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

Summary

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.

Abstract

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.

Introduction

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 process....

Protocol

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 ....

Results

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

Discussion

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 .......

Materials

List of materials used in this article

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