This research is supported by Genentech royalties to City of Hope. This work is not supported in whole or in part by the National Institutes of Health.

1. Preparation of Reagents
<ol>
	<li>Make 18.5% paraformaldehyde (PFA) solution. Add 0.925 g of PFA, 4.8 mL of water (use ultrapurified water throughout the protocol) and 35 &#181;L of 1 M KOH in a 50 mL conical plastic tube. Close the cap tightly and heat the tube in a 400-600 mL glass beaker containing approximately 200 mL of water using a microwave. Remove the tube before the water starts boiling and vortex the tube to dissolve PFA. Allow PFA to cool to room temperature and store on ice. 
	NOTE: Repeat heating and mixing until PFA dissolves completely. Prepare PFA solution immediately before crosslinking (step 2.1.3).</li>
	<li>Make 1.25 M glycine solution. Dissolve 14.07 g of glycine in 150 mL of water and filter through a 0.22 &#181;m pore-size filter. Store at 4 &#176;C.</li>
	<li>Make ChIP cell lysis buffer. Dissolve 378 mg of PIPES, 10.6 mL of 2 M KCl, and 1.25 g of branched octylphenoxy poly(ethyleneoxy)ethanol in water. Adjust pH to 8.0 with 1 M KOH at 4 &#176;C and make up to 250 mL. Filter through a 0.22 &#181;m pore-size filter and store at 4 &#176;C.</li>
	<li>Make micrococcal nuclease (MNase) digestion buffer. To make 1 mL, mix 0.1 mL of 10x MNase digestion buffer, 10 &#181;L of 100x BSA solution, 10 &#181;L of 0.1 M dithiothreitol and 0.88 mL of water.</li>
	<li>Make 1 M NaHCO3. Dissolve 4.2 g of NaHCO3 in 50 mL of water and filter through a 0.22 &#181;m pore-size filter. Store at room temperature.</li>
	<li>Make 10% sodium dodecyl sulfate (SDS). Dissolve 5 g of SDS in 50 mL of water. Store at room temperature.</li>
	<li>Make elution buffer. Mix 15 &#181;L of 1 M NaHCO3, 15 &#181;L of 10% SDS, and 120 &#181;L of water to obtain 150 &#181;L of elution buffer for one sample. 
	NOTE: Increase the volumes proportionally depending on the number of samples.</li>
	<li>Make 3 M sodium acetate (pH 5.2) solution. Dissolve 24.6 g of sodium acetate anhydrous in water. Adjust pH to 5.2 with acetic acid and make up 100 mL. Filter through a 0.22 &#181;m pore-size filter and store at room temperature.</li>
</ol>
2. Determination of MNase Digestion Conditions
NOTE: In the step 2 of protocol, an example using VCaP, human prostate cancer cells is presented. Any mammalian cell lines can be used; see Note at the steps.
<ol>
	<li>Preparation of crosslinked chromatin
	<ol>
		<li>Maintain VCaP cells in DME-high glucose supplemented with 10% fetal bovine serum (FBS) at 37 &#176;C in a carbon dioxide incubator. Seed VCaP cells at 4 x 106 cells in six 6 cm dishes in 4 mL of culture media and culture for 3 days. 
		NOTE: Use appropriate culture media for each cell line. Up to 10 x 106 cells can be seeded in a 6 cm or 10 cm dish. For suspended cells, seed cells in T25 flasks. The number of dishes or flasks depends how many doses are tested for MNase digestion. If 4 doses are tested, prepare 6 dishes or flasks. See also step 2.2.</li>
		<li>Detach VCaP cells from one dish using trypsin and count the cell. Calculate cell number in one dish. For suspended cells, remove a small amount of cell suspension from one flask and count cell number.</li>
		<li>Add 0.229 mL of 18.5% PFA to a 6 cm dish in 4 mL of culture media at a final concentration of 1%. Gently but thoroughly swirl a dish or flask to distribute PFA evenly. Incubate at room temperature for exactly 10 min. 
		NOTE: In this step, chromatin and proteins are crosslinked. As PFA is toxic, carry out this step in a chemical fume hood and use proper personal protective equipment.</li>
		<li>After 10 min, add 0.47 mL of 1.25 M glycine solution at a final concentration of 125 mM. Incubate at room temperature for 5 min. 
		NOTE: Glycine neutralizes excess PFA to stop further crosslinking.</li>
		<li>Aspirate formaldehyde/glycine/culture media. Wash the cells by adding 4 mL of phosphate-buffered saline (PBS) twice. For suspended cells, transfer the cell suspension to a 15 mL conical tube and centrifuge at 300 x g for 5 min at room temperature. Aspirate the supernatant and add one medium volume of PBS and fully suspend. Repeat this step. Dispose liquid wastes properly since they contain formaldehyde.</li>
		<li>Prepare PBS containing protease and phosphatase inhibitor cocktail (PIC) by adding 1/100 volume of 100x PIC to PBS. Aspirate PBS from a dish and add 1 mL per dish of PBS containing PIC. Scrape the cells and transfer the cell suspension to a 1.5 mL microcentrifuge tube. For suspended cells, simply transfer the suspension to a 1.5 mL tube.</li>
		<li>Centrifuge tubes at 3,000 x g for 5 min at room temperature to recover the cell pellet. Remove the PBS completely using a pipette. Record the cell number per tube and store the cell pellet at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Cell lysis and MNase digestion 
	NOTE: The reagent volume in the following steps is based on one tube containing 6 x 106 cells. Adjust reagent volume proportionally depending on cell number; when one tube contains 2 x 106 cells, use 100 &#181;L of ChIP cell lysis buffer (step 2.2.1), MNase digestion buffer (step 2.2.3), and ChIP dilution buffer (step 2.2.5), and 10 &#181;L of 0.5 M EDTA (pH 8.0) (step 2.2.4).
	<ol>
		<li>Thaw the stored cell pellet (VCaP cells, containing 6 x 106 cells per tube) prepared in step 2.1.7 on ice. Prepare ChIP cell lysis buffer supplemented with PIC by adding 1/100 volume of 100x PIC to the buffer. Add 300 &#181;L of ChIP cell lysis buffer supplemented with PIC and resuspend the pellet thoroughly. Vortex the tube for 15 s and incubate the suspension on ice for 10 min.</li>
		<li>Centrifuge at 9,000 x g for 3 min at 4 &#176;C and remove the supernatant completely. Resuspend the pellet in 300 &#181;L of MNase digestion buffer.</li>
		<li>Dilute MNase (2,000 gel unit/&#181;L) with MNase digestion buffer to give 50 gel units/&#181;L. Add 0, 0.5, 1, 2, 4 &#181;L of 50 gel units/&#181;L of MNase to the suspension and incubate at 37 &#176;C for exactly 10 min, mixing by inversion every 2.5 min. 
		NOTE: Table 1 shows optimal amounts of MNase in several cell lines.</li>
		<li>Add 30 &#181;L of 0.5 M EDTA (pH 8.0) to terminate MNase digestion and vortex briefly. Incubate for 5 min on ice. Centrifuge at 9,000 x g for 5 min at 4 &#176;C and remove the supernatant completely.</li>
		<li>Prepare ChIP dilution buffer supplemented with PIC by adding 1/100 volume of 100x PIC to the buffer. Resuspend the pellet in 300 &#181;L of ChIP dilution buffer supplemented with PIC.</li>
		<li>Sonicate the suspension on ice using a sonicator equipped with a microtip probe. Use the following sonication conditions: amplitude 2, processed time 15 s, pulse ON 5 s, pulse OFF 30 s. Take 1 &#181;L of the suspension and spot onto a slide glass and observe them using a microscope. Ensure that the cell structure is almost broken. 
		NOTE: Figure 1A shows representative microphotographs of the VCaP cell suspension before and after sonication. This step is for releasing digested chromatin into the supernatant by breaking nuclear membranes. A power setting should be adjusted to less than 5% of maximum power and try sonication for 5 s three times with more than a 30 s interval. If wattage control is available, adjust power setting to 5 W and process the samples for total 15 s as mentioned above to give total power of 70-75 J. Check whether the cell structure is broken as mentioned above. If not enough, repeat one more time. The condition described above is practically applied to all cell lines tested.</li>
		<li>Centrifuge at 9,000 x g for 10 min at 4 &#176;C, transfer the supernatant to a new 1.5 mL microcentrifuge tube, and save 20 &#181;L of the digested chromatin for step 2.3. Store the remainder at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Reverse crosslinking, purification of DNA, and analysis of digested chromatin
	<ol>
		<li>Add 75 &#181;L of water, 4 &#181;L of 5 M NaCl, and 1 &#181;L of proteinase K to a 1.5 mL screw tube. Add 20 &#181;L of digested chromatin from various MNase digestion conditions prepared in step 2.2.7 to tubes containing water, NaCl, and proteinase K. Close the cap tightly, mix completely, and incubate the tube at 65 &#176;C overnight. 
		NOTE: This step is for the removal of crosslinking between protein and DNA.</li>
		<li>Prepare two 1.5-mL microcentrifuge tubes per condition. Add 100 &#181;L of phenol:chloroform:isoamylalcohol (25:24:1) (PCI) to one 1.5 mL tube. Add 10 &#181;L of 3 M sodium acetate (pH 5.2) and 2 &#181;L of glycogen to another tube. 
		NOTE: The use of chloroform:isoamylalcohol is not necessary7. Increasing volume may result in low DNA recovery after ethanol precipitation8.</li>
		<li>Centrifuge the tube containing digested chromatin from step 2.3.1 briefly and add 100 &#181;L of PCI. Close the cap tightly, and vortex vigorously to form emulsion. Centrifuge the tube at maximum speed (e.g., 20,000 x g) for 30 s at room temperature.</li>
		<li>Carefully take the upper phase containing DNA and add to the tube containing PCI prepared in step 2.3.2. Vortex vigorously and centrifuge the tube as step 2.3.3. 
		NOTE: If lower phase is contaminated, centrifuge the tube again, or remove most of the lower phase first, centrifuge the tube, and take the upper phase.</li>
		<li>Carefully take the upper phase as described in step 2.3.4 and add to the tube containing sodium acetate and glycogen prepared in step 2.3.2. Add 250 &#181;L of ethanol. Mix by inversion and incubate for 10 min at room temperature. Centrifuge the tube at maximum speed (e.g., 20,000 x g) for 30 min at 4 &#176;C. 
		NOTE: Incubation of the solution at room temperature does not affect DNA recovery8,9.</li>
		<li>Confirm the pellets on the bottom of the tube. Carefully remove the supernatant so as not to disturb the pellet and add 500 &#181;L of 70% ethanol. Centrifuge the tube at maximum speed (e.g., 20,000 x g) for 5 min at 4 &#176;C.</li>
		<li>Remove the supernatant completely and dry the pellet for approximately 5 min at room temperature. Dissolve the pellet in 20 &#181;L of 10 mM Tris&#183;HCl/1 mM EDTA (pH 8.0) (TE). Measure DNA concentration using a UV spectrophotometer.</li>
		<li>Mix 0.5 &#181;g of DNA with a gel loading dye and apply to 2% agarose gel. Electrophorese the samples at 100 V in tris-acetate-EDTA buffer until purple dye migrated to the two-third of the gel and stain a gel with 0.5 &#181;g/mL ethidium bromide for 10 min. Take a picture of the gel. 
		NOTE: See Figure 2 for detail.</li>
	</ol>
	</li>
</ol>
3. ChromatinImmunoprecipitation
<ol>
	<li>Preparation of digested chromatin
	<ol>
		<li>Prepare thecells. For adherent cells, seed 2-10 x 106 cells per dish in 2 dishes (6 cm or 10 cm) per treatment group and allow the cells to attach to the bottom of dishes for more than 1 day. For suspended cells, seed in 2 T25 flasks per treatment group. Treat the cells as desired.</li>
		<li>Take one dish or flask from each group and count cell number as described in step 2.1.2. Prepare crosslinked chromatin as mentioned in steps 2.1.3-2.1.7.</li>
		<li>Lyse the cell pellet and MNase digestion as described in steps 2.2.1-2.2.7. Use the optimal amount of MNase to digest chromatin as determined in step 2. Store digested chromatin at -80 &#176;C.</li>
