组蛋白3赖氨酸 79 Dimethylation 标记沉淀后神经祖细胞的分离培养
Summary
我们提出一种有效和可重复的方法, 分离和培养胚胎和产后脑组织中的神经祖细胞染色质沉淀 (芯片) 的组蛋白3赖氨酸 79 dimethylation (H3K79me2)-一个组蛋白标记位于组蛋白3的球状域。
Abstract
大脑发育是一个复杂的过程, 它是由来说的梯度和不同的转录程序控制在颞空间的方式。此外, 后生染色质修饰, 如组蛋白甲基化, 有一个重要的作用, 建立和维护特定的细胞命运在这个过程中。大多数组蛋白甲基化发生在灵活的组蛋白尾部, 这是组蛋白修饰剂、橡皮擦和组蛋白的阅读蛋白。相比之下, H3K79 甲基化位于组蛋白3的球状领域, 并与不同的发育功能有牵连。H3K79 甲基化是进化保守的, 可以从智人到酿酒酵母的种类繁多的物种中发现。改变发生在不同的细胞数量的生物体内, 包括神经祖。H3K79 甲基化在组蛋白3的球状域中的位置使得难以评估。在这里, 我们提出的方法分离和培养皮层祖细胞 (党) 从胚胎皮质脑组织 (E11.5-E14.5) 或小脑颗粒神经元祖 (CGNPs) 从产后组织 (P5-P7), 并有效 immunoprecipitateH3K79me2 定量 PCR (qPCR) 和全基因组测序。
Introduction
大脑的感官、运动和认知功能非常复杂, 容易受到物理和环境变化的影响。大脑由后、中、前脑的三一般部分组成, 它们是紧密相连的。前脑内, 端可分为背部端 (DT) 和腹端 (VT)。小鼠的 DT 由六皮质层组成, 它们是在 E11.5 和 E18.5 之间形成的, 以 “内而外” 的方式1。VT 包括节主教在发育中, 后来形成基底节2,3。几种细胞类型可以归类在哺乳动物中枢神经系统, 如神经元, 星形胶质细胞, 或突4, 在颞空间的方式5发展。首先, 神经祖细胞 (npc) 产生不同类型的神经元, 神经元在 VT, 和投射神经元在 DT, 后来到神经胶质细胞 (例如, 星形细胞6)。在皮质发育过程中, 首先形成了含有卡哈尔 Retzius 细胞的最浅层 (I 层)。然后, 在 E12.5 和 E14.5 之间, npc 产生更深的神经元层 (VI, V), 而在14.5 和16.5 之间, 祖细胞引起上层 (IV-II) 神经元7,8。神经元的身份是由不同的形态诱导的颞空间转录程序和另外的后生程序2指定的。
小脑, 这是牵连在运动协调, 位于后脑和发展之间的 E10 和粗略 P20 在小鼠9。它包含小脑皮质和小脑核10。成人小脑皮质包括三层, 最外层的分子层, 浦肯野细胞层, 和包含颗粒状神经元的最里层的颗粒层10。小脑颗粒细胞是最小的神经元, 代表了脊椎动物大脑中大约80% 的神经元11。它们从位于外部生发区的前驱体发展而来, 并通过浦肯野细胞层迁移到它们的目标12。象在端, 小脑的发展由几个重要来说调控, 有具体时间和空间相关的作用和创始被定义的转录节目10。
皮质和小脑层的发育由特定来说的转录表达控制, 因此, 由 DNA 的染色质状态。在一个简化的观点, 染色质状态可以被划分成染色质作为转录活跃和异作为转录沈默区域。核作为染色质的基本单位, 包含了每个核心组蛋白 H2A、H2B、H3 和 H4 的两个拷贝, 周围有147碱基对 DNA13。组蛋白是高度后翻译修饰的甲基化, 乙酰化, 磷酸, 泛, sumo, ADP adp-, 脱, 和脯氨酸异构化反应14,15。组蛋白赖氨酸甲基化被认为是最稳定的蛋白修饰, 控制转录, 复制, 重组16, DNA 损伤的反应,17, 和基因印记18。Lysines 可以是单、di 或三甲基化的19 , 并且不仅出现在可访问的组蛋白尾部, 而且还显示在组组的球状域中20。具体的 h3k4 在 H3K4 和 H3K36 主要与染色质, 具体 h3k4 在 H3K9, H3K27, 或 H4K20 主要在异地区发现, 虽然所有残留物位于组蛋白尾部14, 19,21。H3K79 甲基化位于组蛋白球状域内, 并与转录活性有关, 但也与转录惰性基因的区域22有关。由于在酵母、小牛胸腺、鸡肉和人类23中观察到了这种改变, 因此在进化上是保守的。H3K79 单, di, 和 trimethylation (H3K79me1, me2, me3) 是由组蛋白甲基 DOT1L24,25和核集合域含蛋白质 2 (Nsd2)26。DOT1L 与增殖、DNA 修复和细胞 reprograming27有牵连。Dot1l在小鼠中的丢失导致在发育阶段 E10.528,29周围的产前死亡。在心脏发育和 myocardiocyte 分化期间, DOT1L 是基因表达调控的关键30。在中枢神经系统中, DOT1L 功能可能牵连神经管发育31, 它涉及在前脑发育32期间抑制Tbr1表达, 并可在 ER 应力调节中起作用响应基因33。H3K79me 的上下文相关激活或镇压行动, 特别是与在体内情况象中央神经系统的发展, 是到目前为止仅部份地被了解的32。由于 H3K79 甲基化位于组蛋白3的球状域中, 因此与灵活的组蛋白尾23的修饰相比, 阻的可访问性较差。为了了解 H3K79 甲基化的作用, 需要可靠和可重复的分析方法来确定其位置和基因组环境。在本方法中, 我们提出了不同的神经祖细胞的分离方法 (党用于小脑的皮质和 CGNPs), 有效的 DOT1L 抑制剂治疗, 以及在不同时间通过 qPCR 或测序来分析 H3K79 甲基化的芯片方法在皮质和小脑发育过程中的点。有关协议及其可能性的概述, 请参见图 1。
Protocol
