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>

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 ChIP7. Even supposedly identical 7A6 antibodies from different manufacturers exhibited significant differences with respect to their performance in ChIP. A major challenge is the potential disruption of target protein epitopes by the fixation process used in XChIP protocols25.
The chromatin shearing procedure is also critical for acquiring appropriate results in XChIP. A fragment size of 200-500 bp as assessed by agarose gel electrophoresis was found to be optimal in this NFAT2 ChIP protocol7. Inappropriate shearing conditions resulting in a shortage of DNA starting material or DNA fragments of smaller or larger size typically lead to suboptimal results when performing this assay. Suboptimal shearing was also observed when using frozen and rethawed PBMCs. Hence, thawing of PBMCs resulted in a lower yield of DNA from cells as well as an increase in excessive shearing of DNA-fragments.
The availability of a broad variety of ChIP kits and ChIP-grade antibodies is challenging, but also offers the possibility to use the technique in a wide context. Thus, a diversity of transcription factors can be investigated in different cell types. For example, other members of the NFAT family (NFAT1 and NFAT4) have been investigated recently using ChIP35,36,37.
The ChIP kit used here also had to be modified to suit our needs. First, the time of fixation was adjusted to avoid masking of the epitope recognized by the used antibody and to enable optimal shearing. The volume of buffers used for lysis and shearing was also changed to reduce the loss of cells during the different washing steps. Additionally, the shearing conditions were optimized to obtain DNA fragments in the range of 200-500 bp. The protease inhibitor and the IgG-control antibody were exchanged with other commercially available reagents as they proved to be comparable and more cost-effective. The step involving the carrier provided by the manufacturer was omitted as it interfered with the precipitation of the DNA. In the end, the amount of buffer used to elute the DNA was adapted to yield a sufficient volume to be used in the qRT-PCR.
There are several other kits available from various companies and there is also the possibility to perform the ChIP without a kit. However, testing and establishment are very challenging, as there are many factors to be considered, like the compatibility of analyzed cells and proteins with different fixation and shearing methods. The mentioned kit was used as it was well-established in our laboratory.
A major restriction of this method is its limited applicability to the large-scale investigations in intact model organisms because appropriate antibodies have to be identified or generated for each individual transcription factor. The analysis of genes which are expressed only at low levels, in a small number of cells or during a restricted time window can also be challenging using ChIP.
While ChIP is the gold standard to demonstrate a direct interaction of a given transcription factor with its respective target genes in intact eukaryotic cells25, there is&#160;a number of other techniques to investigate an interaction between proteins and DNA. EMSA is a useful method to detect low-abundance DNA binding proteins in cell lystates24. It can also be used to characterize the binding affinity of a particular protein through a systematic DNA probe mutational analysis. A major advantage of EMSA in comparison to ChIP is the fact that it is generally substantially less time-consuming to establish the respective assay. DNA pull-down assays, microplate capture and detection assays and reporter assays are other techniques to analyze protein-DNA interactions23.
More recent developments have made it possible to apply the ChIP assay to genome-wide approaches by its combination with&#160;microarray technology (ChIP-on-chip)38,39,40 or&#160;next-generation sequencing (ChIP-Seq)41,42,43.

