Name: A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia (Video) | JoVE Journal
Uploaded: 2018-12-04T13:00:00.000+00:00
Duration: 9 min 52 s
Description: Learn chromatin immunoprecipitation to identify NFAT2 target genes in chronic lymphocytic leukemia cells. This method reveals protein-DNA interactions to advance therapeutic strategies.

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This work was supported by the DFG grant MU 3340/1-1 and the Deutsche Krebshilfe grant 111134 (both awarded to M.R.M.). We thank Elke Malenke for excellent technical assistance).

All experiments conducted with&#160;human material were approved by the Ethics Committee of the University of T&#252;bingen and written informed consent was obtained from all patients who contributed samples to this study.
1. Isolation and Stimulation of Jurkat cells
NOTE: To optimize the protocol, use the Jurkat cell line which is known to express the high levels of NFAT2. All steps are performed under a laminar flow hood.
<ol>
	<li>Prepare 50 mL of RPMI 1640 supplemented with 10% FCS and 1% penicillin/streptomycin by warming it to 37 &#176;C in a water bath.</li>
	<li>Culture Jurkat cells for 1-2 d in a cell culture flask at 37 &#176;C and 5% CO2 in 20 mL of RPMI 1640 supplemented with 10% FCS and 1% penicillin/streptomycin to a density of approximately 2 x 106 cells/mL (cells are counted with Trypan blue staining and a Neubauer chamber under the microscope) and harvest them by centrifuging for 5 min at 200 x g.</li>
	<li>Remove the supernatant with a pipette and resuspend the cells in 10 mL of RPMI 1640 supplemented with 10% FCS and 1% penicillin/streptomycin. Subsequently, count the cells with a conventional trypan blue staining and a Neubauer chamber under the microscope.</li>
	<li>Centrifuge the cells from step 1.3 for 5 min at 200 x g and remove the supernatant by aspiration. Then, resuspend the cells at a density of 2 x 107 cells/mL in RPMI 1640 supplemented with 10% FCS and 1% penicillin/streptomycin and transfer 1 mL in a new conventional 5 mL tube by pipetting.</li>
	<li>Stimulate the cells by adding 32.4 nM phorbol 12-myristate 13-acetate (PMA) and 1 &#181;M ionomycin. Then incubate the cells for 16 h at 37 &#176;C and 5% CO2 with the lid of the tube opened (to avoid contamination, a sealing film permeable to air can be used).</li>
</ol>
2. Isolation and Stimulation of Primary CLL Cells from Human Patients
NOTE: Patient samples were acquired and stimulated as previously described7. All steps are performed under a laminar flow hood.
<ol>
	<li>Prepare the equipment necessary to draw blood from patients and to perform a density gradient separation: 21-G needles, tourniquet, NH4-heparin-blood collection syringes (e.g., S-monovettes), 1x PBS, and density gradient medium (density 1.077 g/mL).</li>
	<li>Upon venipuncture, draw the blood from CLL patients (ca. 40 mL blood per patient) into the syringes. Then transfer the blood in 50 mL conical tubes. Wash the syringe with 10 mL PBS and pool the blood-PBS-suspension in the 50 mL tubes (adjust the number of tubes to the volume of blood).</li>
	<li>Prepare 15 mL of the density gradient medium in a fresh 50 mL conical tube. Subsequently, layer 35 mL of the blood-PBS-suspension carefully on the density gradient medium. For this purpose, hold the tube at a flat angle and slowly pipette the blood-PBS-suspension against the bottom wall of the 50 mL conical tube. As more of the blood-PBS-suspension is accumulating in the 50 mL conical tube, increase the angle of the tube up to a right angle. Repeat this step according to the volume of the blood-PBS-suspension.</li>
	<li>Perform centrifugation at 560 x g for 20 min (without brake), then extract the mononuclear cell phase which contains the peripheral blood mononuclear cells (PBMCs). To do so, collect the white interphase of the density gradient separation with a 5 mL serological pipette and transfer it to a new 50 mL conical tube.</li>
	<li>Fill the cell-suspension to 50 mL with PBS and centrifuge for 5 min at 360 x g. Remove the supernatant by aspiration. Repeat this step with centrifugation for 5 min at 275 x g. Then, pool the cells of one patient in 5-10 mL of PBS in a 50 mL conical tube.</li>
	<li>Count the cells as described in 1.3. 
	NOTE: It depends on the proportion of CLL cells in the isolated PBMCs, if the cells can be used directly for this procedure or a B cell isolation has to be conducted, e.g., via magnetic cell sorting (MACS). The B cell isolation is recommended but can be omitted if the proportion of CLL cells is above 95% of total lymphocytes (determined via flow cytometry). Blood of CLL patients is fixed and lysed according to the manufacturer&#39;s instructions. Then a staining with CD19-FITC and CD5-PE antibodies is performed according to the manufacturer&#39;s instructions and the cells are analyzed via flow cytometry. One exemplary patient is shown in Figure 1, in which the proportion of CLL cells is 89.03%. Thus, a B cell isolation has to be performed in this patient. The proportion of physiological B cells is negligible (3.72% in the example).</li>
	<li>Wash the cells with 10 mL of pre-warmed (37 &#176;C) RPMI 1640 supplemented with 10% FCS, 1% penicillin/streptomycin, 100 mM non-essential amino acids, 1 mM sodium pyruvate and 50 mM &#946;-mercaptoethanol for 5 min at 200 x g and remove the supernatant by aspiration.</li>
