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>

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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. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+protein-DNA+interactions+in+vivo:+distribution+of+RNA+polymerase+on+specific+bacterial+genes.">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><li>Gilmour, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lis+J.T.+In+vivo+interactions+of+RNA+polymerase+II+with+genes+of+Drosophila+melanogaster.">Lis J.T. In vivo interactions of RNA polymerase II with genes of Drosophila melanogaster.</a> Mol Cell Biol. 5 (8), 2009-2018 (1985).</li><li>Mundade, R., Ozer, H. 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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=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. 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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 to DNA, and to characterize the DNA sequence to which the protein binds. The latter information can help in the identification of genes regulated by a specific protein of interest and is achieved by the use of ChIP-on-chip, ChIP-seq, or cloning strategies7,8,9. One of the major advantages of ChIP versus other methods of demonstrating DNA-protein binding, such as EMSA or DNase I footprinting, is that, in the ChIP technology, the binding is captured in vivo, while with the others, it is performed in vitro. Hence, ChIP provides insight into DNA-protein binding within the cellular context, while the other techniques represent an isolated system.
However, ChIP analysis is complex, involves multiple steps that can impact the results, and requires optimization and experience. One of the first steps that is critical for successful ChIP is the crosslink step (step 2.2). UV crosslinking is irreversible and therefore unsuitable for ChIP. Formaldehyde crosslinking is preferred, but formaldehyde concentration and crosslinking time can both influence the efficiency of chromatin shearing and antigen precipitation. In general, lower formaldehyde concentrations (1% w/v) and shorter crosslinking times (5-10 min) are preferable, as they improve the shearing efficiency. However, formaldehyde is not efficient at protein-protein crosslinking and therefore is suboptimal for proteins that do not directly bind to DNA16. For such cases, ChIP can be done in a two-step approach, in which protein-protein crosslinking is done first using crosslinkers such as disuccinimidyl glutarate, followed by formaldehyde-mediated DNA-protein crosslinking17.
The next critical step is chromatin shearing. In our study, we used enzymatic shearing using micrococcal nuclease. Enzymatic shearing is especially useful for noncrosslinked native ChIP (N-ChIP), when sonication would disrupt the DNA-protein binding. N-ChIP is predominantly used for the analysis of histones and histone modifiers18. While micrococcal nuclease is considered a relatively non-specific endo-exonuclease, it has been shown to elicit sequence-specific cleavage19. This can result in a sequence-dependent bias in the resulting fragments, with overrepresentation of certain gene loci20. Sonication creates randomly sized DNA fragments, without sequence bias, and is generally preferred for crosslinked ChIP (X-ChIP). However, sonication conditions must be determined empirically for each cell or tissue type and sonicator model, and resulting DNA fragments are generally larger than with enzymatic shearing16. Also, over-sonication or emulsification of the sample can result in the loss of antibody epitopes due to protein denaturation and degradation.
The immunoprecipitation step is another critical step that can greatly influence the results of ChIP. Agarose beads bind DNA nonspecifically, and variation in the number of added beads can affect the specific signal-to-background ratio. Hence, it is important to keep the agarose bead &#34;slurry&#34; well-suspended when it is added to the DNA-protein samples. In regard to the antibody used for the immunoprecipitation, in general, ChIP-grade antibodies should be used and, in cases where these are not available, at least immunoprecipitation-grade antibodies should be preferred. As specific epitopes can be masked during crosslinking, polyclonal antibodies are advantageous, as they recognize several epitopes. The amount of antibody added should be in excess of the factor being precipitated and thus should be determined empirically for each factor/antibody. Also, as the kinetics for reaching the equilibrium of antibody binding differ for each antibody, the optimal incubation conditions must be determined for each antibody.
Several controls can and should be included in the experimental setup. In cases where the induction of a specific DNA-protein binding reaction is evaluated, it is important to include an input DNA control to demonstrate equal template DNA amounts in the final analysis (i.e.&#160;PCR or qRT-PCR). An antibody control is required to confirm the specificity of the immunoprecipitation of the protein of interest. Usually isotype-matched immunoglobulins are well-suited as negative controls, but agarose beads can also be used. Occasionally, positive controls are used to confirm a functional experimental flow of the ChIP, and anti-histone antibodies are frequently used for this purpose. In stimulation experiments, a preferable positive control is immunoprecipitation of the protein of interest, with subsequent sequence analysis for a known DNA region the proteins bind to upon stimulation. In our representative results, the SBE within the PAI-1 promoter was used as a known target for TGF-&#946;1-induced SMAD binding. In experiments without stimulated protein binding, a DNA sequence known not to be a target for the protein of interest can be used for the subsequent DNA analysis. In regard to the DNA analysis, it is important to include a reaction without template DNA to rule out contamination.
ChIP is an excellent technique to directly demonstrate the binding of a protein of interest to a gene. However, it is not a functional study. In cases in which a protein of interest is thought of having a regulatory role, it is imperative to also perform functional assays, such as reporter gene-based assays (e.g., luciferase). In these protocols, the gene of interest is cloned in a regulatory position of a reporter gene. Induction of the gene of interest results in the expression of the reporter gene if it has a regulatory function. For further confirmation of the regulatory role of the specific protein of interest, either knock-down or knock-in cells can be generated in which the DNA-binding protein is genetically silenced. As an additional control, the protein binding site in the regulatory gene can be mutated to prevent the binding of the protein of interest. In either of the latter two experimental designs, cell stimulation will not result in the expression of the marker gene.