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

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+/CD5- B cells is 3.72% in the respective patient.
Figure 2 shows examples of antibody performance in Jurkat cells fixed for different time periods as assessed by SDS-PAGE and Western Blot. Different antibodies from different manufacturers were analyzed. Figure 2a shows the performance of the &#945;NFAT2-antibody (clone 7A6) from different manufacturers detected with a green fluorescent anti-mouse antibody. The antibody of manufacturer 1 showed binding to its epitope without fixation (band A) and even when Jurkat cells were fixed for 2.5 min or 5 min (band B or C, respectively). The &#945;NFAT2-antibody (clone 7A6) from manufacturer 2, on the other hand, showed a weaker performance even in Jurkat cells not fixed (band D). Upon fixation, the antibody of this manufacturer achieved even weaker results (bands E (2.5 min fixation) and F (5 min fixation)). Figure 2b shows the performance of the &#945;NFAT2-antibody (clone D15F1) from a different manufacturer detected with a red fluorescent anti-rabbit antibody. This antibody also performed weakly in unfixed samples (band A) and the absence of a band indicates the masking of its epitope by the fixation (bands B (2.5 min fixation) and C (5 min fixation)).
Figure 3 shows examples of sheared chromatin from Jurkat cells obtained after different fixation and shearing times of good and poor quality as assessed by gel electrophoresis. Bands A and B (0 min fixation or 2.5 min fixation, respectively) show good quality sheared chromatin, which is characterized by a DNA fragment size of 200-500 bp which can be detected on an agarose gel as a typical smear. Sheared chromatin of poor quality, on the other hand, can be recognized by almost complete or complete absence of the expected DNA smear (band C) as well as a smear in a larger or smaller than expected fragment size range (bands D-F). The complete absence of the smear indicates the use of insufficient amounts of starting material. The detection of smaller DNA fragments hints to excessive DNA shearing (band E) while larger DNA fragments hint to insufficient shearing.
Figure 4 shows the results of a typical experiment with Jurkat cells. LCK was analyzed as a potential NFAT2 target. IL-2 which is a well-defined target gene of NFAT2 in T cells was used as a positive control. An IgG-control antibody was utilized for normalization. Figure 4a shows an experiment with optimal results documented by significant enrichment of the IL-2 positive control. From this experiment, it can be concluded that there is a significant enrichment of LCK sequences in the precipitated DNA demonstrating that LCK is a direct NFAT2 target in Jurkat cells. Figure 4b shows an experiment performed with poor quality DNA due to missing fixation or a suboptimal shearing procedure causing a low degree of enrichment in the IL-2 positive control.
Figure 5 shows the results of an experiment performed with primary human CLL cells. CD40L which is a known NFAT2 target in B cells served as the positive control and LCK was analyzed as the experimental target. Figure 5a shows an experiment with a considerable level of enrichment of CD40L DNA in the positive control. From the even stronger enrichment of LCK DNA, it could be concluded that LCK is also a direct NFAT2 target in primary human CLL cells. Figure 5b shows an experiment performed with poor quality DNA most likely due to inadequate shearing. No substantial enrichment of CD40L DNA could be detected in the positive control rendering the experimental data meaningless.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58270/58270fig1v2.jpg" /> 
Figure 1: Exemplary Flow cytometry analysis of CLL patients. (a) Samples of CLL patients were analyzed via flow cytometry after staining with CD19-FITC and CD5-PE antibodies and lymphocytes were gated. (b) Subsequently, the proportion of CD19+/CD5+ CLL cells and CD19+/CD5- B cells were determined. Here, CD19+/CD5+ CLL cells account for 89.03% of lymphocytes, whereas CD19+/CD5- B cells represent 3.72% of lymphocytes. One exemplary patient is shown. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58270/58270fig1large.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/58270/58270fig2v2.jpg" /> 