	<li>Adjust the cells to 2 x 107 cells/mL in RPMI 1640 supplemented with 10% FCS, 1% penicillin/streptomycin, 100 mM non-essential amino acids, 1 mM sodium pyruvate and 50 mM &#946;-mercaptoethanol (37 &#176;C) and fill 1 mL of the cell suspension into a 5 mL tube. 
	NOTE: The &#223;-mercaptoethanol is employed to counteract oxidative stress.</li>
	<li>Stimulate the cells by adding 32.4 nM PMA and 1 &#181;M ionomycin. Then incubate the cells for 16 h at 37 &#176;C and 5% CO2 with the lid of the tube opened (to avoid contamination, a sealing film permeable to air can be used). 
	NOTE: To reduce the level of stress the cells can be rested for 1 h at 37 &#176;C and 5% CO2 in the incubator prior to the 16 h of stimulation.</li>
</ol>
3. Fixation, Cell Lysis, and Chromatin Shearing
NOTE: Patient samples and Jurkat cells are fixed and lysed with a commercially available ChIP kit according to the manufacturer&#39;s instructions with modifications as described previously7. The fixation is performed under a laminar flow hood.
<ol>
	<li>Prepare a 1.5 mL tube with 1 mL of 37% formaldehyde, a 1.5 mL tube with 1 mL of 1.25 M glycine, a 5 mL tube with 4 mL of 1x PBS and a box with ice for the fixation of the cells.</li>
	<li>After stimulation of the cells for the respective incubation time in step 1.5 or 2.9, spin the 5 mL tubes at 200 x g for 5 min and resuspend the cells in 500 &#181;L of PBS in the 5 mL tubes.</li>
	<li>Add 340 &#181;M formaldehyde (13.5 &#181;L of 37% formaldehyde) to the cells. After brief mixing by carefully pipetting up and down incubate the suspension for 2.5 min (Jurkat cells) or 5 min (patient sample) at room temperature. 
	NOTE: Prolonged fixation time is needed for primary cells to assure optimal shearing.</li>
	<li>Add 125 mM glycine (57 &#181;L of 1.25 M glycine) to stop the fixation and incubate for another 5 min. Put the cells on ice immediately thereafter and centrifuge at 500 x g for 5 min at 4 &#176;C. Remove the supernatant by aspiration.</li>
	<li>Wash the cells twice with 1 mL of ice-cold PBS at 200 x g for 5 min at 4 &#176;C and remove the supernatant every time by aspiration. 
	NOTE: After the fixation, the protocol can be paused. Fixed cells should be stored at -80 &#176;C. To test, if the epitope of the antibody intended to use in the following protocol is not masked via fixation, additional samples can be used for analysis via SDS-PAGE gel electrophoresis and Western blot. These steps are performed with 100 &#181;g of protein according to manufacturer&#39;s instructions. All &#945;NFAT2 antibodies are used at a dilution of 1:1000, the GAPDH antibody at a dilution of 1:10000. An exemplary image of fixed samples from Jurkat cells with appropriate and inappropriate antibodies is shown in Figure 2.</li>
	<li>Resuspend the cell pellet in 5 mL of commercially available lysis buffer 1 by pipetting up and down. Then, place the samples on the ice on a shaker and incubate them for 10 min while shaking. 
	NOTE: Prior to lysis, the cells can be disintegrated for 10 s in liquid nitrogen. This is useful for Jurkat cells but should be avoided in primary patient samples. For the disintegration, place the 5 mL tubes for 5 s in 5-10 mL of liquid nitrogen in a specific container. Wear safety glasses and appropriate safety gloves.</li>
	<li>Subsequently, centrifuge the tubes at 500 x g for 5 min at 4 &#176;C and remove the supernatant carefully by pipetting.</li>
	<li>Homogenize the cells in 5 mL of commercially available lysis buffer 2 by pipetting up and down and incubate for 10 min on ice while shaking. Then centrifuge the tubes at 500 x g for 5 min at 4 &#176;C and remove the supernatant carefully by pipetting.</li>
	<li>Resuspend the cell pellet in 500 &#181;L (Jurkat cells) or 140 &#181;L (patient samples) of shearing buffer 1 containing 1x protease inhibitor (5 &#181;L or 1.4 &#181;L, respectively) and incubate the mixture for 10 min on ice.</li>
	<li>For chromatin shearing, transfer 140 &#181;L of the cell-suspension from step 3.9 into sonicator tubes (avoid producing air bubbles and repeat this step if necessary due to sample volume). Place the tubes in the focused ultra-sonicator at a temperature of 7 &#176;C and shear for 10 min (cell line) or 7.5 min (patient samples) with an average incident power of 9.375 W to obtain 200-500 bp DNA fragments.</li>
	<li>Finally, centrifuge at 15700 x g for 10 min at 4 &#176;C in the small centrifuge and collect the supernatant in a new 1.5 mL tube.</li>
	<li>To test for the appropriate fragment sizes, analyze 20 &#181;L of the sheared chromatin via gel-electrophoresis in 1.5% TBE agarose gel. An exemplary image of sheared chromatin from Jurkat cells of good and bad quality is shown in Figure 3. At this point, the protocol can be potentially paused. Sheared chromatin should be stored at -80 &#176;C.</li>
</ol>
4. Chromatin Immunoprecipitation
NOTE: The chromatin immunoprecipitation was performed with a commercially available ChIP kit according to the manufacturer&#39;s instructions with modifications7.
<ol>
	<li>Calculate the number of immunoprecipitations (70&#160;&#181;L (Jurkat cells) or 15 &#181;L (patient samples) of sheared chromatin plus one antibody) and prepare 20 &#181;L of protein A coated beads per precipitation in a 1.5 mL tube. Wash the beads in 40 &#181;L of 1x ChIP buffer 1 per precipitation by pipetting up and down, let the beads rest in a magnetic rack for 1 min and remove the supernatant by pipetting. Perform this step four times in total. Eventually, resuspend the beads in the original volume with 1x ChIP buffer 1.</li>
