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Abstract

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Protocol

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Acknowledgements

Materials

References

Cancer Research

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

Published: December 4th, 2018

DOI:

10.3791/58270

1Dept. of Hematology, Oncology and Immunology, University of Tübingen, 2Dept. of Endocrinology, Diabetology, Clinical Pathology and Metabolism, University of Tübingen

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 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 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 αNFAT2 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.

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 therapies2,3. Nuclear factor of activated T cells (NFAT) is a family of transcription factors controlling various developmental and activation process....

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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 sup.......

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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

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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 .......

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

....

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

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