弗莱堡大学的动物福利委员会和地方当局批准了以下协议中提到的所有动物实验 (G12/13, G16/11)。 1. 准备工作 党分离制剂 建立定时交配, 以获得胚胎在不同阶段的皮层发育 (E11.5 和 E14.5)。使用小鼠的应变 NMRI (海军医学研究所), 这是至少8周的老。交配后, 考虑一个积极的阴道堵塞在 E0.5。 要有足够的材料, 在 E12.5 或 E14.5 的皮质上用一小块 NMRI…
Representative Results
神经祖分离、培养、H3K79me2 芯片和芯片分析方法的一般方案:图 1显示了一个流程图, 用于在胚胎发育期或产后阶段的小脑颗粒神经元祖细胞的不同时间点执行 H3K79me2 芯片。作为第一步, 大脑必须被孤立, 端 (在 E11.5 和 E14.5 之间) 或小脑 (P5-P7) 必须被检索。通过将端划分为 DT 和 VT, 可以分析不同区域。随后组织将匀浆和神经祖细胞分离。现?…
Discussion
有两种主要的方法来执行染色质沉淀, 以检测组蛋白的修饰, 转录因子的染色体的占有率, 蛋白的读者, 作家, 或橡皮擦。一种是用核酸消化的原生芯片法, 沉淀的原染色质, 另一种是采用粉煤灰固定的, 被剪切的染色质, 其中核和其他 dna 附着的蛋白质与 dna 共价键结合的方法。39. 本机芯片具有较高的抗体检测率, 应适用于组蛋白甲基化, 因为染色质和组蛋白 h3k4 在整个芯片中相对稳?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢亨利埃特 Bertemes 在实验室中帮助建立 CGN 种植协议。本方法通过对电视的资助, 由 DFG 资助的 CRC992 医学遗传学研究支持。作者承认弗莱堡星系小组的支持: Pavankumar Videm, bj Grüning 和 Prof., 生物信息学, 德国弗赖堡大学, 由合作研究中心资助的992医学遗传学 (Backofen 补助金 DFG 992/1 2012)并且德国联邦教育和研究部 (BMBF 授予 031 A538A RBC (de。NBI))。
Materials
| Anti-GAPDH | Abcam | ab8245 | Category: Antibody Abbreviation/Comment: Immunoblot dilution 1:5000 |
| Anti-H3 | Abcam | ab1791 | Category: Antibody Abbreviation/Comment: Immunoblot dilution 1:3000 |
| Anti-H3K79me2 | Diagenode | pAb-051-050 | Category: Antibody Abbreviation/Comment: ChIP antibody |
| Anti-H3K79me2 | Abcam | ab-051-050 | Category: Antibody Abbreviation/Comment: Immunoblot dilution 1:1000 |
| Anti-rabbit-IgG | Diagenode | C15410206 | Category: Antibody Abbreviation/Comment: ChIP Ctrl antibody |
| Anti-Tubulin alpha | Abcam | ab108629 | Category: Antibody Abbreviation/Comment: Immunoblot dilution 1:3000 |
| Apo-Transferrin (1 mg/ml) | Sigma-Aldrich | T1147 | Category: Cell culture Abbreviation/Comment: For CCM |
| B27 Supplement (50x) | Life Technologies | 17504044 | Category: Cell culture Abbreviation/Comment: For CCM |
| Bioanalyzer | Agilent technologies | G2940CA | Category: ChIP Abbreviation/Comment: For analysis of sheared chromatin |
| Bioruptor NextGen | Diagenode | B01020001 | Category: ChIP Abbreviation/Comment: Ultrasonicator |
| Boric acid pH 8.4 | Sigma Aldrich | B6768 | Category: Cell culture Abbreviation/Comment: For CPC culturing |
| CFX Connect RT PCR Detection System | Bio-Rad | 1855201 | Category: ChIP Analysis Abbreviation/Comment: Detection system for qPCR |
| DMEM-F12 | Life Technologies | 11320-033 | Category: Cell culture Abbreviation/Comment: For CGM |