	<li>For the immunoprecipitation itself, use 10 &#181;g of the &#945;NFAT2 antibody (7A6) to capture NFAT2 with its bound target sequences. Use 2.5 &#181;g of a rabbit IgG antibody (DA1E) as an IgG-control. Prepare the reactions&#160;in fresh 1.5 mL tubes as indicated in Table 1. 
	NOTE: It is important to use a monoclonal antibody with high affinity whose epitope is not masked during fixation for this protocol. Polyclonal antibodies introduce an additional level of variation making it significantly more difficult to appropriately interpret the received results.</li>
	<li>Incubate the mixture from step 4.2 overnight at 4 &#176;C on a rotating wheel at 6 rpm.</li>
	<li>On the next day spin the tubes for 5 s at 7,000 x g in the small centrifuge, incubate them in a magnetic rack for 1 min and remove the supernatant by aspiration. Wash the beads once with each of the wash buffers 1, 2, 3 and 4 consecutively.</li>
	<li>To do so, resuspend the beads in 350 &#181;L of wash buffer and incubate them for 5 min at 4 &#176;C on a rotating wheel at 6 rpm.</li>
	<li>Thereafter, spin the tubes for 5 s at 7000 x g in the small centrifuge. Then, place them for 1 min in a magnetic rack, remove the supernatant and add the next wash buffer.</li>
	<li>After the last washing step, remove the supernatant by pipetting, take up the beads in 100 &#181;L of elution buffer 1 and incubate them for 30 min on a rotating wheel at 6 rpm.</li>
	<li>Spin the tubes shortly (5 s at 7000 x g in the small centrifuge) and place them in a magnetic rack for 1 min.</li>
	<li>Then transfer the supernatant by pipetting to a new 1.5 mL tube and add 4 &#181;L of elution buffer 2.</li>
	<li>To create the input control, mix 1 &#181;L of the sheared chromatin with 99 &#181;L of elution buffer 1 and 4 &#181;L of elution buffer 2 (in this setup, there are three tubes per patient: one input control, one IgG-control and one &#945;NFAT2 sample).</li>
	<li>Incubate the samples for 4 h at 65 &#176;C and 1300 rpm in a 1.5 mL tube shaker. Add 100 &#181;L of 100% isopropanol, vortex and spin the samples for 5 s at 7000 x g in the small centrifuge.</li>
	<li>Resuspend the available magnetic beads by thoroughly vortexing, add 10 &#181;L of the beads to every sample and incubate for 1 h at room temperature on a rotating wheel at 6 rpm.</li>
	<li>After the incubation, spin the tubes for 5 s at 7000 x g in the small centrifuge and place them for 1 min in a magnetic rack. Remove the supernatant by aspiration.</li>
	<li>Resuspend the beads in 100 &#181;L of wash buffer 1. Invert the tubes to mix and incubate for 5 min at room temperature on a rotating wheel at 6 rpm.</li>
	<li>Spin the tubes for 5 s at 7000 x g in the small centrifuge, place them for 1 min in a magnetic rack and remove the supernatant by pipetting. Repeat the steps 4.13-4.14 with wash buffer 2.</li>
	<li>Subsequently, remove the wash buffer by aspiration, resuspend the beads in 55 &#181;L of elution buffer and incubate for 15 min at room temperature on the rotating wheel at 6 rpm to elute the precipitated DNA. Finally, spin the tubes for 5 s at 7000 x g in the small centrifuge, place them for 1 min in a magnetic rack and transfer the supernatant into new 1.5 mL tubes. 
	NOTE: Here the protocol can be potentially paused. The eluted DNA should then be stored at -20 &#176;C.</li>
</ol>
5. Detection of NFAT target genes in CLL cells.
<ol>
	<li>Conduct the qRT-PCR reaction in duplicate for every sample as indicated in Table 2. 
	NOTE: For the detection of target genes a quantitative real-time PCR (qRT-PCR) is conducted using a commercially available qRT-PCR Mix with a Real-time PCR instrument.</li>
	<li>Use white 96-well reaction plates for the reactions and the RT-PCR instrument. The cycling conditions are as follows: 95 &#176;C for 30 s, [95 &#176;C for 5 s, 60 &#176;C for 30 s] x 40 cycles. 
	NOTE: A serial dilution of the input (undiluted or diluted 1:10, 1:100 and 1:1000 in nuclease-free water) is used to calculate the relative enrichment. In Jurkat cells, IL-2 was used as a positive control32. CD40L, a well-known target of NFAT2 in B cells, was used as a positive control in CLL cells and LCK as an example for a novel target gene of NFAT2 in CLL33,34. The primers for the CD40L, the IL-2 and LCK promoter region are described in the table of specific reagents and equipment.</li>
</ol>
6. Normalization and Data Analysis
NOTE: Relative enrichment for the promoter regions of interest (IL-2, CD40L or&#160;LCK) is calculated using the IgG-control for normalization.
<ol>
	<li>First, determine Ct (threshold cycle) values for the input serial dilution, the IL-2, CD40L and the LCK promoter region via the evaluation software from qRT-PCR data.</li>
	<li>Define a standard curve for the concentrations via the serial dilution of the input. With this standard curve calculate the concentration for the IL-2, CD40L and the LCK promoter region for each duplicate of every sample.</li>
	<li>Next, calculate the mean of the duplicates. Then, set the IgG immunoprecipitation sample as a reference. Define the relative enrichment for this sample as 1.0 and compare the other sample (from the NFAT2 immunoprecipitation) to this reference.</li>
	<li>Finally, calculate the relative enrichment by dividing the concentration of the samples by the concentration of the IgG reference.</li>
</ol>

