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

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 specific treatment for many years. In contrast, some patients show rapid progression requiring immediate therapeutic interventions with immune-chemotherapy or other targeted therapies^2,3. Nuclear factor of activated T cells (NFAT) is a family of transcription factors controlling various developmental and activation processes in numerous cell types^4,5,6. 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 process^9,10,11,12. 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 cancer^19,20. Hence, identification of transcription factors and their respective targets might offer novel therapeutic avenues^21,22. To investigate this intriguing field several methods are available like ChIP, electrophoretic mobility shift assay (EMSA), various DNA pull-down assays and reporter-assays^{8,11,12,23,24}.

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 genes^29,30. 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 employed^8,11,12. By combining ChIP with 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 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.

All experiments conducted with human material were approved by the Ethics Committee of the University of Tübingen 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.

1. Prepare 50 mL of RPMI 1640 supplemented with 10% FCS and 1% penicillin/streptomycin by warming it to 37 °C in a water bath.
2. Culture Jurkat cells for 1-2 d in a cell culture flask at 37 °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.
3. 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.
4. 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.
5. Stimulate the cells by adding 32.4 nM phorbol 12-myristate 13-acetate (PMA) and 1 µM ionomycin. Then incubate the cells for 16 h at 37 °C and 5% CO₂ with the lid of the tube opened (to avoid contamination, a sealing film permeable to air can be used).

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.

1. 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).
2. 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).
3. 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.
4. 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.
5. 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.
6. 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's instructions. Then a staining with CD19-FITC and CD5-PE antibodies is performed according to the manufacturer'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).
7. Wash the cells with 10 mL of pre-warmed (37 °C) RPMI 1640 supplemented with 10% FCS, 1% penicillin/streptomycin, 100 mM non-essential amino acids, 1 mM sodium pyruvate and 50 mM β-mercaptoethanol for 5 min at 200 x g and remove the supernatant by aspiration.
8. 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 β-mercaptoethanol (37 °C) and fill 1 mL of the cell suspension into a 5 mL tube.
   NOTE: The ß-mercaptoethanol is employed to counteract oxidative stress.
9. Stimulate the cells by adding 32.4 nM PMA and 1 µM ionomycin. Then incubate the cells for 16 h at 37 °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 °C and 5% CO₂ in the incubator prior to the 16 h of stimulation.

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's instructions with modifications as described previously⁷. The fixation is performed under a laminar flow hood.

1. 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.
2. 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 µL of PBS in the 5 mL tubes.
3. Add 340 µM formaldehyde (13.5 µL 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.
4. Add 125 mM glycine (57 µL 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 °C. Remove the supernatant by aspiration.
5. Wash the cells twice with 1 mL of ice-cold PBS at 200 x g for 5 min at 4 °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 °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 µg of protein according to manufacturer's instructions. All αNFAT2 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.
6. 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.
7. Subsequently, centrifuge the tubes at 500 x g for 5 min at 4 °C and remove the supernatant carefully by pipetting.
8. 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 °C and remove the supernatant carefully by pipetting.
9. Resuspend the cell pellet in 500 µL (Jurkat cells) or 140 µL (patient samples) of shearing buffer 1 containing 1x protease inhibitor (5 µL or 1.4 µL, respectively) and incubate the mixture for 10 min on ice.
10. For chromatin shearing, transfer 140 µL 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 °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.
11. Finally, centrifuge at 15700 x g for 10 min at 4 °C in the small centrifuge and collect the supernatant in a new 1.5 mL tube.
12. To test for the appropriate fragment sizes, analyze 20 µL 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 °C.

4. Chromatin Immunoprecipitation

NOTE: The chromatin immunoprecipitation was performed with a commercially available ChIP kit according to the manufacturer's instructions with modifications⁷.