| Dynabeads Protein A | Invitrogen | 10001D | Category: ChIP Abbreviation/Comment: Magnetic beads, for ChIP |
| EPZ-5676 | Selleckchem | S7062 | Category: DOT1L inhibition Abbreviation/Comment: For DOT1L inhibition in cell culture |
| Ethylenediamine tetraacetic acid | SERVA | 39760.01 | Category: ChIP Abbreviation/Comment: EDTA |
| Fetal Bovine Serum 10% (v/v) | Gibco | 10082147 | Category: Cell culture Abbreviation/Comment: For CPC isolation and CGM |
| Glucose | Sigma-Aldrich | G5767 | Category: Cell culture Abbreviation/Comment: For CGNP isolation |
| Glutathione (1.25 mg/ml) | Sigma-Aldrich | G4251 | Category: Cell culture Abbreviation/Comment: For CCM |
| Glycine | Carl Roth | 3187 | Category: ChIP Abbreviation/Comment: For cell fixation |
| GoTaq mastermix | Promega | A6002 | Category: ChIP Analysis Abbreviation/Comment: DNA polymerase master mix for qPCR |
| Hank’s Balanced Salt Solution | Life Technologies | 14025-100 | Category: Cell culture Abbreviation/Comment: HBSS |
| L-glutamine (200 mM) | Life Technologies | 25030081 | Category: Cell culture Abbreviation/Comment: For CCM |
| Laminin | Sigma-Aldrich | L2020 | Category: Cell culture Abbreviation/Comment: For CPC culturing |
| Lithium chloride | Sigma-Aldrich | L4408 | Category: ChIP Abbreviation/Comment: LiCl |
| N2 supplement | Life Technologies | 17502048 | Category: Cell culture Abbreviation/Comment: For CGM |
| NanoDrop 3300 | Thermo Fisher | 3300 | Category: ChIP Abbreviation/Comment: Fluorospectrometer for DNA quantification |
| NEB Next Ultra DNA Library Prep Kit for Illumina | NEB | E7645S | Category: ChIP Analysis Abbreviation/Comment: Kit for Library preparation |
| NEBNext Multiplex Oligos for Illumina | NEB | E7335 | Category: ChIP Analysis Abbreviation/Comment: Oligos for Library preparation |
| Neurobasal medium | Gibco | 21103049 | Category: Cell culture Abbreviation/Comment: For CCM |
| NP-40 Alternative | Calbiochem | 492016 | Category: ChIP Abbreviation/Comment: For ChIP buffer |
| Paraformaldehyde | Carl Roth | 335 | Category: ChIP Abbreviation/Comment: PFA, for cell fixation |
| Penicillin-Streptomycin-Neomycin 1% (v/v) | Life Technologies | 15640055 | Category: Cell culture Abbreviation/Comment: PSN, for CCM and CGM |
| Phosphate buffered saline | Life Technologies | 10010023 | Category: Cell culture Abbreviation/Comment: PBS, for CPC isolation |