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<li>Fujita, T., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biochemical+Analysis+of+Genome+Functions+Using+Locus-Specific+Chromatin+Immunoprecipitation+Technologies%5BTitle%5D)+AND+%22Gene+Regulation+and+Systems+Biology%22%5BJournal%5D)">Biochemical Analysis of Genome Functions Using Locus-Specific Chromatin Immunoprecipitation Technologies.</a> Gene Regulation and Systems Biology. 10 (Suppl 1), 1-9 (2016).</li>
<li>O'Neill, L. P., Turner, B. M. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/12893176">Immunoprecipitation of native chromatin: NChIP.</a> Methods. 31 (1), 76-82 (2003).</li>
<li>Gilmour, D. S., Lis, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detecting+protein-DNA+interactions+in+vivo%3A+distribution+of+RNA+polymerase+on+specific+bacterial+genes%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Detecting protein-DNA interactions in vivo: distribution of RNA polymerase on specific bacterial genes.</a> Proceedings of the National Academy of Sciences of the United States of America. 81 (14), 4275-4279 (1984).</li>
<li>Gilmour, D. S., Lis, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+interactions+of+RNA+polymerase+II+with+genes+of+Drosophila+melanogaster%5BTitle%5D)+AND+%22Molecular+and+Cellular+Biology%22%5BJournal%5D)">In vivo interactions of RNA polymerase II with genes of Drosophila melanogaster.</a> Molecular and Cellular Biology. 5 (8), 2009-2018 (1985).</li>
<li>Solomon, M. J., Larsen, P. L., Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mapping+protein-DNA+interactions+in+vivo+with+formaldehyde%3A+evidence+that+histone+H4+is+retained+on+a+highly+transcribed+gene%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Mapping protein-DNA interactions in vivo with formaldehyde: evidence that histone H4 is retained on a highly transcribed gene.</a> Cell. 53 (6), 937-947 (1988).</li>
<li>Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Discovery+of+cellular+regulation+by+protein+degradation%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Discovery of cellular regulation by protein degradation.</a> Journal of Biological Chemistry. 283 (50), 34469-34489 (2008).</li>
<li>Hebbes, T. R., Thorne, A. W., Crane-Robinson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+direct+link+between+core+histone+acetylation+and+transcriptionally+active+chromatin%5BTitle%5D)+AND+%22EMBO+Journal%22%5BJournal%5D)">A direct link between core histone acetylation and transcriptionally active chromatin.</a> EMBO Journal. 7 (5), 1395-1402 (1988).</li>
<li>Decker, E. L., Skerka, C., Zipfel, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+early+growth+response+protein+%28EGR-1%29+regulates+interleukin-2+transcription+by+synergistic+interaction+with+the+nuclear+factor+of+activated+T+cells%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">The early growth response protein (EGR-1) regulates interleukin-2 transcription by synergistic interaction with the nuclear factor of activated T cells.</a> Journal of Biological Chemistry. 273 (41), 26923-26930 (1998).</li>
<li>Grammer, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+CD40+ligand+expressed+by+human+B+cells+costimulates+B+cell+responses%5BTitle%5D)+AND+%22Journal+of+Immunology%22%5BJournal%5D)">The CD40 ligand expressed by human B cells costimulates B cell responses.</a> Journal of Immunology. 154 (10), 4996-5010 (1995).</li>
<li>Pham, L. V., Tamayo, A. T., Yoshimura, L. C., Lin-Lee, Y. C., Ford, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Constitutive+NF-kappaB+and+NFAT+activation+in+aggressive+B-cell+lymphomas+synergistically+activates+the+CD154+gene+and+maintains+lymphoma+cell+survival%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Constitutive NF-kappaB and NFAT activation in aggressive B-cell lymphomas synergistically activates the CD154 gene and maintains lymphoma cell survival.</a> Blood. 106 (12), 3940-3947 (2005).</li>
<li>Zhou, Z. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+acid-sensing+ion+channel%2C+ASIC2%2C+promotes+invasion+and+metastasis+of+colorectal+cancer+under+acidosis+by+activating+the+calcineurin%2FNFAT1+axis%5BTitle%5D)+AND+%22Journal+of+Experimental+and+Clinical+Cancer+Research%22%5BJournal%5D)">The acid-sensing ion channel, ASIC2, promotes invasion and metastasis of colorectal cancer under acidosis by activating the calcineurin/NFAT1 axis.</a> Journal of Experimental and Clinical Cancer Research. 36 (1), 130(2017).</li>
<li>Estrada-Aviles, R., Rodriguez, G., Zarain-Herzberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+cardiac+calsequestrin+gene+transcription+is+modulated+at+the+promoter+by+NFAT+and+MEF-2+transcription+factors%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">The cardiac calsequestrin gene transcription is modulated at the promoter by NFAT and MEF-2 transcription factors.</a> PloS One. 12 (9), e0184724(2017).</li>
<li>Kadziolka, B., Lesniak, W., Filipek, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+CacyBP%2FSIP+expression+by+NFAT1+transcription+factor%5BTitle%5D)+AND+%22Immunobiology%22%5BJournal%5D)">Regulation of CacyBP/SIP expression by NFAT1 transcription factor.</a> Immunobiology. 222 (8-9), 872-877 (2017).</li>
<li>Lieb, J. D., Liu, X., Botstein, D., Brown, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Promoter-specific+binding+of+Rap1+revealed+by+genome-wide+maps+of+protein-DNA+association%5BTitle%5D)+AND+%22Nature+Genetics%22%5BJournal%5D)">Promoter-specific binding of Rap1 revealed by genome-wide maps of protein-DNA association.</a> Nature Genetics. 28 (4), 327-334 (2001).</li>
<li>Ren, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-wide+location+and+function+of+DNA+binding+proteins%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Genome-wide location and function of DNA binding proteins.</a> Science. 290 (5500), 2306-2309 (2000).</li>
<li>Iyer, V. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genomic+binding+sites+of+the+yeast+cell-cycle+transcription+factors+SBF+and+MBF%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBF.</a> Nature. 409 (6819), 533-538 (2001).</li>
<li>Johnson, D. S., Mortazavi, A., Myers, R. M., Wold, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-wide+mapping+of+in+vivo+protein-DNA+interactions%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Genome-wide mapping of in vivo protein-DNA interactions.</a> Science. 316 (5830), 1497-1502 (2007).</li>
<li>Robertson, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-wide+profiles+of+STAT1+DNA+association+using+chromatin+immunoprecipitation+and+massively+parallel+sequencing%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing.</a> Nature Methods. 4 (8), 651-657 (2007).</li>
<li>Schmid, C. D., Bucher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ChIP-Seq+data+reveal+nucleosome+architecture+of+human+promoters%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">ChIP-Seq data reveal nucleosome architecture of human promoters.</a> Cell. 131 (5), author reply 832-833 831-832 (2007).</li>
</ol>