1. Calculate the number of immunoprecipitations (70 µL (Jurkat cells) or 15 µL (patient samples) of sheared chromatin plus one antibody) and prepare 20 µL of protein A coated beads per precipitation in a 1.5 mL tube. Wash the beads in 40 µL 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.
2. For the immunoprecipitation itself, use 10 µg of the αNFAT2 antibody (7A6) to capture NFAT2 with its bound target sequences. Use 2.5 µg of a rabbit IgG antibody (DA1E) as an IgG-control. Prepare the reactions 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.
3. Incubate the mixture from step 4.2 overnight at 4 °C on a rotating wheel at 6 rpm.
4. 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.
5. To do so, resuspend the beads in 350 µL of wash buffer and incubate them for 5 min at 4 °C on a rotating wheel at 6 rpm.
6. 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.
7. After the last washing step, remove the supernatant by pipetting, take up the beads in 100 µL of elution buffer 1 and incubate them for 30 min on a rotating wheel at 6 rpm.
8. Spin the tubes shortly (5 s at 7000 x g in the small centrifuge) and place them in a magnetic rack for 1 min.
9. Then transfer the supernatant by pipetting to a new 1.5 mL tube and add 4 µL of elution buffer 2.
10. To create the input control, mix 1 µL of the sheared chromatin with 99 µL of elution buffer 1 and 4 µL of elution buffer 2 (in this setup, there are three tubes per patient: one input control, one IgG-control and one αNFAT2 sample).
11. Incubate the samples for 4 h at 65 °C and 1300 rpm in a 1.5 mL tube shaker. Add 100 µL of 100% isopropanol, vortex and spin the samples for 5 s at 7000 x g in the small centrifuge.
12. Resuspend the available magnetic beads by thoroughly vortexing, add 10 µL of the beads to every sample and incubate for 1 h at room temperature on a rotating wheel at 6 rpm.
13. 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.
14. Resuspend the beads in 100 µL of wash buffer 1. Invert the tubes to mix and incubate for 5 min at room temperature on a rotating wheel at 6 rpm.
15. 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.
16. Subsequently, remove the wash buffer by aspiration, resuspend the beads in 55 µL 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 °C.

5. Detection of NFAT target genes in CLL cells.

1. 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.
2. Use white 96-well reaction plates for the reactions and the RT-PCR instrument. The cycling conditions are as follows: 95 °C for 30 s, [95 °C for 5 s, 60 °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 CLL^33,34. The primers for the CD40L, the IL-2 and LCK promoter region are described in the table of specific reagents and equipment.

6. Normalization and Data Analysis

NOTE: Relative enrichment for the promoter regions of interest (IL-2, CD40L or LCK) is calculated using the IgG-control for normalization.

1. 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.
2. 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.
3. 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.
4. Finally, calculate the relative enrichment by dividing the concentration of the samples by the concentration of the IgG reference.

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 αNFAT2-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 αNFAT2-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 αNFAT2-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.

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. Please click here to view a larger version of this figure.

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 αNFAT2-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 αNFAT2-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). Please click here to view a larger version of this figure.

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). Please click here to view a larger version of this figure.

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 ± SEM (Student's t-test, *P < 0.05; **P < 0.01 ***P < 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 ± SEM (Student's t-test, *P < 0.05; **P < 0.01 ***P < 0.001). Please click here to view a larger version of this figure.

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 ± SEM (Student's t-test, *P < 0.05; **P < 0.01 ***P < 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 ± SEM (Student's t-test, *P < 0.05; **P < 0.01 ***P < 0.001) Please click here to view a larger version of this figure.

Table 1: Schedule for pipetting the chromatin immunoprecipitation. The chromatin immunoprecipitation was performed by preparing the reactions as indicated in the table.

Table 2: Schedule for pipetting the qRT-PCR. The qRT-PCR was performed by preparing the reactions as indicated in the table.

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 αNFAT2 antibody proved to be particularly challenging during the development of this protocol. While there are several αNFAT2 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 ChIP^35,36,37.

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 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 microarray technology (ChIP-on-chip)^38,39,40 or next-generation sequencing (ChIP-Seq)^41,42,43.