| PicoGreen Kit | Thermo Fisher | P11496 | Category: ChIP Analysis Abbreviation/Comment: Visualizing dye for DNA quantification |
| Poly-D-lysine | Sigma-Aldrich | P6407 | Category: Cell culture Abbreviation/Comment: For CGNP isolation |
| Poly-L-ornithine hydrobromide | Sigma Aldrich | P3655 | Category: Cell culture Abbreviation/Comment: For CPC culturing |
| Potassium chloride | Thermo Fisher | AM9640G | Category: Cell culture Abbreviation/Comment: KCl, for CGM |
| Protease inhibitor | Roche | 4693159001 | Category: ChIP Abbreviation/Comment: For ChIP |
| Proteinase K | Sigma-Aldrich | 3115879001 | Category: ChIP Abbreviation/Comment: For ChIP |
| Qiagen MinElute | Qiagen | 28004 | Category: ChIP Abbreviation/Comment: Kit for DNA purification |
| RNAse | Sigma-Aldrich | R6513 | Category: ChIP Abbreviation/Comment: For ChIP |
| SGC0946 | Selleckchem | S7079 | Category: DOT1L inhibition Abbreviation/Comment: For DOT1L inhibition in cell culture |
| Sodium bicarbonate | Carl Roth | 8551.1 | Category: ChIP Abbreviation/Comment: for Elution buffer |
| Sodium chloride | Carl Roth | 9265 | Category: ChIP Abbreviation/Comment: NaCl, for ChIP buffer |
| Sodium deoxycholate | Sigma-Aldrich | 30970 | Category: ChIP Abbreviation/Comment: For ChIP |
| Sodium dodecylsulfate | Carl Roth | 183 | Category: ChIP Abbreviation/Comment: SDS, for ChIP |
| Sonic hedgehock (SHH) | Sigma-Aldrich | SRP6004 | Category: Cell culture Abbreviation/Comment: For CGNP isolation |
| Superoxide dismutase (1mg/ml) | Sigma-Aldrich | S7571 | Category: Cell culture Abbreviation/Comment: For CCM |
| Tris(hydroxymethyl)aminomethane | Carl Roth | 9090 | Category: ChIP Abbreviation/Comment: TRIS, for ChIP buffer |
| Triton X-100 | Carl Roth | X100 | Category: ChIP Abbreviation/Comment: For ChIP buffer |
| Trypsin-EDTA 0,05% (w/v) | Sigma Aldrich | 59417C | Category: Cell culture Abbreviation/Comment: For CPC isolation |
| Tween20 | Carl Roth | 28320 | Category: ChIP Abbreviation/Comment: For bead preparation |
| Other Lab devices: Neubauer counting chamber, Incubator, Rotator, Shaker, Disection set, Water bath | |||
| CCM: Cortical cell medium | |||
| CGM: CGNP cell culture medium | |||
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Citer Cet Article
Bovio, P., Roidl, D., Heidrich, S., Vogel, T., Franz, H. Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark. J. Vis. Exp. (131), e56631, doi:10.3791/56631 (2018).