The authors have nothing to disclose.

The critical steps of performing a successful ChIP assay are the selection of an appropriate antibody and the optimization of the chromatin shearing process25. The selection of the &#945;NFAT2 antibody proved to be particularly challenging during the development of this protocol. While there are several &#945;NFAT2 antibodies commercially available and the majority of these works fine for western blotting and other applications, clone 7A6 was the only antibody which could be successfully used for ...

Chronic lymphocytic leukemia (CLL) represents the most common leukemia in adults in western countries, exhibiting distinct accumulation of CD19, CD23, and CD5 expressing mature B cells1. Most patients exhibit an indolent disease course, which does not necessitate&#160;specific treatment for many years. In contrast, some patients show rapid progression requiring immediate therapeutic interventions with immune-chemotherapy or other targeted therapies2,3. Nuclear factor of activated T cells (NFAT) is a family of transcription factors controlling various developmental and activation processes in numerous cell types4,5,6. We recently demonstrated overexpression and constitutional activation of NFAT2 in CLL cells from patients with indolent disease7. Here, it regulates an unresponsive state to B cell receptor stimulation called anergy7. To demonstrate that NFAT2 binds to the lymphocyte-specific protein tyrosine kinase (LCK) promoter and regulates LCK expression in human CLL cells, a specific chromatin immunoprecipitation assay (ChIP) was developed and employed.
ChIP is one of the several techniques to investigate the role of transcription factors in gene expression8. Gene expression is tightly orchestrated in a very complex manner by several regulators with transcription factors taking an irreplaceable part in this process9,10,11,12. Transcription factors regulating the gene expression in a spatial and temporal context have been identified in numerous species (e.g., for development and differentiation)13,14,15,16,17,18. Errors in the intricate control mechanisms involving transcription factors can lead to a variety of pathologic processes including cancer19,20. Hence, identification of transcription factors and their respective targets might offer novel therapeutic avenues21,22. To investigate this intriguing field several methods are available like ChIP, electrophoretic mobility shift assay (EMSA), various DNA pull-down assays and reporter-assays8,11,12,23,24.
To demonstrate that a certain transcription factor interacts with specific regions of the genome in vivo, ChIP is an ideal technique25. For this purpose, DNA and associated proteins in living cells are cross-linked using UV irradiation or formaldehyde (cross-linked ChIP, XChIP). This step is omitted to obtain better DNA and protein recovery in the so-called native ChIP (NChIP)26. The DNA-protein complexes are subsequently sheared by sonication into fragments of approximately 200-500 base pairs (bp) and immunoprecipitated from the cell debris using a specific antibody against the transcription factor of interest. The associated DNA fragments are then purified and characterized by PCR, molecular cloning, and sequencing. Alternative techniques use microarrays (ChIP-on-Chip) or the next-generation sequencing (ChIP-Seq) to analyze the immunoprecipitated DNA.
ChIP was first introduced by Gilmour and Lis in 1984 when they used UV light to covalently cross-link DNA and bound proteins in living bacteria27. Upon cell lysis and immunoprecipitation of bacterial RNA polymerase, specific probes of known genes were used to map the in vivo distribution and density of RNA polymerase. The method was subsequently used by the same investigators to analyze the distribution of eukaryotic RNA polymerase II on heat shock protein genes in Drosophila28. The XChIP assay was further refined by Varshavsky and coworkers who first used formaldehyde cross-linking to study the association of histone H4 with heat shock protein genes29,30. The NChIP approach, which carries the advantage of a better DNA and protein recovery due to naturally intact epitopes and, therefore, greater antibody specificity, was first described by Hebbes and colleagues in 198831.
The advantage of ChIP in comparison to other techniques to analyze DNA-protein interactions is in fact, that the actual interaction of a transcription factor can be investigated in vivo and no probes or artificial conditions created by buffers or gels are employed8,11,12. By combining ChIP with&#160;next-generation sequencing, multiple targets can be identified simultaneously.
Major limitations of this technique are its limited applicability to large-scale assays in intact organisms25. The analysis of&#160;differential gene expression patterns can also be challenging using ChIP techniques if the respective proteins are expressed only at low levels or during narrow time windows. Another potentially limiting factor is the availability of an appropriate antibody suited for ChIP11.
The ChIP protocol presented here can be employed for the in vivo identification of target genes of a transcription factor by quantitative real-time PCR (qRT-PCR). Specifically, the goal was to identify novel target genes of NFAT2 in CLL. ChIP was chosen because of its potential to directly demonstrate the binding of NFAT2 to the promoter regions of different target genes under natural conditions in human CLL patient cells.