		<li>Reverse crosslink 20 &#181;L of digested chromatin and purify DNA as described in step 2.3.8. Measure DNA concentration as described in step 2.3.7. Electrophorese the samples in 2% agarose gel to check the size of digested chromatin as mentioned in step 2.3. 
		NOTE: The DNA concentration is identical to that of stored digested chromatin prepared in step 3.1.3.</li>
		<li>Dilute a reverse cross-linked digested chromatin sample from step 3.1.4 to give 10 ng/&#181;L with water and serially dilute to make concentrations of 1, 0.1, and 0.01 ng/&#181;L. Store at -20 &#176;C. 
		NOTE: These samples are used for real-time PCR standards.</li>
	</ol>
	</li>
	<li>Immunoprecipitation 
	NOTE: In the steps 3.2-3.5 of protocol, anti-trimethylated histone H3 lysine 4 (H3K4me3) occupancy in VCaP cells is determined. See also Figure 3.
	<ol>
		<li>Thaw digested chromatin from step 3.1.3. Keep all samples on ice.</li>
		<li>Prepare ChIP dilution buffer supplemented with PIC by adding 1/100 volume of 100x PIC to the buffer. Dilute digested chromatin to 5 &#181;g/500 &#181;L with ChIP dilution buffer supplemented with PIC. Prepare 1,000 &#181;L plus extra for (1) IP with nonimmune IgG (Control IgG), (2) IP with H3K4me3.</li>
		<li>Add 5 &#181;L of 5 &#181;g/500 &#181;L digested chromatin to a 1.5 mL screw tube as an input sample (1% of one IP). Store the sample at -80 &#176;C.</li>
		<li>Add 500 &#181;L each of 5 &#181;g/500 &#181;L digested chromatin to a 1.5 mL screw tube as an IP sample. Add 2 &#181;g of antibody to the tube. Close the cap and incubate the tube at 4 &#176;C overnight with gentle mixing using a rocking platform. 
		NOTE: Although optimal antibody amount should be determined empirically, 2 &#181;g per IP is recommended at first.</li>
		<li>Add 30 &#181;L of ChIP-grade protein G magnetic beads per IP. Incubate the tube at 4 &#176;C for 2 h with gentle mixing using a rocking platform. 
		NOTE: Prewash of magnetic beads is not necessary.</li>
	</ol>
	</li>
	<li>Washing immune complex and reversing crosslinking
	<ol>
		<li>Spin down the tube from step 3.2.5 briefly. Place the tube in a polyethylene rack containing neodinium magnets for 1 min. Carefully remove the supernatant by aspiration.</li>
		<li>Add 0.5 mL per tube of low salt immune complex wash buffer. Disperse the beads by gently tapping or briefly vortexing the tube. Incubate the tube at 4 &#176;C for 5 min with gentle mixing using a rocking platform. After 5 min, repeat step 3.3.1.</li>
		<li>Add 0.5 mL of high salt immune complex wash buffer per tube. Disperse and wash the beads as step 3.3.2. After washing, repeat step 3.3.1.</li>
		<li>Add 0.5 mL of LiCl immune complex wash buffer per tube. Disperse and wash the beads as step 3.3.2. After washing, repeat step 3.3.1.</li>
		<li>Place the tube in a magnetic rack for 1 min. Remove the remaining supernatant completely.</li>
		<li>Add 150 &#181;L of elution buffer to the tube. Vortex the tube to disperse the beads completely. Close the cap and incubate the tube at 65 &#176;C for 30 min, mixing by inversion or vortexing every 5 min to disperse the beads thoroughly.</li>
		<li>During the incubation, prepare a 1.5 mL screw tube and add 6 &#181;L of 5 M NaCl and 2 &#181;L of proteinase K. Thaw 1% input sample prepared in step 3.2.3. Add 150 &#181;L of elution buffer, 6 &#181;L of 5 M NaCl and 2 &#181;L of proteinase K to 1% input sample.</li>
		<li>After the incubation, spin down the tube. Place the tube in a magnetic rack for 1 min and transfer the supernatant to the screw tube containing NaCl and proteinase K prepared in step 3.3.7.</li>
		<li>Close the cap and vortex all IP and input samples to mix completely. Incubate the tube at 65 &#176;C overnight.</li>
	</ol>
	</li>
	<li>DNA purification
	<ol>
		<li>Prepare the tube as described in 2.3.2 and add 150 &#181;L of PCI instead of 100 &#181;L. In another tube, add 12 &#181;L of 3 M sodium acetate (pH 5.2) and 2 &#181;L of glycogen.</li>
		<li>Remove the tube from the incubator. Spin down the tube and add 150 &#181;L of PCI.</li>
		<li>Vortex vigorously the tube to form emulsion. Centrifuge the tube at maximum speed (e.g. 20,000 x g) for 30 s at room temperature.</li>
		<li>Carefully transfer 140 &#181;L of the upper phase to the tube containing PCI prepared in step 3.4.1. Repeat step 3.4.3. 
		NOTE: See also Note in step 2.3.4.</li>
		<li>Carefully transfer 120 &#181;L of upper phase to the tube containing sodium acetate and glycogen prepared in step 3.4.1. 
		NOTE: See also Note in step 2.3.4.</li>
		<li>Add 300 &#181;L of ethanol. Mix by inversion and incubate for 10 min at room temperature.</li>
		<li>Centrifuge the tube at maximum speed (e.g., 20,000 x g) for 30 min at 4 &#176;C. Wash the pellet as described in step 2.3.6.</li>
		<li>After drying the pellet as described in step 2.3.7, dissolve the pellet in 50 &#181;L of TE. Store at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Detection of DNA fragments using real-time PCR
	<ol>
		<li>Thaw the following samples: (1) IP with Control IgG, (2) IP with anti-H3K4me3, (3) 1% input sample. Thaw also 4 doses of standards prepared in step 3.1.5 (total of 7 samples). 
		NOTE: Use standards from the same group for the quantification of DNA amounts in the input and IP samples.</li>
		<li>Make PCR working solution for 8 samples. Mix 40 &#181;L of 2x real-time PCR supermix, 8 &#181;L each of 4 &#181;M forward and reverse primers, and 8 &#181;L of water. 
		NOTE: One PCR reaction mixture contains 5 &#181;L of 2x real-time PCR supermix, 1 &#181;L each of 4 &#181;M forward and reverse primers, and 1 &#181;L of water. Primer sequences are listed in Table 2.</li>
		<li>Aliquot 8 &#181;L of PCR working solution into one well of a PCR plate. Add 2 &#181;L of samples from step 3.4.8 or standards from step 3.1.5.</li>
		<li>Seal the PCR plate and run PCR using the following conditions: 1) 95 &#176;C for 3 min for initial denaturation, 2) 95 &#176;C for 10 s for denaturation, 3) 56 &#176;C for 30 s for annealing, 4) 72 &#176;C for 30 s for extension and data collection, repeat steps 2) to 4) for 55 cycles. After cycling, measure a melting curve of the PCR product. Analyze the data. 
		NOTE: Raw data for Figure 3 are shown in Table 3. Calculate starting quantity (SQ) of the samples using a standard curve. Multiply SQ in 1% input by 100 to adjust a SQ of 1% input sample to one IP to give adjusted SQ in Input (Eq. 1). Divide SQ in IP sample by adjusted SQ in Input to give % input value (percent input method, Eq. 2). To calculate fold enrichment, divide SQ in IP with anti-H3K4me3 by SQ in IP with control IgG (fold enrichment method, Eq. 3). 
		Adjusted SQ in Input = (SQ in 1% input) x 100 (1) 
		Percent input of H3K4me3 = 100 x (SQ in IP with anti-H3K4me3) / (Adjusted SQ in Input) (2) 
		Fold enrichment of H3K4me3 = (SQ in IP with anti-H3K4me3) / (SQ in IP with control IgG) (3)</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Although sonication is commonly used to obtain fragmented chromatin, it is time-consuming and cumbersome to identify reproducible conditions. In this protocol, we used MNase digestion because enzyme digestion should be easier to identify reproducible conditions. A brief sonication step after MNase digestion (see step 2.2) was necessary to break the cell membrane and to release the digested chromatin. Therefore, the sonication power in our protocol should be as low as possible. We use the same sonication conditions for all cells we employed (see Figure 1 and Table 1) to obtain complete breakdown of the membrane.
Digestion of chromatin by MNase is a critical step in our protocol, and thus we have exerted great effort to optimize the conditions for the digestion of chromatin inside cells. Digestion activity is determined by the amounts of enzyme and substrate (chromatin) and incubation time. In addition, since ploidy varies among different cells, the optimum conditions for MNase digestion must be identified for each cell type. Once established, the same conditions can be applied irrespective of the cell treatments. We always check the chromatin digestion patterns in each experiment to ensure that the digestion patterns are suitable for ChIP assays, as shown in Figure 2A, lane 5.
Some factors affect the results of ChIP assays. It is important to use the right amount of digested chromatin in our protocol. In many protocols, the cell number but not the amount of chromatin for one IP is shown15,16. These experimental conditions produce high variability in the amount of chromatin among cells due to ploidy differences. We use 5 &#181;g of chromatin and 2 &#181;g of antibody per one IP in the assay, thus our protocol is more straightforward and clearer than other protocols, although optimum amounts of antibody for IP may be needed. The selection of antibodies is also important; use ChIP assay-validated antibodies.
There are two methods to analyze ChIP PCR data: the percent input method and the fold enrichment method. In the percent input method, signals from IP samples are divided by signals from total chromatin in the IP sample. In the fold enrichment method, signals from IP with a specific antibody such as H3K4me3 are divided by signals from IP with the control IgG. The later method is only applicable when signals from IP with the control IgG are similar and reproducible at multiple targets or identical targets under the different physiological conditions. In practice, the signal levels vary so the fold enrichment value has high variability as shown in Figure 4C. Therefore, we do not recommend using the fold enrichment method to represent ChIP data in our method.
MNase favors an euchromatin &#8216;open&#8217; environment and is not accessible to a heterochromatin structure, suggesting that MNase digestion may produce some bias. It has been reported that enrichment of lamin A-interacting chromatin domains is different in between sonication-sheared and MNase-digested chromatin preparations17. Thus, MNase digestion in ChIP assay may not be suitable to analyze nuclear structure-associated molecules such as lamin A/C and Special AT-rich Sequence Binding Protein 1 (SATB1).
In ChIP assay protocols designed for downstream microarray (ChIP-on-chip) or sequencing (ChIP-seq) analyses, RNase is used during the DNA purification step, although not in protocols designed for PCR analyses18. We have not tested whether our protocol is compatible with ChIP-on-chip and ChIP-seq analyses, but we assume that our protocol is applicable when samples are treated with RNase. If RNase treatment is needed, use 2 &#181;L of 10 mg/mL DNase-free RNase A instead of proteinase K in step 3.3 and incubate at 65 &#176;C overnight. Before purifying DNA (step 3.4), add proteinase K and incubate for an additional 1 h at 60 &#176;C.
We routinely carry out ChIP assays with modified histone and transcription factors using the method described here and have shown that the chromatin remodeling factor, AT-rich interaction domain 5B, regulates AR gene expression by changing the occupancy of PolII(pS5) and H3K4me3 in the AR promoter19. We believe that our protocol is technically easier than other ChIP assays and is widely acceptable in molecular biology research.

