一种检测慢性淋巴细胞白血病新 nfat2 靶基因的染色质免疫沉淀法
Summary
慢性淋巴细胞白血病 (cll) 是西方世界最常见的白血病。nfat 转录因子是多种细胞类型发育和激活的重要调节剂。在这里, 我们提出了一个在人的 cll 细胞中使用染色质免疫沉淀 (chip) 来识别 nfat2 的新靶向基因的协议。
Abstract
慢性淋巴细胞白血病 (clones) 的特点是恶性 b 细胞克隆的扩张, 是西方国家最常见的白血病。大多数 cll 患者表现出疾病的懒散过程, 以及他们的白血病细胞的过敏性表型, 指的是 b 细胞受体对外部刺激没有反应。我们最近已经证明, 转录因子 nfat2 是 cll 过敏的关键调节剂。在分析转录因子在不同疾病中的作用方面的一个主要挑战是确定其特定的目标基因。这对于阐明发病机制和潜在的治疗干预措施具有重要意义。染色质免疫沉淀 (chip) 是一种典型的技术, 以证明蛋白质与 dna 的相互作用, 因此, 可以用来识别直接的目标基因转录因子在哺乳动物细胞。在这里, chip 被用来鉴定lck作为人 cll 细胞中 nfat2 的直接靶向基因。dna 和相关蛋白质使用甲醛交联, 然后通过超声剪断成大约200-500 碱基对 (bp) 的 dna 片段。然后, 使用α-nfat2 抗体从细胞碎片中选择性地进行免疫沉淀。纯化后, 通过定量实时 pcr (qrt-pcr) 检测相关的 dna 片段。具有明显富集的 dna 序列代表了 nfat2在体内所针对的基因组区域。适当的 dna 剪切和所需抗体的选择对这种方法的成功应用特别关键。该方案是证明 nfat2 与目标基因直接相互作用的理想方案。其主要局限性在于难以在大规模检测中应用 chip 分析完整生物体内多种转录因子的靶向基因。
Introduction
慢性淋巴细胞白血病 (cll) 是西方国家成年人中最常见的白血病, 表现出明显的 cd19、cd23 和 cd5 积累, 表达成熟的 b 细胞1。大多数患者表现出懒散的疾病过程, 这不需要多年的具体治疗。相反, 一些患者表现出快速进展, 需要立即采取免疫化疗或其他靶向治疗的治疗干预 2,3。活化 t 细胞 (nfat) 的核因子 (nfat) 是控制 4、5、6 种多种细胞类型的各种发育和活化过程的转录因子家族。我们最近从懒散病患者7中证明了 nfat2 在 cll 细胞中的过度表达和宪法激活。在这里, 它调节 b 细胞受体刺激的无反应状态称为过敏 7。为了证明 nfat2 与淋巴细胞特异性蛋白酪氨酸激酶 (lck) 启动子结合并调节人 cll 细胞中lck 的表达, 研制并应用了一种特异性染色质免疫沉淀法 (chip)。
chip 是研究转录因子在基因表达中的作用的几种技术之一.基因表达是由几个调控者以非常复杂的方式紧密协调的, 转录因子在这一过程中占不可替代的作用 9、10、11、12。在许多物种中 (例如, 用于发育和分化), 已经确定了在空间和时间范围内调节基因表达的转录因子 (例如,用于发育和分化)13、14、15、 16,17,18。涉及转录因子的复杂控制机制中的错误可导致各种病理过程, 包括癌症19,20。因此, 转录因子及其各自靶点的鉴定可能提供新的治疗途径21,22。为了研究这个有趣的领域, 有几种方法可用, 如 chip, 电泳移动转移分析 (emsa), 各种 dna 下拉分析和报告检测 8,11, 12,23,24岁
为了证明一定的转录因子与体内基因组的特定区域相互作用, chip 是一种理想的技术25。为此, 使用紫外线照射或甲醛 (交联 chip, xchip) 将活细胞中的 dna 和相关蛋白质交联。这一步被省略, 以获得更好的 dna 和蛋白质恢复在所谓的本地 chip (nchip)26。dna-蛋白质复合物随后通过超声复制被剪切成大约200-500 碱基对 (bp) 的片段, 并利用针对感兴趣的转录因子的特定抗体从细胞碎片中免疫沉淀。然后通过 pcr、分子克隆和测序对相关的 dna 片段进行纯化和定性。替代技术使用微阵列 (芯片上的 ChIP-on-Chip) 或下一代测序 (ChIP-on-Chip) 来分析免疫沉淀的 dna。