<table><tbody><tr><td>1 X PBS</td><td>Sigma Aldrich</td><td>D8537</td><td></td></tr><tr><td>1.5 mL tube shaker Themomixer comfort</td><td>Eppendorf</td><td>5355 000.011</td><td>Can be substituted with similar instruments</td></tr><tr><td>10X Bolt Sample Reducing Agent</td><td>Thermo Scientific</td><td>B0009</td><td></td></tr><tr><td>20X Bolt MES SDS Running Buffer</td><td>Thermo Scientific</td><td>B0002</td><td></td></tr><tr><td>37 % Formaldehyde p.a., ACS</td><td>Roth</td><td>4979.1</td><td></td></tr><tr><td>4X Bolt LDS Sample Buffer</td><td>Thermo Scientific</td><td>B0007</td><td></td></tr><tr><td>Anti-NFAT2 antibody</td><td>Alexis</td><td>1008505</td><td>Clone 7A6</td></tr><tr><td>Anti-NFAT2 antibody</td><td>Cell Signaling</td><td>8032S</td><td>Clone D15F1</td></tr><tr><td>Anti-NFAT2 antibody ChIP Grade</td><td>Abcam</td><td>ab2796</td><td>Clone 7A6</td></tr><tr><td>big Centrifuge</td><td>Eppendorf</td><td>5804R</td><td>Can be substituted with similar instruments</td></tr><tr><td>CD19-FITC mouse Anti-human</td><td>BD Biosciences</td><td>555412</td><td>Clone HIB19</td></tr><tr><td>CD5-PE mouse Anti-human CD5</td><td>BD Biosciences</td><td>555353</td><td>Clone UCHT2</td></tr><tr><td>Density gradient medium Biocoll (Density 1,077 g/ml)</td><td>Merck</td><td>L 6115</td><td></td></tr><tr><td>DNA LoBind Tube 1.5 mL</td><td>eppendorf</td><td>22431021</td><td></td></tr><tr><td>FBS superior</td><td>Merck</td><td>S0615</td><td></td></tr><tr><td>Flow Cytometer</td><td>BD Biosciences</td><td>FACSCalibur</td><td>Can be substituted with similar instruments</td></tr><tr><td>Halt Protease and Phosphatase Inhibitor Cocktail (100X)</td><td>Thermo Scientific</td><td>78440</td><td></td></tr><tr><td>iBlot 2 Gel Transfer Device</td><td>Thermo Scientific</td><td>IB21001</td><td></td></tr><tr><td>iBlot 2 Transfer Stacks, nitrocellulose, regular size</td><td>Thermo Scientific</td><td>IB23001</td><td></td></tr><tr><td>iDeal ChIp-seq kit for Histones</td><td>Diagenode</td><td>C01010059</td><td></td></tr><tr><td>Ionomycin calcium salt</td><td>Sigma Aldrich</td><td>I3909</td><td></td></tr><tr><td>IRDye 680LT Donkey anti-Rabbit IgG (H + L), 0.5 mg</td><td>LI-COR Biosciences</td><td>926-68023</td><td></td></tr><tr><td>IRDye 800CW Goat anti-Mouse IgG (H + L), 0.1 mg</td><td>LI-COR Biosciences</td><td>925-32210</td><td></td></tr><tr><td>LI-COR Odyssey Infrared Imaging System</td><td>LI-COR Biosciences</td><td>B446</td><td></td></tr><tr><td>LightCycler 480 Multiwell Plate 96, white</td><td>Roche</td><td>4729692001</td><td>Can be substituted with other plates in different real-time PCR instruments</td></tr><tr><td>Lysing Solution OptiLyse B</td><td>Beckman Coulter</td><td>IM1400</td><td></td></tr><tr><td>M220 AFA-grade water</td><td>Covaris</td><td>520101</td><td></td></tr><tr><td>M220 Focused-ultrasonicator</td><td>Covaris</td><td>500295</td><td></td></tr><tr><td>Magnetic rack, DynaMag-15 Magnet</td><td>Thermo Scientific</td><td>12301D</td><td>Can be substituted with similar instruments</td></tr><tr><td>MEM Non-Essential Amino Acids Solution 100X</td><td>Thermo Scientific</td><td>11140050</td><td></td></tr><tr><td>Microscope Axiovert 25</td><td>Zeiss</td><td>451200</td><td>Can be substituted with