Gene expression in mammalian cells is tightly and dynamically regulated, and transcription is one of the key steps. Gene transcription is mainly regulated by transcription factors and histones. A transcription factor is a protein that binds to specific DNA sequences and controls gene transcription. These factors either promote or inhibit the recruitment of RNA polymerase II (PolII), which initiates mRNA synthesis from genomic DNA as a template1. Histone modifications such as acetylation and methylation of histone tail residues positively and negatively affect gene transcription by changing the chromatin structure2. Since alterations in gene expression affect the cellular context, it is essential to examine the molecular mechanisms by which transcription is regulated.
To date, several methods for investigating the regulation of gene transcription are available. Electrophoretic mobility shift assay (EMSA), also called a gel shift assay, is used for analyzing a protein-DNA interaction3. A nuclear extract from cells of interest is incubated with a radioactive isotope (for example, 32P)-labeled DNA probe and electrophoresed on a polyacrylamide gel. Its autoradiogram shows that the DNA-protein complex migrates slower than the probe in a gel. In the presence of an antibody against the protein, the DNA-protein-antibody complex migrates in a gel more slowly than the DNA-protein complex. This supershifted band reveals specific binding between the DNA and protein. However, EMSA only determines a specific DNA-protein interaction in a cell-free system, and therefore it remains unknown whether the interaction controls transcription in living cells. The transient reporter assay, commonly called luciferase reporter assay, was developed to address gene expression regulation in cells. Typically, an upstream genomic region of a gene of interest is inserted into a reporter plasmid, transiently transfected into cells, and the reporter activity is measured. A variety of deletion mutants allows the identification of regions that are responsible for gene regulation. Even though a reporter assay is a useful tool for identifying transcription factors and binding DNA sequences controlling transcription, this method has a major disadvantage in that a reporter plasmid is free of chromatin structure and does not reflect &#8220;real&#8221; transcription machinery. In addition, changes in histone modifications cannot be determined by the system.
The development of the chromatin immunoprecipitation (ChIP) method was based on Jackson and Chalkley&#8217;s reports that &#8220;whole cell&#8221; fixation with formaldehyde preserved chromatin structure4,5. Since then, many related techniques have been developed and improved6. In ChIP assays, cells are fixed with formaldehyde to cross-link DNA and proteins. The chromatin is fragmented and then immunoprecipitated with antibodies of interest. The immune complex is washed, and DNA is purified. PCR amplification with primers targeted to a particular region of the genome reveals the occupancy of proteins of interest in the genome.
Although ChIP is a powerful tool to identify the interactions of proteins such as transcription factors and modified histones with DNA, the method involves some difficulties, such as a chromatin fragmentation step, in practice. Sonication has been widely used for shearing chromatin; however, it is cumbersome to identify reproducible conditions. Micrococcal nuclease (MNase) treatment is an alternative method for chromatin shearing. MNase is an endo-exonuclease that digests double-stranded, single-stranded, circular and linear DNA and RNA. It is relatively easy to determine the conditions, including the amounts of chromatin and enzyme, temperature, and incubation time, for optimum chromatin fragmentation. We modified and simplified the existing protocols, and we established a straightforward and reproducible method. This paper provides the protocol for a ChIP assay using MNase in mammalian cells.