Figure 2: Examples of antibody performance in fixed Jurkat cells assessed by SDS-PAGE and Western-Blot. (a) Jurkat cells were fixed for 0 min (bands A and D), 2.5 min (bands B and E), or 5 min (C and F) and the &#945;NFAT2-antibody (clone 7A6) from different manufacturers (bands A-C = manufacturer 1, bands D-F = manufacturer 2) was compared. The antibody of manufacturer 1 showed a better overall performance, binding with the higher affinity even to fixed samples (compare bands A-C and bands D-F). (b) The &#945;NFAT2-antibody (clone D15F1) from a different manufacturer was used. This antibody showed only a poor performance in unfixed samples (band A) and the epitope was masked upon fixation. Therefore, no binding of the antibody could be detected after fixation (bands B and C). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58270/58270fig2large.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/58270/58270fig3v2.jpg" /> 
Figure 3: Examples of chromatin of good and poor shearing quality from Jurkat cells assessed by gel electrophoresis. The Jurkat cells were fixed and sheared for different time periods. Fixation was done for 0 min (bands A and D), 2.5 min (bands B and E), or 5 min (C and F). Shearing was performed either for 10 min (bands A-C) or 20 min (bands D-F). Chromatin of good shearing quality is characterized by a DNA fragment size of 200-500 bp which can be detected as a smear in the respective region (bands A and B). Chromatin of poor quality can be recognized either by an almost complete or complete absence of the DNA smear due to an insufficient amount of starting material used (band C) or by a smear in a smaller or larger size region because of inappropriate shearing conditions (bands D-F). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58270/58270fig3large.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/58270/58270fig4v2.jpg" /> 
Figure 4: ChIP of Jurkat cells assessed by qRT-PCR. (a) NFAT2 binds to LCK and the IL-2 promoter in Jurkat cells. The relative enrichment of LCK and IL-2 promoter regions precipitated with the NFAT2 antibody is shown as analyzed by qRT-PCR. Three independent ChIP-experiments are shown as mean &#177; SEM (Student&#39;s t-test, *P &#60; 0.05; **P &#60; 0.01 ***P &#60; 0.001). (b) DNA from samples with poor quality sheared chromatin was used. No relevant binding of NFAT2 to the target sequences could be detected. A similar picture can be detected if there was no fixation or the incubation time with the NFAT2 antibody was insufficient. Three independent ChIP-experiments are shown as mean &#177; SEM (Student&#39;s t-test, *P &#60; 0.05; **P &#60; 0.01 ***P &#60; 0.001). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58270/58270fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58270/58270fig5v2.jpg" /> 
Figure 5: ChIP of primary human CLL cells assessed by qRT-PCR. (a) NFAT2 specifically binds to the LCK and CD40L promoters in primary human CLL cells from patients with an indolent course of the disease. The diagram shows the relative enrichment of LCK and CD40L promoter regions precipitated with the NFAT2 antibody as analyzed by qRT-PCR. Three independent ChIP-experiments are shown as mean &#177; SEM (Student&#39;s t-test, *P &#60; 0.05; **P &#60; 0.01 ***P &#60; 0.001). (b) DNA from patient samples with poor quality sheared chromatin was used. No binding of NFAT2 to the respective target sequences could be observed. Three independent ChIP-experiments are shown as mean &#177; SEM (Student&#39;s t-test, *P &#60; 0.05; **P &#60; 0.01 ***P &#60; 0.001) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58270/58270fig5v2large.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>item</td>
			<td>volume (&#181;L) per IP</td>
		</tr>
		<tr>
			<td>5% BSA</td>
			<td>6</td>
		</tr>
		<tr>
			<td>100 x protease inhibitor</td>
			<td>3</td>
		</tr>
		<tr>
			<td>5 x ChIP buffer 1</td>
			<td>56</td>
		</tr>
		<tr>
			<td>sheared chromatin</td>
			<td>x (15&#160;&#181;L or 70&#160;&#181;L)</td>
		</tr>
		<tr>
			<td>Protein A coated magnetic beads</td>
			<td>20</td>
		</tr>
		<tr>
			<td>ChIP seq grade water</td>
			<td>185-x-y</td>
		</tr>
		<tr>
			<td>antibody (&#945;NFAT2 or IgG-control)</td>
			<td>y (1.09 &#181;L or 1 &#181;L)</td>
		</tr>
		<tr>
			<td>total</td>
			<td>270</td>
		</tr>
	</tbody>
</table>
Table 1: Schedule for pipetting the chromatin immunoprecipitation. The chromatin immunoprecipitation was performed by preparing the reactions as indicated in the table.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>item</td>
			<td>volume (&#181;L) per qRT-PCR</td>
		</tr>
		<tr>
			<td>immunoprecipitated DNA</td>
			<td>9</td>
		</tr>
		<tr>
			<td>qRT-PCR Mix</td>
			<td>10</td>
		</tr>
		<tr>
			<td>primer forward (LCK/CD40L/IL-2)</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>primer reverse (LCK/CD40L/IL-2)</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>total</td>
			<td>20</td>
		</tr>
	</tbody>
</table>
Table 2: Schedule for pipetting the qRT-PCR. The qRT-PCR was performed by preparing the reactions as indicated in the table.