similar instruments</td></tr><tr><td>microTUBE AFA Fiber Pre-Slit Snap-Cap 6x16mm</td><td>Covaris</td><td>520045</td><td></td></tr><tr><td>Neubauer improved counting chamber</td><td>Karl Hecht GmbH &amp;amp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Co KG</td><td>40442012</td><td>Can be substituted with similar instruments</td></tr><tr><td>NH4 Heparin Monovette</td><td>Sarstedt</td><td>02.1064</td><td></td></tr><tr><td>Nuclease-free water</td><td>Promega</td><td>P1193</td><td></td></tr><tr><td>NuPAGE 4-12% Bis-Tris Protein Gels, 1.0 mm, 15-well</td><td>Thermo Scientific</td><td>NP0323BOX</td><td></td></tr><tr><td>Odyssey&amp;reg; Blocking Buffer (TBS) 500 mL</td><td>LI-COR Biosciences</td><td>927-50000</td><td></td></tr><tr><td>Penicillin/Streptomycin 100X</td><td>Merck</td><td>A2213</td><td></td></tr><tr><td>PerfeCTa SYBR Green FastMix</td><td>Quanta Bio</td><td>95072-012</td><td></td></tr><tr><td>PMA</td><td>Sigma Aldrich</td><td>P1585</td><td></td></tr><tr><td>Primer &lt;em&gt;CD40L&lt;/em&gt; promotor region forward</td><td>Sigma Aldrich</td><td></td><td>5&amp;rsquo;-ACTCGGTGTTAGCCAGG-3&amp;rsquo;</td></tr><tr><td>Primer &lt;em&gt;CD40L&lt;/em&gt; promotor region reverse</td><td>Sigma Aldrich</td><td></td><td>5&amp;rsquo;-GGGCTCTTGGGTGCTATTGT -3&amp;rsquo;</td></tr><tr><td>Primer &lt;em&gt;IL-2&lt;/em&gt; promotor region forward</td><td>Sigma Aldrich</td><td></td><td>5&amp;rsquo;-TCCAAAGAGTCATCAGAAGAG-3&amp;rsquo;</td></tr><tr><td>Primer &lt;em&gt;IL-2&lt;/em&gt; promotor region reverse</td><td>Sigma Aldrich</td><td></td><td>5&amp;rsquo;-GGCAGGAGTTGAGGTTACTGT-3&amp;rsquo;</td></tr><tr><td>Primer &lt;em&gt;LCK&lt;/em&gt; promotor region forward</td><td>Sigma Aldrich</td><td></td><td>5&amp;rsquo;-CAGGCAAAACAGGCACACAT-3&amp;rsquo;</td></tr><tr><td>Primer &lt;em&gt;LCK&lt;/em&gt; promotor region reverse</td><td>Sigma Aldrich</td><td></td><td>5&amp;rsquo;-CCTCCAGTGACTCTGTTGGC-3&amp;rsquo;</td></tr><tr><td>Rabbit mAb IgG XP Isotype Control</td><td>Cell Signaling</td><td># 3900S</td><td>Clone DA1E</td></tr><tr><td>Real-time PCR instrument</td><td>Roche</td><td>LightCycler 480</td><td>Can be substituted with similar instruments</td></tr><tr><td>Roller mixers</td><td>Phoenix Instrument</td><td>RS-TR 5</td><td></td></tr><tr><td>RPMI 1640 Medium, GlutaMAX Supplement</td><td>Thermo Scientific</td><td>61870010</td><td></td></tr><tr><td>Safety-Multifly-needle 21G</td><td>Sarstedt</td><td>851638235</td><td></td></tr><tr><td>SeeBlue Plus2 Pre-stained Protein Standard</td><td>Thermo Scientific</td><td>LC5925</td><td></td></tr><tr><td>Shaker Duomax 1030</td><td>Heidolph Instruments</td><td>543-32205-00</td><td>Can be substituted with similar instruments</td></tr><tr><td>small Centrifuge</td><td>Thermo Scientific</td><td>Heraeus Fresco 17</td><td>Can be substituted with similar instruments</td></tr><tr><td>Sodium Pyruvate</td><td>Thermo Scientific</td><td>11360070</td><td></td></tr><tr><td>&amp;szlig;-Mercaptoethanol</td><td>Thermo Scientific</td><td>21985023</td><td></td></tr><tr><td>Tris Buffered Saline (TBS-10X)</td><td>Cell Signaling</td><td>#12498</td><td></td></tr><tr><td>Trypan Blue solution</td><td>Sigma Aldrich</td><td>93595-50ML</td><td></td></tr></tbody></table>