<table><tbody><tr><td>0.5 M EDTA (pH 8.0)</td><td>Thermo Scientific</td><td>AM9010</td><td></td></tr><tr><td>2 M KCl</td><td>Thermo Scientific</td><td>AM9010</td><td></td></tr><tr><td>2x iQ SYBR Green supermix</td><td>Bio-Rad</td><td>1706862</td><td></td></tr><tr><td>5 M NaCl</td><td>Thermo Scientific</td><td>AM9010</td><td></td></tr><tr><td>50 bp DNA ladder</td><td>New England Biolabs</td><td>N3236S</td><td></td></tr><tr><td>Agarose</td><td>Research Product International</td><td>A20090</td><td></td></tr><tr><td>Branched octylphenoxy poly(ethyleneoxy)ethanol</td><td>Millipore Sigma</td><td>I8896</td><td>IGEPAL CA-630</td></tr><tr><td>ChIP-grade protein G magnetic beads</td><td>Cell signaling technology</td><td>9006S</td><td></td></tr><tr><td>Chromatin Immunoprecipitation (ChIP) Dilution Buffer</td><td>Millipore Sigma</td><td>20-153</td><td>Buffer composition: 0.01% SDS, 1.1% Triton X- 100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl.</td></tr><tr><td>Gel Loading Dye Purple (6x)</td><td>New England Biolabs</td><td>B7024S</td><td></td></tr><tr><td>Glycine</td><td>Bio-Rad</td><td>161-0724</td><td>Electropheresis grade</td></tr><tr><td>Glycogen</td><td>Millipore Sigma</td><td>G1767</td><td>19-22 mg/mL</td></tr><tr><td>Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100x)</td><td>Thermo Scientific</td><td>78445</td><td></td></tr><tr><td>High Salt Immune Complex Wash Buffer</td><td>Millipore Sigma</td><td>20-155</td><td>Buffer composition: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl.</td></tr><tr><td>Histone H3K4me3 antibody (pAb)</td><td>Active Motif</td><td>39915</td><td></td></tr><tr><td>LiCl Immune Complex Wash Buffer</td><td>Millipore Sigma</td><td>20-156</td><td>Buffer composition: 0.25 M LiCl, 1% IGEPAL CA630, 1% deoxycholic acid (sodium salt), 1 mM EDTA, 10 mM Tris, pH 8.1.</td></tr><tr><td>Low Salt Immune Complex Wash Buffer</td><td>Millipore Sigma</td><td>20-154</td><td>Buffer composition: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl.</td></tr><tr><td>Magna GrIP Rack (8 well)</td><td>Millipore Sigma</td><td>20-400</td><td>Any kind of magnetic separation stands that are compatible with a 1.5 mL tube is fine.</td></tr><tr><td>Micrococcal nuclease</td><td>New England Biolabs</td><td>M0247S</td><td>comes with 10x buffer (500 mM Tris-HCl, 50 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, pH 7.9 at 25 &amp;deg;C) and 100x BSA (10 mg/mL)</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>JT Baker</td><td>3506-01</td><td></td></tr><tr><td>Normal rabbit IgG</td><td>Millipore Sigma</td><td>12-370</td><td></td></tr><tr><td>PIPES</td><td>Millipore Sigma</td><td>P6757</td><td></td></tr><tr><td>Proteinase K</td><td>Millipore Sigma</td><td>3115887001</td><td></td></tr><tr><td>Real-time PCR system</td><td>Bio-Rad</td><td>CFX96, C1000</td><td></td></tr><tr><td>RNA pol II CTD phospho Ser5 antibody</td><td>Active Motif</td><td>39749</td><td></td></tr><tr><td>SDS</td><td>Boehringer Mannheim</td><td>100155</td><td>Electropheresis grade</td></tr><tr><td>sodium acetate</td><td>Millipore Sigma</td><td>S5636</td><td></td></tr><tr><td>Sonicator equipped with a microtip probe</td><td>QSONICA</td><td>Q700</td><td>Any kind of sonicators that are compatible with a 1.5 mL tube is fine.</td></tr><tr><td>UltraPure Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)</td><td>Thermo Scientific</td><td>15593031</td><td>pH 8.05</td></tr></tbody></table>