Watch this Scientific Journal Video about A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia at JoVE.com

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

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

Nanyang Technological University

Research

JoVE Journal

Cancer Research

7.4K Views.  University of Tübingen. Deze methode kan helpen om belangrijke informatie op het gebied van oncologie te verstrekken, zoals de identificatie van vermeende doelgenen en pathogene mechanismen om potentiële therapeutische interventies te ontwikkelen. Het belangrijkste voordeel van deze techniek is dat men de interactie van NFAT2 en andere transcriptiefactoren met bekende en nieuwe doelgenen kan detecteren. Over het algemeen individuen nieuw op deze methode zal worstelen, omdat optimale fixatie en scheren voorwaarden z...

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

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

The role of long noncoding RNAs (lncRNAs) in cancer is coming to the forefront due to growing interest in understanding their mechanistic functions during cancer development and progression. Despite this, the global epigenetic regulation of lncRNAs and repetitive sequences in cancer has not been well investigated, particularly in chronic lymphocytic leukemia (CLL). This study focuses on a unique approach: the immunoprecipitation-based capture of double-stranded, methylated DNA fragments using methyl-binding domain (MBD) proteins, followed by next-generation sequencing (MBD-seq). CLL patient samples belonging to two prognostic subgroups (5 IGVH mutated samples + 5 IGVH unmutated samples) were used in this study. Analysis revealed 5,800 hypermethylated and 12,570 hypomethylated CLL-specific differentially methylated genes (cllDMGs) compared to normal healthy controls. Importantly, these results identified several CLL-specific, differentially methylated lncRNAs, repetitive elements, and protein-coding genes with potential prognostic value. This work outlines a detailed protocol for an MBD-seq and bioinformatics pipeline developed for the comprehensive analysis of global methylation profiles in highly CpG-rich regions using CLL patient samples. Finally, a protein-coding gene and an lncRNA were validated using pyrosequencing, which is a highly quantitative method to analyze CpG methylation levels to further corroborate the findings from the MBD-seq protocol.

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.

The role of long noncoding RNAs (lncRNAs) in cancer is coming to the forefront due to growing interest in understanding their mechanistic functions during ...

Genetics

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

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and validated for the quantification of extracellular purine nucleotides and nucleosides produced by purified chronic lymphocytic leukemia (CLL) cells under different culture conditions. The chromatographic separation of adenosine 5&#39;-monophosphate (AMP), adenosine (ADO) and inosine (INO) is performed at RT on a silica-based, reversed-phase column that is used for polar compound retention. The method includes a binary mobile phase, which consists of 7 mM ammonium acetate and acetonitrile with a flow rate of 1.00 ml/min. The eluates are monitored using a Photodiode Array UV detector set at 260 nm. A standard calibration curve is generated to calculate the equation for the analytical quantification of each purine compound. System control, data acquisition and analysis are then performed. Applying this protocol, AMP, INO and ADO elute at 7, 11 and 11.9 min, respectively, and the total run time for each sample is 20 min. This protocol may be applied to different cell types and cell lines (both suspension and adherent), using culture media as matrix. The advantages are easy and fast sample preparation and the requirement of a small amount of supernatant for analysis. Furthermore, the use of a serum-free medium allows skipping the protein precipitation step with acetonitrile that impacts the final concentration of purine compounds. One of the limitations of the method is the requirement of the equilibration column run before each single sample run, making the total run time of the experiment longer and preventing high throughput screening applications.

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.

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and ...

Chemistry

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

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant cellular localization disrupting their mechanism of action. The survival of chronic lymphocytic leukaemia (CLL) cells, among other cancer cells, is assisted by the deregulation of nuclear to cytoplasmic shuttling, at least in part through deregulation of the transport receptor XPO1 and the constitutive activation of PI3K-mediated signaling pathways. It is essential to understand the role of individual proteins in the context of their intracellular location to gain a deeper understanding of the role of such proteins in the pathobiology of the disease. Furthermore, identifying processes that underlie cell stimulation and the mechanism of action of specific pharmacological inhibitors, in the context of subcellular protein trafficking, will provide a more comprehensive understanding of the mechanism of action. The protocol described here enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These fractions can be used to determine changes in protein trafficking between the nuclear and cytoplasmic fractions upon cell stimulation and drug treatment. The data can be quantified and presented in parallel with immunofluorescent images, thus providing robust and quantifiable data.

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.

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant ...

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

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as &#8220;spots&#8221; on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.

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.

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a ...

