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 sup...

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

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

<table>
	<tbody>
		<tr>
			<td>1 X PBS</td>
			<td>Sigma Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL tube shaker Themomixer comfort</td>
			<td>Eppendorf</td>
			<td>5355 000.011</td>
			<td>Can be substituted with similar instruments</td>
		</tr>
		<tr>
			<td>10X Bolt Sample Reducing Agent</td>
			<td>Thermo Scientific</td>
			<td>B0009</td>
			<td></td>
		</tr>
		<tr>
			<td>20X Bolt MES SDS Running Buffer</td>
			<td>Thermo Scientific</td>
			<td>B0002</td>
			<td></td>
		</tr>
		<tr>
			<td>37 % Formaldehyde p.a., ACS</td>
			<td>Roth</td>
			<td>4979.1</td>
			<td></td>
		</tr>
		<tr>
			<td>4X Bolt LDS Sample Buffer</td>
			<td>Thermo Scientific</td>
			<td>B0007</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-NFAT2 antibody</td>
			<td>Alexis</td>
			<td>1008505</td>
			<td>Clone 7A6</td>
		</tr>
		<tr>
			<td>Anti-NFAT2 antibody</td>
			<td>Cell Signaling</td>
			<td>8032S</td>
			<td>Clone D15F1</td>
		</tr>
		<tr>
			<td>Anti-NFAT2 antibody ChIP Grade</td>
			<td>Abcam</td>
			<td>ab2796</td>
			<td>Clone 7A6</td>
		</tr>
		<tr>
			<td>big Centrifuge</td>
			<td>Eppendorf</td>
			<td>5804R</td>
			<td>Can be substituted with similar instruments</td>
		</tr>
		<tr>
			<td>CD19-FITC mouse Anti-human</td>
			<td>BD Biosciences</td>
			<td>555412</td>
			<td>Clone &#160;HIB19</td>
		</tr>
		<tr>
			<td>CD5-PE mouse Anti-human CD5&#160;</td>
			<td>BD Biosciences</td>
			<td>555353</td>
			<td>Clone &#160;UCHT2</td>
		</tr>
		<tr>
			<td>Density gradient medium Biocoll&#160; (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&#160;&#160;&#160;&#160;&#160; 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 &#38;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 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&#174; 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 CD40L promotor region forward</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td>5&#8217;-ACTCGGTGTTAGCCAGG-3&#8217;</td>
		</tr>
		<tr>
			<td>Primer CD40L promotor region reverse</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td>5&#8217;-GGGCTCTTGGGTGCTATTGT -3&#8217;</td>
		</tr>
		<tr>
			<td>Primer IL-2 promotor region forward</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td>5&#8217;-TCCAAAGAGTCATCAGAAGAG-3&#8217;</td>
		</tr>
		<tr>
			<td>Primer IL-2 promotor region reverse</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td>5&#8217;-GGCAGGAGTTGAGGTTACTGT-3&#8217;</td>
		</tr>
		<tr>
			<td>Primer LCK promotor region forward</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td>5&#8217;-CAGGCAAAACAGGCACACAT-3&#8217;</td>
		</tr>
		<tr>
			<td>Primer LCK promotor region reverse</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td>5&#8217;-CCTCCAGTGACTCTGTTGGC-3&#8217;</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>&#223;-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<sup...

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 ...