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.

Digesting chromatin is one of the important steps for a ChIP assay. We used MNase to digest chromatin to obtain a mixture of nucleosome oligomers. In the MNase digestion step, MNase can go through the nuclear membrane and digest chromatin. However, the digested chromatin cannot go through the membrane and remains in the nuclei. To release the digested chromatin from the nuclei, brief sonication is needed. Figure 1A shows microphotographs before and after sonication of VCaP cell suspension. Without sonication, the cell structure remains intact, indicating that the chromatin is present in the nuclei. A brief sonication breaks the cell structure, and checking the cells in microphotographs helps to determine the brief sonication conditions. We also represented other examples for brief sonication in 293T cells (Figure 1B) the human B-cell acute lymphoblastic leukemia cell line, REH cells (Figure 1C) and the human prostate cancer cell line, LNCaP (Figure 1D).
Figure 2A shows chromatin fragmentation after treatment with different amounts of MNase in VCaP cells. We treated 6 x 106 crosslinked VCaP cell pellets with 0, 50, 100, 200 gel units of MNase in 300 &#181;L of digestion buffer for 10 min at 37 &#176;C. After purification of the digested chromatin, 500 ng of DNA was analyzed on 2% agarose gel and stained with ethidium bromide. Without adding MNase, a smear pattern with a very high molecular weight appeared (lane 1). The addition of MNase gave a ladder pattern (N; a mononucleosome unit), showing that MNase digests internucleosome (lanes 2-5). Figure 2B shows an inappropriate digestion pattern. Overdigestion mainly resulted in mononucleosome production (Figure 2B, lane 7). We should find the proper conditions that produce chromatin fragments up to 900 bp (one to five nucleosomes; e.g., lane 5).
To check whether the ChIP assay is performed properly, it is essential to have appropriate controls in the assay. For immunoprecipitation, nonimmune IgGs from the same species as the antibodies of interest are used as a control that shows nonspecific binding to the same region (see discussion). In addition, it is recommended to measure the binding of the proteins (occupancy) in both positive and negative regions. It has been widely accepted that H3K4me3 occupancy is distributed between approximately one kilobase (kb) upstream and downstream of transcription start sites10,11. We measured H3K4me3 occupancy in the AR genome spanning approximately 20 kb upstream through 12 kb downstream of the AR transcription start site (AR-TSS) in AR-positive VCaP cells. Digestion pattern of chromatin in VCaP cells used in this experiment was shown in Figure 3A, indicating the proper digestion of chromatin. The highest occupancy of H3K4me3 was observed around the AR-TSS and 0.5 kb and 1 kb upstream of the AR-TSS (Figure 3B). As long as genes are transcriptionally active, TSSs can be &#8220;positive regions&#8221;. Regions located at 19 kb and 8 kb upstream and 12 kb downstream of AR-TSS, however, had little occupancy of H3K4me3 (Figure 3B), indicating that these can be used as &#8220;negative regions&#8221;.
It has been shown that an androgen increases RNA polymerase II occupancy in the PSA promoter and enhancer in LNCaP cells using sheared chromatin by sonication12,13. We therefore tested the validity of our protocol by measuring active RNA polymerase II occupancy (phosphorylated RNA polymerase II at serine 5; PolII(pS5)) in the cells. We performed the same experiment to check the reproducibility of our method. LNCaP cells were cultured in steroid-starved medium for 3 days and stimulated with a vehicle or 10 nM dihydrotestosterone (DHT) for 4 h. Active RNA polymerase II occupancy was measured by immunoprecipitation with anti-PolII(pS5), followed by real-time PCR. Figure 4A shows a reproducible digestion pattern of chromatin from LNCaP cells in three independent experiments. As shown in Figure 4B, DHT significantly increased PolII(pS5) occupancy in the PSA promoter and enhancer when using percent input method. We also calculated the occupancy using fold enrichment method (Figure 4C) and found that no significant difference in PolII(pS5) in the PSA promoter was observed with or without DHT treatment. DHT did not affect occupancy in the GAPDH promoter as previously published14. Importantly, our data were similar to that obtained from sonication-sheared chromatin samples12,13.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59375/59375fig1.jpg" /> 
Figure 1: Representative microphotographs of crosslinked cell pellets before and after sonication. Crosslinked VCaP (A), 293T (B), REH (C) and LNCaP (D) cell pellets were treated with MNase, and pellets were resuspended in ChIP dilution buffer. Before and after sonication, pictures of the suspensions were taken. Scale bar = 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59375/59375fig1large.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/59375/59375fig2.jpg" /> 
Figure 2: Representative agarose gel analysis of digested chromatin. (A) Crosslinked chromatin was prepared from VCaP cells and digested with various amounts of MNase as described in step 2.2. Digested chromatin was reverse crosslinked, purified, and analyzed in a 2% agarose gel. N; a mononucleosome unit. (B) Chromatin of VCaP cells was digested with 250 gel units of MNase per 2 x 106 cells at 37 &#176;C for 20 min and analyzed (as described in A). Larger amounts of MNase and longer incubation times caused almost complete digestion of chromatin to form mononucleosomes (150 bp). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59375/59375fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59375/59375fig3.jpg" /> 
Figure 3: H3K4me3 occupancy in the AR genome. Digested chromatin was prepared from VCaP cells. (A) Digestion pattern was analyzed using an agarose gel. (B) 5 &#181;g of digested chromatin was immunoprecipitated with 2 &#181;g of either normal rabbit IgG or anti-H3K4me3 antibody as mentioned in step 3.1 and step 3.2. Immune complexes were washed and eluted from beads, and reverse crosslinked. Purified DNA fragments were analyzed using real-time PCR with the primer sets listed in Table 2. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59375/59375fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59375/59375fig4.jpg" /> 
Figure 4: Androgen increased active RNA polymerase II occupancy in the PSA promoter and enhancer. Steroid-starved LNCaP cells were treated with or without 10 nM DHT for 4 h, and digested chromatin was prepared. (A) Digestion pattern of chromatin from LNCaP cells in three independent experiments. (B,C) Digested chromatin was immunoprecipitated with an anti-PolII(pS5) antibody, and DNA fragments were purified as described for Figure 3. The occupancy of active RNA polymerase II in the PSA promoter, enhancer, and GAPDH promoter as a percent input (B) and fold enrichment (C) was determined using real-time PCR with the primer sets listed in Table 2. The results shown are mean &#177; SE of three independent experiments. (*); p&#60;0.05, (**); p &#60; 0.01 versus 0 nM DHT treatment. NS; not significant versus 0 nM DHT. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59375/59375fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell line</td>