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 subsequently regulate the expression of the gene of interest10. To test if the c-KIT ligand SCF is transcriptionally regulated by the direct TGF-&#946;1-induced binding of SMAD2 to its promoter, we analyzed the SCF promoter-containing, 2.4-kb, 5&#39;-flanking region of the SCF gene for the SMAD2 binding motif, 5&#39;-AGAC-3&#39; (SBE)13,14. We identified 7 putative SBEs upstream of the SCF start codon (Figure 1A). To confirm the TGF-&#946;1-induced binding of SMAD2 to the SCF promoter, the ChIP assay described here was performed. The TGF-&#946;1 treatment resulted in SMAD2 binding to the SCF promoter in human liver cancer lines, HepG2 and Hep3B, cells (Figure 1B). In the absence of TGF-&#946;1-stimulation, no SMAD binding was noted. As a positive control for TGF-&#946;1-induced SMAD activation, ChIP for PAI-1 was performed. The PAI-1 promoter is a known target of TGF-&#946;/SMAD13. To confirm the specificity of the SMAD2 immunoprecipitation, unspecific IgG antibodies were used; no SMAD2 binding to the SBE was noted after immunoprecipitation with IgG.
For another demonstration of the ChIP technology, we analyzed the 5&#39;-flanking region of the TGF-&#946; ligand gene for the STAT3 consensus binding motifs 5&#39;-TT(N4)AA-3&#39; and 5&#39;-TT(N5)AA-3&#39;15. We identified two putative STAT3 binding sites upstream of the TGFB start codon at positions -4384/-4373 (STB-1) and -5365/-5357 (STB-2) (Figure 2A). To confirm the TGF-&#946;1-induced binding of STAT3 to the TGF-&#946; ligand gene, we performed ChIP assays using TGF-&#946;1-treated HepG2 and Hep3B cells. TGF-&#946;1 treatment resulted in STAT3 binding to the second putative STAT3 binding site (STB-2) of the TGFB gene, but not to the first one (STB-1) (Figure 2B). In this example, the positive STAT3 binding to STB-2 serves as an internal positive control. Similar to the above ChIP experiment, STAT3 binding to the STB-2 was not seen after immunoprecipitation using IgG.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55689/55689fig1.jpg" /> 
Figure 1: TGF-&#946;-induced SMAD2 Binding to the SCF Promoter. (A) Schematic representation of the SCF promoter, with putative SBEs shown as boxes (white boxes = AGAC) with their relative position to the start codon. The ChIP primer location is shown below, with the relative positions to the SCF start codon and the PCR product size. (B) ChIP for SMAD2 binding to the SCF promoter. Chromatin-protein complexes of TGF-&#946;1-treated and untreated HepG2 and Hep3B cells were immunoprecipitated with anti-SMAD2 antibody. PCR was performed using SCF-specific primers. On the left, PCR results using input DNA are shown. In the middle, PCR results after immunoprecipitation with unspecific IgG are shown for the confirmation of SMAD2 immunoprecipitation specificity. Shown in the bottom panel, ChIP for SMAD2 binding to PAI-1 was used as a positive control. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55689/55689fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55689/55689fig2.jpg" /> 
Figure 2: TGF-&#946;-induced STAT3 Binding to the TGF-&#946; Gene. (A) Schematic of the TGF-&#946; gene, with putative STAT3 binding sites shown as gray boxes with their relative position to the start codon. ChIP primer locations are shown with their relative positions to the TGF-&#946; start codon and the PCR product sizes indicated below. (B) ChIP for TGF-&#946;1-induced STAT3 binding to the TGF-&#946; gene. Chromatin-protein complexes of TGF-&#946;1-treated and untreated HepG2 and Hep3B cells were immunoprecipitated with anti-STAT3 antibody. PCR was performed using TGF-&#946;-specific primers. On the left, the PCR results using input DNA are shown. In the middle, the PCR results after immunoprecipitation with unspecific IgG are shown for confirmation of the STAT3 immunoprecipitation specificity. The upper panel shows the PCR results using primers specific for STAT3 binding site 1 (STB-1), and the lower panel shows the results for STAT3 binding to STAT3 binding site 2 (STB-2). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55689/55689fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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. ...