This method can help to provide key information in the field of oncology such as the identification of putative target genes and pathogenic mechanisms to develop potential therapeutic interventions. The main advantage of this technique is that one can detect the interaction of NFAT2 and other transcription factors with known and novel target genes. Generally individuals new to this method will struggle because optimal fixation and shearing conditions are difficult to establish.To begin fill a 1.5-milliliter tube with one milliliter of 37%formaldehyde. Then fill another 1.5-milliliter tube with one milliliter of 1.25 molar glycine in a five-milliliter tube with four milliliters of PBS. Next, after the stimulation of the cells spin the cells according to the text protocol, and re-suspend the cells in 500 microliters of PBS and place the tube in an ice-filled box.Add 13.5 microliters of 37%formaldehyde to the cells and mix by pipetting up and down. Then incubate the suspension at room temperature. After this, add 57 microliters of 1.25 molar glycine to stop the fixation.And allow the suspension to incubate at room temperature for five minutes. Immediately following the incubation period place the cells on ice and centrifuge the cells at 500 times G for five minutes at four degrees Celsius. Then aspirate the supernatant.Next, wash the cells twice with one milliliter of ice-cold PBS at 200 times G for five minutes at four degrees Celsius and remove the supernatant. Resuspend the cell pellet in five milliliters of lysis buffer one by pipetting up and down. Then put the samples on ice and incubate them with shaking for 10 minutes.After this, centrifuge the tubes at 500 times G for five minutes at four degrees Celsius. Then use a pipette to carefully remove the supernatant. Homogenize the cells in five milliliters of lysis buffer two and pipette up and down to mix.Then incubate the cells on ice for 10 minutes with shaking. After the incubation period centrifuge the tubes at 500 times G for five minutes at four degrees Celsius, and carefully remove the supernatant. Next, resuspend the cell pellet in shearing buffer one with 1X protease inhibitor.Then incubate the cell suspension on ice for 10 minutes. Transfer 140 microliters of the cell suspension to sonicator tubes taking care to avoid forming air bubbles. Place the tubes in a focused-ultrasonicator at seven degrees Celsius and shear for 10 to seven and 1/2 minutes.Transfer the cell suspension into 1.5-milliliter tubes. Centrifuge the samples at 15, 700 times G for 10 minutes at four degrees Celsius, and collect the supernatant in a new tube. First, prepare 20 microliters of protein A-coated beads for each precipitation in one 1.5-milliliter tube.Allow the beads to settle on a magnetic rack for one minute, and remove the supernatant. Then wash the beads with 40 microliters of 1X chip buffer one for each 20 microliters of protein A-coated beads by pipetting up and down. Allow the beads to settle on a magnetic rack for one minute and remove the supernatant.Repeat this washing process three more times before re-suspending the beads in their original volume of 1X chIP buffer one. Add BSA protease inhibitor, 5X chIP buffer, and ChIP-seq grade water to the beads. Next, add the sheared chromatin.Use 10 micrograms of anti-NFAT2 antibody to capture NFAT2, and 2.5 micrograms of IgG antibody as a control. Incubate the mixtures overnight at four degrees Celsius on a wheel rotating at six RPM. The next day spin the tubes for five seconds at 7, 000 times G and incubate them on the magnetic rack for one minute.Then aspirate the supernatant and wash the beads once with wash buffers one, two, three, and four consecutively. Spin the tubes for five seconds at 7000 times G before incubating the tubes on the magnetic rack for one minute. After this, remove the supernatant and add the next wash buffer.After the last wash, spin the tubes for five seconds at 7000 times G.Incubate the tubes on the magnetic rack for one minute. Remove the supernatant and take up the beads in 100 microliters of elution buffer one. Then incubate the tubes on the rotating wheel for 30 minutes.Next, briefly spin the tubes and place them on a magnetic rack for one minute. Transfer the supernatant to a new 1.5-milliliter tube and add four microliters of elution buffer two. To create the input control mix one microliter of the sheared chromatin with 99 microliters of elution buffer one and four microliters of elution buffer two.Then incubate the samples for four hours at 65 degrees Celsius with shaking. After this, add 100 microliters of 100%isopropanol to the tubes. Vortex and spin the samples for five seconds at 7000 times G.Then add 10 microliters of magnetic beads to each sample.Vortex and incubate the samples on a rotating wheel at room temperature for an hour. Next, spin the tubes briefly and place them on a magnetic rack for one minute. Aspirate the supernatant and resuspend the beads in 100 microliters of wash buffer one.Invert the tubes to mix and incubate them for five minutes on a rotating wheel at room temperature. Next, briefly centrifuge the tubes. Place them in a magnetic rack for one minute, and use a pipette to remove the supernatant.Then add 100 microliters of wash buffer two. Invert the tubes to mix and incubate them for five minutes on a rotating wheel at room temperature. After this, briefly centrifuge the tubes and place them in a magnetic rack for one minute.Remove the wash buffer two via aspiration and resuspend the beads in 55 microliters of elution buffer. To elute the precipitated DNA incubate the samples in a rotating wheel at room temperature for 15 minutes. After this, spin the tubes briefly and place them in a magnetic rack for one minute.Finally, transfer the supernatant to new 1.5-milliliter tubes. This protocol was first performed with Jurkat cells analyzing LCK as a potential NFAT2 target. As shown here, optimal results were documented by significant enrichment of the IL-2 positive control and LCK DNA.Suboptimal shearing or missing fixation may lead to poor quality DNA. Finally, this protocol was performed with primary human CLL cells with CD40L serving as the positive control and LCK as the experimental target. As demonstrated by the strong enrichment of LCK DNA, LCK is a direct NFAT2 target in primary human CLL cells.Inadequate shearing resulting in poor quality DNA can return suboptimal results. While attempting this procedure it's important to remember that the fixation and sonication steps are crucial for the success of this method and might have to be adjusted to the cells being analyzed.

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Cancer Research

7.1K visualizações.  University of Tübingen. Esse método pode ajudar a fornecer informações-chave no campo da oncologia, como a identificação de genes-alvo putativos e mecanismos patogênicos para desenvolver possíveis intervenções terapêuticas. A principal vantagem dessa técnica é que se pode detectar a interação de NFAT2 e outros fatores de transcrição com genes de destino conhecidos e novos. Geralmente, indivíduos novos neste método lutarão porque as condições ideais de fixação e desarquivamento são difíceis d...