			<td>gel units per two million cells in 100 &#181;L of buffer, 37 &#176;C for 10 min</td>
		</tr>
		<tr>
			<td>LNCaP</td>
			<td>267</td>
		</tr>
		<tr>
			<td>VCaP</td>
			<td>66.7</td>
		</tr>
		<tr>
			<td>293T</td>
			<td>450</td>
		</tr>
		<tr>
			<td>REH</td>
			<td>134</td>
		</tr>
		<tr>
			<td>22Rv1</td>
			<td>400</td>
		</tr>
	</tbody>
</table>
Table 1: Optimum amount of micrococcal nuclease in various cell lines. The value represents the amounts of MNase per 2 x 106 cells in 100 &#181;L of buffer, 37 &#176;C for 10 min.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Primer name</td>
			<td></td>
			<td>Sequence</td>
		</tr>
		<tr>
			<td rowspan="2">AR (-18.8kb)</td>
			<td>FWD</td>
			<td>ATTTGGAACTGGGAACATCT</td>
		</tr>
		<tr>
			<td>REV</td>
			<td>CACCTTCTCTCCTCCACTCT</td>
		</tr>
		<tr>
			<td rowspan="2">AR (-8.8kb)</td>
			<td>FWD</td>
			<td>TAACAGCTTTGCATCCAAGT</td>
		</tr>
		<tr>
			<td>REV</td>
			<td>TGAAATCTGGGACTAAAGCA</td>
		</tr>
		<tr>
			<td rowspan="2">AR (-8.2kb)</td>
			<td>FWD</td>
			<td>CAGTGCTATTCCCTTGTGAC</td>
		</tr>
		<tr>
			<td>REV</td>
			<td>TTGGACTGGCTCTATCTTGA</td>
		</tr>
		<tr>
			<td rowspan="2">AR-TSS (0 kb)</td>
			<td>FWD</td>
			<td>GCAAACTGTTGCATTTGCTC</td>
		</tr>
		<tr>
			<td>REV</td>
			<td>GGCCCTTTTTCCCTCTGTC</td>
		</tr>
		<tr>
			<td rowspan="2">AR (0.6 kb)</td>
			<td>FWD</td>
			<td>CACGACCCGCCTGGTTAG</td>
		</tr>
		<tr>
			<td>REV</td>
			<td>TGAAGACCTGACTGCCTTTTC</td>
		</tr>
		<tr>
			<td rowspan="2">AR (+1.0kb)</td>
			<td>FWD</td>
			<td>CCGCAAGTTTCCTTCTCTGG</td>
		</tr>
		<tr>
			<td>REV</td>
			<td>CTTCCCAGCCCTAACTGCAC</td>
		</tr>
		<tr>
			<td rowspan="2">AR (+11.8kb)</td>
			<td>FWD</td>
			<td>CCTTGCTTGTGGAACTGTAG</td>
		</tr>
		<tr>
			<td>REV</td>
			<td>TTTATTGTCTGGTGCTAGGC</td>
		</tr>
		<tr>
			<td rowspan="2">PSA promoter</td>
			<td>FWD</td>
			<td>CCTAGATGAAGTCTCCATGAGCTACA</td>
		</tr>
		<tr>
			<td>REV</td>
			<td>GGGAGGGAGAGCTAGCACTTG</td>
		</tr>
		<tr>
			<td rowspan="2">PSA enhancer</td>
			<td>FWD</td>
			<td>GCCTGGATCTGAGAGAGATATCATC</td>
		</tr>
		<tr>
			<td>REV</td>
			<td>ACACCTTTTTTTTTCTGGATTGTTG</td>
		</tr>
		<tr>
			<td rowspan="2">GAPDH</td>
			<td>FWD</td>
			<td>TACTAGCGGTTTTACGGGCG</td>
		</tr>
		<tr>
			<td>REV</td>
			<td>TCGAACAGGAGGAGCAGAGAGCGA</td>
		</tr>
	</tbody>
</table>
Table 2: Paired Primer sequences used for ChIP assay.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AR (-18.8kb)</td>
			<td>Cq</td>
			<td>SQ</td>
			<td>Adjusted to one IP</td>
			<td>% Input</td>
			<td>Fold enrichment</td>
		</tr>
		<tr>
			<td>1% Input</td>
			<td>23.49</td>
			<td>0.815</td>
			<td>81.5</td>
			<td>100</td>
			<td></td>
		</tr>
		<tr>
			<td>IP with Control IgG</td>
			<td>27.68</td>
			<td>0.051</td>
			<td>0.051</td>
			<td>0.062</td>
			<td>1</td>
		</tr>
		<tr>
			<td>IP with anti-H3K4me3</td>
			<td>22.48</td>
			<td>1.590</td>
			<td>1.590</td>
			<td>1.951</td>
			<td>31.4</td>
		</tr>
		<tr>
			<td>AR (-8.8kb)</td>
			<td>Cq</td>
			<td>SQ</td>
			<td>Adjusted to one IP</td>
			<td>% Input</td>
			<td>Fold enrichment</td>
		</tr>
		<tr>
			<td>1% Input</td>
			<td>23.22</td>
			<td>0.586</td>
			<td>58.6</td>
			<td>100</td>
			<td></td>
		</tr>
		<tr>
			<td>IP with Control IgG</td>
			<td>26.81</td>
			<td>0.052</td>
			<td>0.052</td>
			<td>0.088</td>
			<td>1</td>
		</tr>
		<tr>
			<td>IP with anti-H3K4me3</td>
			<td>23.74</td>
			<td>0.414</td>
			<td>0.414</td>
			<td>0.706</td>
			<td>8.0</td>
		</tr>
		<tr>
			<td>AR (-8.2kb)</td>
			<td>Cq</td>
			<td>SQ</td>
			<td>Adjusted to one IP</td>
			<td>% Input</td>
			<td>Fold enrichment</td>
		</tr>
		<tr>
			<td>1% Input</td>
			<td>23.19</td>
			<td>0.643</td>
			<td>64.3</td>
			<td>100</td>
			<td></td>
		</tr>
		<tr>
			<td>IP with Control IgG</td>
			<td>26.99</td>
			<td>0.048</td>
			<td>0.048</td>
			<td>0.075</td>
			<td>1</td>
		</tr>
		<tr>
			<td>IP with anti-H3K4me3</td>
			<td>23.63</td>
			<td>0.477</td>
			<td>0.477</td>
			<td>0.742</td>
			<td>9.9</td>
		</tr>
		<tr>
			<td>AR-TSS (0 kb)</td>
			<td>Cq</td>
			<td>SQ</td>
			<td>Adjusted to one IP</td>
			<td>% Input</td>
			<td>Fold enrichment</td>
		</tr>
		<tr>
			<td>1% Input</td>
			<td>25.06</td>
			<td>0.657</td>
			<td>65.7</td>
			<td>100</td>
			<td></td>
		</tr>
		<tr>
			<td>IP with Control IgG</td>
			<td>28.63</td>
			<td>0.050</td>
			<td>0.050</td>
			<td>0.077</td>
			<td>1</td>
		</tr>
		<tr>
			<td>IP with anti-H3K4me3</td>
			<td>20.70</td>
			<td>15.064</td>
			<td>15.064</td>
			<td>22.944</td>
			<td>299.8</td>
		</tr>
		<tr>
			<td>AR (0.6 kb)</td>
			<td>Cq</td>
			<td>SQ</td>
			<td>Adjusted to one IP</td>
			<td>% Input</td>
			<td>Fold enrichment</td>
		</tr>
		<tr>
			<td>1% Input</td>
			<td>23.86</td>
			<td>0.716</td>
			<td>71.6</td>
			<td>100</td>
			<td></td>
		</tr>
		<tr>
			<td>IP with Control IgG</td>
			<td>26.67</td>
			<td>0.106</td>
			<td>0.106</td>
			<td>0.147</td>
			<td>1</td>
		</tr>
		<tr>
			<td>IP with anti-H3K4me3</td>
			<td>19.15</td>
			<td>17.787</td>
			<td>17.787</td>
			<td>24.840</td>
			<td>168.6</td>
		</tr>
		<tr>
			<td>AR (+1.0kb)</td>
			<td>Cq</td>
			<td>SQ</td>
			<td>Adjusted to one IP</td>
			<td>% Input</td>
			<td>Fold enrichment</td>
		</tr>
		<tr>
			<td>1% Input</td>
			<td>23.51</td>
			<td>0.730</td>
			<td>73.0</td>
			<td>100</td>
			<td></td>
		</tr>
		<tr>
			<td>IP with Control IgG</td>
			<td>25.94</td>
			<td>0.125</td>
			<td>0.125</td>
			<td>0.171</td>
			<td>1</td>
		</tr>
		<tr>
			<td>IP with anti-H3K4me3</td>
			<td>19.06</td>
			<td>18.486</td>
			<td>18.486</td>
			<td>25.335</td>
			<td>147.8</td>
		</tr>
		<tr>
			<td>AR (+11.8kb)</td>
			<td>Cq</td>
			<td>SQ</td>
			<td>Adjusted to one IP</td>
			<td>% Input</td>
			<td>Fold enrichment</td>
		</tr>
		<tr>
			<td>1% Input</td>
			<td>24.54</td>
			<td>0.876</td>
			<td>87.6</td>
			<td>100</td>
			<td></td>
		</tr>
		<tr>
			<td>IP with Control IgG</td>
			<td>29.14</td>
			<td>0.033</td>
			<td>0.033</td>
			<td>0.037</td>
			<td>1</td>
		</tr>
		<tr>
			<td>IP with anti-H3K4me3</td>
			<td>24.47</td>
			<td>0.918</td>
			<td>0.918</td>
			<td>1.048</td>
			<td>27.97</td>
		</tr>
	</tbody>
</table>
Table 3: Raw data from quantitative PCR analysis for Figure 3. Cq: Threshold cycle number, SQ: starting quantity calculated using a standard curve, Adjusted to one IP: multiply SQ in 1% input by 100 as 1% sample volume of one IP is used for PCR, % Input: divide SQ in IP sample by adjusted SQ in Input, Fold enrichment: divide SQ in IP with anti-H3K4me3 by SQ in IP with control IgG.