analysis-c-kit-ligand-promoter-using-chromatin

Nanyang Technological University

Research

JoVE Journal

Genetics

7.7K 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

Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous nature of most mammalian chromatin-modifying complexes. In order to overcome these hurdles, we have adapted a sequential salt extraction (SSE) assay for evaluating the relative binding affinities of chromatin-bound complexes. This easy and straightforward assay can be used by non-experts to evaluate the relative difference in binding affinity of two related complexes, the changes in affinity of a complex when a subunit is lost or an individual domain is inactivated, and the change in binding affinity after alterations to the chromatin landscape. By sequentially re-suspending bulk chromatin in increasing amounts of salt, we are able to profile the elution of a particular protein from chromatin. Using these profiles, we are able to determine how alterations in a chromatin-modifying complex or alterations to the chromatin environment affect binding interactions. Coupling SSE with other in vitro and in vivo assays, we can determine the roles of individual domains and proteins on the functionality of a complex in a variety of chromatin environments.

Sequential salt extraction of chromatin bound proteins is a useful tool for determining the binding properties of large protein complexes. This method can be employed to evaluate the role of individual subunits or domains in the overall affinity of a protein complex to bulk chromatin.

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous ...

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 ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Here, we describe a chromatin immunoprecipitation (ChIP) and ChIP-seq library preparation protocol to generate global epigenomic profiles from low-abundance chicken embryonic samples.

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for spermatogenesis. These techniques allow for visualization of protein and DNA localization patterns during meiotic events such as homologous chromosome pairing, synapsis and DNA repair. While a few protocols have been described in the literature, general chromatin spread techniques using mammalian prophase oocytes are limited and difficult due to the timing of meiosis initiation in fetal ovaries. In comparison, prophase spermatocytes can be collected from juvenile male mice with higher yields without the need for microdissection. However, it is difficult to obtain a pure synchronized population of cells at specific stages due to the heterogeneity of meiotic and post-meiotic germ cell populations in the juvenile and adult testis. For later stages of meiosis, it is advantageous to assess oocytes undergoing meiosis I (MI) or meiosis II (MII), because groups of mature oocytes can be collected from adult female mice and stimulated to resume meiosis in culture. Here, methods for meiotic chromatin spread preparations using oocytes dissected from fetal, neonatal and adult ovaries are described with accompanying video demonstrations. Chromosome missegregation events in mammalian oocytes are frequent, particularly during MI. These techniques can be used to assess and characterize the effects of different mutations or environmental exposures during various stages of oogenesis. As there are distinct differences between oogenesis and spermatogenesis, the techniques described within are invaluable to increase our understanding of mammalian oogenesis and the sexually dimorphic features of chromosome and protein dynamics during meiosis.

Oogenesis in mammals is known to be error-prone, particularly due to chromosome missegregation. This manuscript describes chromatin spread preparation methods for mouse prophase, metaphase I and II-staged oocytes. These fundamental techniques allow for the study of chromatin-bound proteins and chromosome morphology throughout mammalian oogenesis.

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for ...

