACAAGCTG-3&amp;rsquo;</td></tr><tr><td>PCR reverse primer: PAI-1</td><td></td><td></td><td>Sequence: 5&amp;rsquo;-TGCAGCCAGCCACGTGATTGTC-3&amp;rsquo;</td></tr><tr><td>PCR forward primer: SCF</td><td></td><td></td><td>Sequence: 5&amp;rsquo;-CACTGATGTTAATGTTCAGC-3&amp;rsquo;&amp;nbsp;</td></tr><tr><td>PCR reverse primer: SCF</td><td></td><td></td><td>Sequence: 5&amp;rsquo;-GCTCTAATTTAAACCTGGAGC-3&amp;rsquo;</td></tr><tr><td>PCR forward primer: TGF-&amp;beta;1 (STB-1)</td><td></td><td></td><td>Sequence: 5&amp;rsquo;-GAGAGAGACGTGAGTGGCATGTT-3&amp;rsquo;&amp;nbsp;</td></tr><tr><td>PCR reverse primer: TGF-&amp;beta;1 (STB-1)</td><td></td><td></td><td>Sequence: 5&amp;rsquo;-TAGCTTTCTCTGCCTTGGTCTCCCC-3&amp;rsquo;&amp;nbsp;</td></tr><tr><td>PCR forward primer: TGF-&amp;beta;1 (STB-2)</td><td></td><td></td><td>Sequence: 5&amp;rsquo;-GTACTGGGGGAGGAGCGGCATC-3&amp;rsquo;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td></tr><tr><td>PCR reverse primer: TGF-&amp;beta;1 (STB-2)</td><td></td><td></td><td>Sequence: 5&amp;rsquo;-TGCCACTGTCTGGAGAGAGGTGTGTC-3&amp;rsquo;&amp;nbsp;&amp;nbsp;</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...

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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. ...

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Research

JoVE Journal

Genetics

7.8K Views.  The University of Texas MD Anderson Cancer Center. 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.

Video: Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational modifications (PTMs) of histone tails, the exchange of canonical histones with histone variants, and nucleosome eviction. Such regulation requires the binding of signal-sensitive transcription factors (TFs) that recruit chromatin-modifying enzymes at regulatory elements defined as enhancers. Understanding how signaling cascades regulate enhancer activity requires a comprehensive analysis of the binding of TFs, chromatin modifying enzymes, and the occupancy of specific histone marks and histone variants. Chromatin immunoprecipitation (ChIP) assays utilize highly specific antibodies to immunoprecipitate specific protein/DNA complexes. The subsequent analysis of the purified DNA allows for the identification the region occupied by the protein recognized by the antibody. This work describes a protocol to efficiently perform ChIP of histone proteins in a mature mouse T-cell line. The presented protocol allows for the performance of ChIP assays in a reasonable timeframe and with high reproducibility.

Chromatin Immunoprecipitation ChIP in Mouse T-cell Lines

This work describes a protocol for chromatin immunoprecipitation (ChIP) using a mature mouse T-cell line. This protocol is suitable to investigate the distribution of specific histone marks at specific promoter sites or genome-wide.

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational ...

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

Promoter Capture Hi-C: High-resolution, Genome-wide Profiling of Promoter Interactions

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the spatio-temporal expression of their target genes through physical contact, often bridging considerable (in some cases hundreds of kilobases) genomic distances and bypassing nearby genes. The human genome harbors an estimated one million enhancers, the vast majority of which have unknown gene targets. Assigning distal regulatory regions to their target genes is thus crucial to understand gene expression control. We developed Promoter Capture Hi-C (PCHi-C) to enable the genome-wide detection of distal promoter-interacting regions (PIRs), for all promoters in a single experiment. In PCHi-C, highly complex Hi-C libraries are specifically enriched for promoter sequences through in-solution hybrid selection with thousands of biotinylated RNA baits complementary to the ends of all promoter-containing restriction fragments. The aim is to then pull-down promoter sequences and their frequent interaction partners such as enhancers and other potential regulatory elements. After high-throughput paired-end sequencing, a statistical test is applied to each promoter-ligated restriction fragment to identify significant PIRs at the restriction fragment level. We have used PCHi-C to generate an atlas of long-range promoter interactions in dozens of human and mouse cell types. These promoter interactome maps have contributed to a greater understanding of mammalian gene expression control by assigning putative regulatory regions to their target genes and revealing preferential spatial promoter-promoter interaction networks. This information also has high relevance to understanding human genetic disease and the identification of potential disease genes, by linking non-coding disease-associated sequence variants in or near control sequences to their target genes.

DNA regulatory elements, such as enhancers, control gene expression by physically contacting target gene promoters, often through long-range chromosomal interactions spanning large genomic distances. Promoter Capture Hi-C (PCHi-C) identifies significant interactions between promoters and distal regions, enabling the assignment of potential regulatory sequences to their target genes.

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the ...