Watch this Scientific Journal Video about Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells at JoVE.com

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

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 ...

chromatin-immunoprecipitation-assay-using-micrococcal-nucleases

Nanyang Technological University

Research

JoVE Journal

Genetics

14.4K Views.  Beckman Research Institute of City of Hope. In dit protocol kunnen we de interacties tussen eiwitten en DNA identificeren, waardoor de transcriptieregulatie van het gen wordt onthuld. Dit helpt om de moleculaire mechanismen in de cellen te begrijpen. We wijzigen de reeds bestaande protocollen en stellen vervolgens een eenvoudige en duidelijkere testprotocollen op.Je volgt gewoon het protocol vanaf de eerste stap, zodat de test met succes kan worden gedaan. Ter voorbereiding van crosslinked chromatine, in een rookkap, voeg vier m...

Video: Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks

High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, such as transcription, DNA replication, and DNA repair. HMGB1 binds to structurally distorted DNA with higher affinity than to canonical B-DNA. For example, we found that HMGB1 binds to DNA interstrand crosslinks (ICLs), which covalently link the two strands of the DNA, cause distortion of the helix, and if left unrepaired can cause cell death. Due to their cytotoxic potential, several ICL-inducing agents are currently used as chemotherapeutic agents in the clinic. While ICL-forming agents show preferences for certain base sequences (e.g., 5&#39;-TA-3&#39; is the preferred crosslinking site for psoralen), they largely induce DNA damage in an indiscriminate fashion. However, by covalently coupling the ICL-inducing agent to a triplex-forming oligonucleotide (TFO), which binds to DNA in a sequence-specific manner, targeted DNA damage can be achieved. Here, we use a TFO covalently conjugated on the 5&#39; end to a 4&#39;-hydroxymethyl-4,5&#39;,8-trimethylpsoralen (HMT) psoralen to generate a site-specific ICL on a mutation-reporter plasmid to use as a tool to study the architectural modification, processing, and repair of complex DNA lesions by HMGB1 in human cells. We describe experimental techniques to prepare TFO-directed ICLs on reporter plasmids, and to interrogate the association of HMGB1 with the TFO-directed ICLs in a cellular context using chromatin immunoprecipitation assays. In addition, we describe DNA supercoiling assays to assess specific architectural modification of the damaged DNA by measuring the amount of superhelical turns introduced on the psoralen-crosslinked plasmid by HMGB1. These techniques can be used to study the roles of other proteins involved in the processing and repair of TFO-directed ICLs or other targeted DNA damage in any cell line of interest.