a chromatin immunoprecipitation assay to identify novel nfat2 target genes in chronic lymphocytic leukemia

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

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

Figure 1 shows an exemplary flow cytometry analysis of a CLL patient performed after staining with CD19-FITC and CD5-PE antibodies. Figure 1a shows the gating of the lymphocytes, representing the majority of cells in the blood of CLL patients. Figure 1b shows the proportion of CD19+/CD5+ CLL cells, which represent 89.03% of lymphocytes in this example. The proportion of CD19<sup...

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

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

a-chromatin-immunoprecipitation-assay-to-identify-novel-nfat2-target

Research

JoVE Journal

Cancer Research

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

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

This video describes the protocol for chromatin shearing using micrococcal nuclease enzymatic digestion. Chromatin shearing is an essential step in Chromatin immunoprecipitation or ChIp workflow, which helps investigate protein-DNA interaction in human chronic myeloid leukemia cells. In the feature protocol chromatin shearing is performed using sonication.

Chromatin Shearing: A Method to Generate Chromatin Fragments Using Enzymatic Digestion

This video describes protocol for chromatin immunoprecipitation (ChIP) assay, which is a classic technique to demonstrate protein-DNA interactions.This technique is used to identify target genes in human chronic lymphocytic leukemia cells.

Chromatin Immunoprecipitation Assay: A Technique to Study Protein-DNA Interactions in Cells

In this video, we demonstrate the chromatin immunoprecipitation technique to identify protein binding sites on specific regions of the genomic DNA of oligodendrocyte precursor cells via the selective immunoprecipitation of protein-bound chromatin fragments. This method helps to study the interaction of several regulatory proteins, including transcription factors involved in gene regulation.

Chromatin Immunoprecipitation to Identify Target Protein Binding Sites on Genomic DNA

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.

Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

Here we describe a protocol that couples two proteomic techniques, namely 2-dimensional Electrophoresis (2DE) and Mass Spectrometry (MS), to identify differentially expressed/post-translational modified proteins among two or more groups of primary samples. This approach, together with functional experiments, allows the identification and characterization of prognostic markers/therapeutic targets.

From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

The protocol described here represents an easy and reproducible method that employs reverse phase high-performance liquid chromatography (RP-HPLC) to measure purine metabolism on chronic lymphocytic leukemia (CLL) cells cultured under different conditions.

HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells

This work describes an optimized methyl-CpG-binding domain (MBD) sequencing protocol and a computational pipeline to identify differentially methylated CpG-rich regions in chronic lymphocytic leukemia (CLL) patients.

Comprehensive DNA Methylation Analysis Using a Methyl-CpG-binding Domain Capture-based Method in Chronic Lymphocytic Leukemia Patients

Here we present a protocol which is designed to analyze the genome-wide binding of the oligodendrocyte transcription factor 2 (Olig2) in acutely purified brain oligodendrocyte precursor cells (OPCs) by performing low-cell chromatin immunoprecipitation (ChIP), library preparation, high-throughput sequencing and bioinformatic data analysis.

Identifying Transcription Factor Olig2 Genomic Binding Sites in Acutely Purified PDGFR&#945;+ Cells by Low-cell Chromatin Immunoprecipitation Sequencing Analysis

This protocol enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These samples are used to determine protein localization as well as changes in protein trafficking that take place between the nuclear and cytoplasmic compartments upon cell stimulation and drug treatment.

Subcellular Fractionation of Primary Chronic Lymphocytic Leukemia Cells to Monitor Nuclear/Cytoplasmic Protein Trafficking

This manuscript outlines a protocol for preparing an ATAC-seq library of neutrophils from murine bone marrow, aiming to guarantee optimal neutrophil viability and high library quality. It offers step-by-step instructions on BMC preparation, immunomagnetic sorting, and library construction, serving as a valuable guide, especially for newcomers to studying neutrophils.

ATAC-Seq Library Preparation of Murine Bone Marrow-Derived Neutrophils

Learn chromatin immunoprecipitation to identify NFAT2 target genes in chronic lymphocytic leukemia cells. This method reveals protein-DNA interactions to advance therapeutic strategies.

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

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