Medicine

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

Biology

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

In mammalian cells, gene transcription is regulated in a cell type specific manner by the interactions of transcriptional factors with genomic DNA. Lineage-specific transcription factors are considered to play essential roles in cell specification and differentiation during development. ChIP coupled with high-throughput DNA sequencing (ChIP-seq) is widely used to analyze genome-wide binding sites of transcription factors (or its associated complex) to genomic DNA. However, a large number of cells are required for one standard ChIP reaction, which makes it difficult to study the limited number of isolated primary cells or rare cell populations. In order to understand the regulatory mechanism of oligodendrocyte lineage-specific transcription factor Olig2 in acutely purified mouse OPCs, a detailed method using ChIP-seq to identify the genome-wide binding sites of Olig2 (or Olig2 complex) is shown. First, the protocol explains how to purify the platelet-derived growth factor receptor alpha (PDGFR&#945;) positive OPCs from mouse brains. Next, Olig2 antibody mediated ChIP and library construction are performed. The last part describes the bioinformatic software and procedures used for Olig2 ChIP-seq analysis. In summary, this paper reports a method to analyze the genome-wide bindings of transcriptional factor Olig2 in acutely purified brain OPCs.

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.

In mammalian cells, gene transcription is regulated in a cell type specific manner by the interactions of transcriptional factors with genomic DNA. ...

Neuroscience

NF-&#954;B-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells

The dimeric transcription factor NF-&#954;B regulates many cellular response pathways, including inflammatory pathways by inducing the expression of various cytokines and chemokines. NF-&#954;B is constitutively expressed and is sequestered in the cytosol by the inhibitory protein nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (I&#954;B&#945;). Activation of NF-&#954;B requires the degradation of I&#954;B&#945;, which then exposes a nuclear localization signal on NF-&#954;B and promotes its trafficking to the nucleus. Once in the nucleus, NF-&#954;B binds to the promotor region of NF-&#954;B target genes such as interleukin 6 (IL-6) and IL-23, to promote their expression.
The activation of NF-&#954;B occurs independently of transcription or translation. Therefore, the activation state of NF-&#954;B must be measured either by quantifying NF-&#954;B specifically in the nucleus, or by quantifying expression of NF-&#954;B target genes. In this protocol, cells stably transfected with an NF-&#954;B::luciferase reporter construct are assayed for NF-&#954;B activation using in vitro tissue culture techniques. These cells are infected with Salmonella Typhimurium to activate NF-&#954;B, which traffics to the nucleus and binds to &#954;B sites in the promoter region of luciferase, inducing its expression. Cells are lysed and analyzed with the luciferase assay system. The amount of luciferase produced by the cells correlates with the intensity of the luminescence signal, which is detected by a plate reader. The luminescence signal generated by this procedure provides a quick and highly sensitive method by which to assess NF-&#954;B activation under a range of conditions. This protocol also utilizes quantitative reverse transcription PCR (RT-qPCR) to detect relative mRNA levels that are indicative of gene expression.

Here, we present a protocol to quickly and easily measure nuclear factor kappa-light-chain-enhancer of activated B cells (NF-&#954;B) activation in cell lines expressing NF-&#954;B::luciferase reporter constructs, via measurements of luminescence in the cell lysate. Additionally, gene expression is determined via RT-qPCR isolated from cells infected with Salmonella Typhimurium.

The dimeric transcription factor NF-&#954;B regulates many cellular response pathways, including inflammatory pathways by inducing the expression of ...

Immunology and Infection

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 ...

Chromatin Immunoprecipitation to Identify Target Protein Binding Sites on Genomic DNA

Source: Dong, X., et.al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57547">Identifying Transcription Factor Olig2 Genomic Binding Sites in Acutely Purified PDGFR&#945;+ Cells by Low-cell Chromatin Immunoprecipitation Sequencing Analysis.</a>&#160;J. Vis. Exp.&#160;(2018)
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.

Source: Dong, X., et.al., Identifying Transcription Factor Olig2 Genomic Binding Sites in Acutely Purified PDGFR&#945;+ Cells by Low-cell Chromatin ...

JoVE Encyclopedia of Experiments

Biological Techniques

Biomolecular Interaction Detection Techniques

Protein-nucleic acid interactions

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

Source: Fuchs, A. R.,et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/t/58270">A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia.</a> J. Vis. Exp. (2018).
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.

Source: Fuchs, A. R.,et al. A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia. J. Vis. Exp. ...

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

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