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes that add or remove these marks in response to signals received by the cell. These PTMS are key contributors to the regulation of processes such as gene expression control and DNA repair. Chromatin immunoprecipitation (chIP) has been an instrumental approach for dissecting the abundance and localization of many histone PTMs throughout the genome in response to diverse perturbations to the cell. Here, a versatile method for performing chIP of post-translationally modified histones from the budding yeast Saccharomyces cerevisiae (S. cerevisiae) is described. This method relies on crosslinking of proteins and DNA using formaldehyde treatment of yeast cultures, generation of yeast lysates by bead beating, solubilization of chromatin fragments by micrococcal nuclease, and immunoprecipitation of histone-DNA complexes. DNA associated with the histone mark of interest is purified and subjected to quantitative PCR analysis to evaluate its enrichment at multiple loci throughout the genome. Representative experiments probing the localization of the histone marks H3K4me2 and H4K16ac in wildtype and mutant yeast are discussed to demonstrate data analysis and interpretation. This method is suitable for a variety of histone PTMs and can be performed with different mutant strains or in the presence of diverse environmental stresses, making it an excellent tool for investigating changes in chromatin dynamics under different conditions.

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

Here, we describe a protocol for chromatin immunoprecipitation of modified histones from the budding yeast Saccharomyces cerevisiae. Immunoprecipitated DNA is subsequently used for quantitative PCR to interrogate the abundance and localization of histone post-translational modifications throughout the genome.

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

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 ...

Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of a wide range of transcription factors (TFs) and cofactors mediating transcriptional activation or repression via changes in chromatin structure. Chromatin immunoprecipitation (ChIP) is a useful molecular biology approach for mapping histone modifications and profiling transcription factors/cofactors binding to DNA, thus providing a snapshot of the dynamic nuclear changes occurring during different biological processes.
To study transcriptional regulation in adipose tissue, samples derived from in vitro cell cultures of immortalized or primary cell lines are often favored in ChIP assays because of the abundance of starting material and reduced biological variability. However, these models represent a limited snapshot of the actual chromatin state in living organisms. Thus, there is a critical need for optimized protocols to perform ChIP on adipose tissue samples derived from animal models.
Here we describe a protocol for efficient ChIP-seq of both histone modifications and non-histone proteins in brown adipose tissue (BAT) isolated from a mouse. The protocol is optimized for investigating genome-wide localization of proteins of interest and epigenetic markers in the BAT, which is a morphologically and physiologically distinct tissue amongst fat depots.

Here we describe a protocol for efficient chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) of brown adipose tissue (BAT) isolated from a mouse. This protocol is suitable for both mapping histone modifications and investigating genome-wide localization of non-histone proteins of interest in vivo.

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of ...

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene expression. The cellular transcriptional machinery and its chromatin modification proteins coordinate to regulate transcription. To analyze transcriptional regulation at the molecular level, several experimental methods such as electrophoretic mobility shift, transient reporter and chromatin immunoprecipitation (ChIP) assays are available. We describe a modified ChIP assay in detail in this article because of its advantages in directly showing histone modifications and the interactions between proteins and DNA in cells. One of the key steps in a successful ChIP assay is chromatin shearing. Although sonication is commonly used for shearing chromatin, it is difficult to identify reproducible conditions. Instead of shearing chromatin by sonication, we utilized enzymatic digestion with micrococcal nuclease (MNase) to obtain more reproducible results. In this article, we provide a straightforward ChIP assay protocol using MNase.

Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...

Mapping Alzheimer's Disease Variants to Their Target Genes Using Computational Analysis of Chromatin Configuration

Genome-wide association studies (GWAS) have successfully identified hundreds of genomic loci that are associated with human traits and disease. However, because the majority of the genome-wide significant (GWS) loci fall onto the non-coding genome, the functional impact of many remain unknown. Three-dimensional chromatin interactions identified by Hi-C or its derivatives can provide useful tools to annotate these loci by linking non-coding variants to their actionable genes. Here, we outline a protocol to map GWAS non-coding variants to their putative genes using Alzheimer&#39;s disease (AD) GWAS and Hi-C datasets from human adult brain tissue. Putative causal single-nucleotide polymorphisms (SNPs) are identified by application of fine-mapping algorithms. SNPs are then mapped to their putative target genes using enhancer-promoter interactions based on Hi-C. The resulting gene set represents AD risk genes, as they are potentially regulated by AD risk variants. To garner further biological insights into molecular mechanisms underlying AD, we characterize AD risk genes using developmental brain expression data and brain single-cell expression profiles. This protocol can be expanded to any GWAS and Hi-C datasets to identify putative target genes and molecular mechanisms underlying various human traits and diseases.

We present a protocol to identify functional implications of non-coding variants identified by genome-wide association studies (GWAS) using three-dimensional chromatin interactions.

Genome-wide association studies (GWAS) have successfully identified hundreds of genomic loci that are associated with human traits and disease. However, ...