Targeted DNA damage can be achieved by tethering a DNA damaging agent to a triplex-forming oligonucleotide (TFO). Using modified TFOs, DNA damage-specific protein association, and DNA topology modification can be studied in human cells by the utilization of modified chromatin immunoprecipitation assays and DNA supercoiling assays described herein.

High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, ...

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 ...

Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory regions of oncogenes and tumor suppressors can lead to changes in gene expression and play an important role in the pathogenesis of brain tumors. Native chromatin immunoprecipitation (ChIP) is a popular technique that allows the detection of modifications or other proteins tightly bound to DNA. In contrast to cross-linked ChIP, in native ChIP, cells are not treated with formaldehyde to covalently link protein to DNA. This is advantageous because sometimes crosslinking may fix proteins that only transiently interact with DNA and do not have functional significance in gene regulation. In addition, antibodies are generally raised against unfixed peptides. Therefore, antibody specificity is increased in native ChIP. However, it is important to keep in mind that native ChIP is only applicable to study histones or other proteins that bind tightly to DNA. This protocol describes the native chromatin immunoprecipitation on murine brain tumor neurospheres.

Epigenetic mechanisms are frequently altered in glioma. Chromatin immunoprecipitation could be used to study the consequences of genetic alterations in glioma that result from changes in histone modifications which regulate chromatin structure and gene transcription. This protocol describes native chromatin immunoprecipitation on murine brain tumor neurospheres.

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory ...

Cancer Research

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein interactions in yeast. It allows analysis of a target region of interest without the need for prior knowledge about likely proteins bound to the target region. This, in theory, allows HyCCAPP to be used to analyze any genomic region of interest, and it provides sufficient flexibility to work in different cell systems. This method is not meant to study binding sites of known transcription factors, a task better suited for Chromatin Immunoprecipitation (ChIP) and ChIP-like methods. The strength of HyCCAPP lies in its ability to explore DNA regions for which there is limited or no knowledge about the proteins bound to it. It can also be a convenient method to avoid biases (present in ChIP-like methods) introduced by protein-based chromatin enrichment using antibodies. Potentially, HyCCAPP can be a powerful tool to uncover truly novel DNA-protein interactions. To date, the technology has been predominantly applied to yeast cells or to high copy repeat sequences in mammalian cells. In order to become the powerful tool we envision, HyCCAPP approaches need to be optimized to efficiently capture single-copy loci in mammalian cells. Here, we present our adaptation of the initial yeast HyCCAPP capture protocol to human cell lines, and show that single-copy chromatin regions can be efficiently isolated with this modified protocol.

This is a method to identify novel DNA-interacting proteins at specific target loci, relying on sequence-specific capture of crosslinked chromatin for subsequent proteomic analyses. No prior knowledge about potential binding proteins, nor cell modifications are required. Initially developed for yeast, the technology has now been adapted for mammalian cells.

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein ...

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 ...

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo)

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) is widely used to identify genome-wide binding locations of DNA-binding proteins. However, the ChIP-seq method is limited by its heterogeneity in length of sonicated DNA fragments and non-specific background DNA, resulting in low mapping resolution and uncertainty in DNA-binding sites. To overcome these limitations, the combination of ChIP with exonuclease digestion (ChIP-exo) utilizes 5&#8217; to 3&#8217; exonuclease digestion to trim the heterogeneously sized immunoprecipitated DNA to the protein-DNA crosslinking site. Exonuclease treatment also eliminates non-specific background DNA. The library-prepared and exonuclease-digested DNA can be sent for high-throughput sequencing. The ChIP-exo method allows for near base-pair mapping resolution with greater detection sensitivity and reduced background signal. An optimized ChIP-exo protocol for mammalian cells and next-generation sequencing is described below.

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease ChIP-Exo

Precise determination of protein-binding locations across the genome is important for understanding gene regulation. Here we describe a&#160;genomic mapping method that treats chromatin-immunoprecipitated DNA with exonuclease digestion (ChIP-exo) followed by high-throughput sequencing. This method detects protein-DNA interactions with near base-pair mapping resolution and high signal-to-noise ratio in mammalian neurons.

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled ...

Mapping Mammalian 3D Genome Interactions with Micro-C-XL

Three-dimensional (3D) chromosome organization is a major factor in genome regulation and cell-type specification. For example, cis-regulatory elements, known as enhancers, are thought to regulate the activity of distal promoters via interaction in 3D space. Genome-wide chromosome conformation capture (3C)-technologies, such as Hi-C, have transformed our understanding of how genomes are organized in cells. The current understanding of 3D genome organization is limited by the resolution with which the topological organization of chromosomes in 3D space can be resolved. Micro-C-XL measures chromosome folding with resolution at the level of the nucleosome, the basic unit of chromatin, by utilizing micrococcal nuclease (MNase) to fragment genomes during the chromosome conformation capture protocol. This results in an improved signal-to-noise ratio in the measurements, thus facilitating the better detection of insulation sites and chromosome loops compared to other genome-wide 3D technologies. A visually supported, detailed, step-by-step protocol for preparing high-quality Micro-C-XL samples from mammalian cells is presented in this article.

A protocol for mapping the three-dimensional genome organization with nucleosome resolution using the genome-wide chromosome conformation capture method Micro-C-XL is presented here.

Three-dimensional (3D) chromosome organization is a major factor in genome regulation and cell-type specification. For example, cis-regulatory elements, ...

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

Biology

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 ...

Single-Cell Factor Localization on Chromatin using Ultra-Low Input Cleavage Under Targets and Release using Nuclease

Determining the binding locations of a protein on chromatin is essential for understanding its function and potential regulatory targets. Chromatin Immunoprecipitation (ChIP) has been the gold standard for determining protein localization for over 30 years and is defined by the use of an antibody to pull out the protein of interest from sonicated or enzymatically digested chromatin. More recently, antibody tethering techniques have become popular for assessing protein localization on chromatin due to their increased sensitivity. Cleavage Under Targets &amp; Release Under Nuclease (CUT&amp;RUN) is the genome-wide derivative of Chromatin Immunocleavage (ChIC) and utilizes recombinant Protein A tethered to micrococcal nuclease (pA-MNase) to identify the IgG constant region of the antibody targeting a protein of interest, therefore enabling site-specific cleavage of the DNA flanking the protein of interest. CUT&amp;RUN can be used to profile histone modifications, transcription factors, and other chromatin-binding proteins such as nucleosome remodeling factors. Importantly, CUT&amp;RUN can be used to assess the localization of either euchromatic- or heterochromatic-associated proteins and histone modifications. For these reasons, CUT&amp;RUN is a powerful method for determining the binding profiles of a wide range of proteins. Recently, CUT&amp;RUN has been optimized for transcription factor profiling in low populations of cells and single cells and the optimized protocol has been termed ultra-low input CUT&amp;RUN (uliCUT&amp;RUN). Here, a detailed protocol is presented for single-cell factor profiling using uliCUT&amp;RUN in a manual 96-well format.

CUT&#38;RUN and its variants can be used to determine protein occupancy on chromatin. This protocol describes how to determine protein localization on chromatin using single-cell uliCUT&#38;RUN.

Determining the binding locations of a protein on chromatin is essential for understanding its function and potential regulatory targets. Chromatin ...

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

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