A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western countries. The majority of CLL patients show an indolent course of the disease as well as an anergic phenotype of their leukemia cells, referring to a B cell receptor unresponsive to external stimulation. We have recently shown that the transcription factor NFAT2 is a crucial regulator of anergy in CLL. A major challenge in the analysis of the role of a transcription factor in different diseases is the identification of its specific target genes. This is of&#160;great significance for the elucidation of pathogenetic mechanisms and potential therapeutic interventions. Chromatin immunoprecipitation (ChIP) is a classic technique to demonstrate protein-DNA interactions and can, therefore, be used to identify direct target genes of transcription factors in mammalian cells. Here, ChIP was used to identify LCK as a direct target gene of NFAT2 in human CLL cells. DNA and associated proteins are crosslinked using formaldehyde and subsequently sheared by sonication into DNA fragments of approximately 200-500 base pairs (bp). Cross-linked DNA fragments associated with NFAT2 are then selectively immunoprecipitated from cell debris using an &#945;NFAT2 antibody. After purification, associated DNA fragments are detected via quantitative real-time PCR (qRT-PCR). DNA sequences with evident enrichment represent regions of the genome which are targeted by NFAT2 in vivo. Appropriate shearing of the DNA and the selection of the required antibody are particularly crucial for the successful application of this method. This protocol is ideal for the demonstration of direct interactions of NFAT2 with target genes. Its major limitation is the difficulty to employ ChIP in large-scale assays analyzing the target genes of multiple transcription factors in intact organisms.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. NFAT transcription factors are important regulators of development and activation in numerous cell types. Here, we present a protocol for the use of chromatin immunoprecipitation (ChIP) in human CLL cells to identify novel target genes of NFAT2.

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western ...

Cancer Research

Assays for Validating Histone Acetyltransferase Inhibitors

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene expression. KATs, such as CBP/p300, are under intense investigation as therapeutic targets due to their critical role in tumorigenesis of diverse cancers. The development of novel small molecule inhibitors targeting the histone acetyltransferase (HAT) function of KATs is challenging and requires robust assays that can validate the specificity and potency of potential inhibitors.
This article outlines a pipeline of three methods that provide rigorous in vitro validation for novel HAT inhibitors (HATi). These methods include a test tube HAT assay, Chromatin Hyperacetylation Inhibition (ChHAI) assay, and Chromatin Immunoprecipitation-quantitative PCR (ChIP-qPCR). In the HAT assay, recombinant HATs are incubated with histones in a test tube reaction, allowing for acetylation of specific lysine residues on the histone tails. This reaction can be blocked by a HATi and the relative levels of site-specific histone acetylation can be measured via immunoblotting. Inhibitors identified in the HAT assay need to be confirmed in the cellular environment.
The ChHAI assay uses immunoblotting to screen for novel HATi that attenuate the robust hyperacetylation of histones induced by a histone deacetylase inhibitor (HDACi). The addition of an HDACi is helpful because basal levels of histone acetylation can be difficult to detect via immunoblotting.
The HAT and ChHAI assays measure global changes in histone acetylation, but do not provide information regarding acetylation at specific genomic regions. Therefore, ChIP-qPCR is used to investigate the effects of HATi on histone acetylation levels at gene regulatory elements. This is accomplished through selective immunoprecipitation of histone-DNA complexes and analysis of the purified DNA through qPCR. Together, these three assays allow for the careful validation of the specificity, potency, and mechanism of action of novel HATi.

Inhibitors of histone acetyltransferases (HATs, also known as lysine acetyltransferases), such as CBP/p300, are potential therapeutics for treating cancer. However, rigorous methods for validating these inhibitors are needed. Three in vitro methods for validation include HAT assays with recombinant acetyltransferases, immunoblotting for histone acetylation in cell culture, and ChIP-qPCR.

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene ...

A Comprehensive Approach to Studying the Promoter Interactome in Scarce Cells

An Integrated Workflow to Study the Promoter-Centric Spatio-Temporal Genome Architecture in Scarce Cell Populations

Spatiotemporal gene transcription is tightly regulated by distal regulatory elements, such as enhancers and silencers, which rely on physical proximity with their target gene promoters to control transcription. Although these regulatory elements are easy to identify, their target genes are difficult to predict, since most of them are cell-type specific and may be separated by hundreds of kilobases in the linear genome sequence, skipping over other non-target genes. For several years, Promoter Capture Hi-C (PCHi-C) has been the gold standard for the association of distal regulatory elements to their target genes. However, PCHi-C relies on the availability of millions of cells, prohibiting the study of rare cell populations such as those commonly obtained from primary tissues. To overcome this limitation, low input Capture Hi-C (liCHi-C), a cost-effective and customizable method to identify the repertoire of distal regulatory elements controlling each gene of the genome, has been developed. liCHi-C relies on a similar experimental and computational framework as PCHi-C, but by employing minimal tube changes, modifying&#160;the reagent concentration and volumes, and swapping or eliminating steps, it accounts&#160;for minimal material loss during library construction. Collectively, liCHi-C enables the study of gene regulation and spatiotemporal genome organization in the context of developmental biology and cellular function.

Author Spotlight: An Integrated Workflow to Study the Promoter-Centric Spatio-Temporal Genome Architecture in Scarce Cell Populations  

Gene expression is regulated by interactions of gene promoters with distal regulatory elements. Here, we descirbe how low input Capture Hi-C (liCHi-C) allows the identification of these interactions in rare cell types, which were previously unmeasurable.

Spatiotemporal gene transcription is tightly regulated by distal regulatory elements, such as enhancers and silencers, which rely on physical proximity ...

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 ...

Biology

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Bioengineering

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 ...