chip 最初是由 gilmour 和 lis 在1984年引入的, 当时它们利用紫外光在活细菌27中共价交联 dna 和结合蛋白.在细菌 rna 聚合酶的细胞裂解和免疫沉淀时, 利用已知基因的特异性探针绘制 rna 聚合酶的体内分布和密度图。该方法随后被同一研究人员用于分析真核rna 聚合酶 ii 在果蝇28热休克蛋白基因上的分布。varshavsky 和同事们进一步完善了 xchip 检测方法, 他们首先使用甲醛交联研究组蛋白 h4 与热休克蛋白基因29、30 的相关性。nchip 方法, 具有更好的 dna 和蛋白质恢复的优势, 由于自然完整的表位, 因此, 更大的抗体特异性, 首次由 hebbes 和他的同事在 1988年31年描述。
与其他分析 dna-蛋白质相互作用的技术相比, chip 的优势在于, 转录因子的实际相互作用可以在体内进行研究, 而不是由缓冲液或凝胶产生的探针或人工条件。雇用8,11,12。通过将 chip 与下一代测序相结合, 可以同时识别多个目标。
这种技术的主要局限性在于它对完整生物体中的大规模检测的适用性有限25。差异基因表达模式的分析也可以挑战使用 chip 技术, 如果各自的蛋白质只在低水平或在狭窄的时间窗口表达。另一个潜在的限制因素是是否有适合 chip11的合适抗体。
本文提出的 chip 协议可用于定量实时 pcr (qrt-pcr) 在体内识别转录因子的靶基因。具体而言, 目标是在 cll 中识别 nfat2 的新靶向基因。选择 chip 是因为它有可能直接证明 nfat2 在人 cll 患者细胞自然条件下与不同靶向基因的启动子区域的结合。
Protocol
所有使用人类材料进行的实验都得到了图宾根大学道德委员会的批准, 并获得了为这项研究提供样本的所有患者的书面知情同意。 1. 陪审员室的分离和刺激 注: 若要优化协议, 请使用已知表示高级别 nfat2 的 jurkat 细胞系。所有步骤都是在层流罩下执行的。 在水浴中将 rpmi 1640 加入50毫升, 并加入10% 的 fcs 和1% 的青霉素/链霉素。 在37°c 的细胞?…
Representative Results
图 1显示了用 cd19-fitc 和 cd5-pe 抗体染色后对 cll 患者进行的典型流式细胞仪分析。图 1a显示了淋巴细胞的门控, 代表了 cll 患者血液中的大多数细胞。图 1b显示了 cd19+/cd5+ cll 细胞的比例, 在本例中占淋巴细胞的8.9.03%。cd19+/cd5-b 细胞在患者中的比例为3.72。 <p class="jove_content…
Discussion
执行 chip 分析成功的关键步骤是选择合适的抗体和优化染色质剪切过程25。在该协议的开发过程中, α-nfat2 抗体的选择被证明是特别具有挑战性的。虽然有几种α-nfat2 抗体在商业上可以买到, 其中大多数适用于西方印迹和其他应用, 克隆7a6 是唯一可以成功用于 chip7的抗体。即使是来自不同制造商的相同的7a6 抗体在 chip 中的性能也有显著差异。一个主要的挑战是在…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了 dfg 赠款 mu 3340/1-1 和德国 krebshilfe 赠款111134的支持 (均授予 m. r. m.)。我们感谢艾克·马伦克提供的出色技术援助)。
Materials
| 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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Cite This Article
Fuchs, A. R., Märklin, M., Heitmann, J. S., Futterknecht, S., Haap, M., Wirths, S., Kopp, H., Hinterleitner, C., Dörfel, D., Müller, M. R. A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia. J. Vis. Exp. (142), e58270, doi:10.3791/58270 (2018).