心脏分化的表观遗传调控胚胎干细胞和组织
Summary
基因转录的微调调控underlies胚胎细胞命运决定。在此，我们描述了用于研究干细胞和小鼠胚胎的心脏发育的两个心脏分化的表观遗传调控染色质免疫沉淀测定法。
Abstract
具体的基因转录是一个重要的生物学过程，在胚胎发育过程中underlies细胞命运决定。生物过程是通过其结合基因组的调节区，包括增强子和心组成的基因的启动子的转录因子介导的。的DNA是围绕经受化学修饰组蛋白包裹。组蛋白的修饰进一步导致压抑，激活或泰然自若的基因转录，从而使基因转录微调调控的另一个层次。胚胎干细胞（ES细胞）概括胚状体（ 即 ，细胞聚集体）内，或在2D培养心脏发育的早期步骤。它们提供原则上足够的材料为染色质免疫沉淀（ChIP），一个技术广泛用于鉴定基因的调节区。此外，人ES细胞代表心脏发生的人类细胞的模型。在发育的后期阶段，小鼠胚胎组织允许调查测定细胞特性所需的特定后生景观。在此，我们描述然后进行PCR或使用胚胎干细胞，胚状体和心肌特异性胚胎地区的ChIP测序沉淀，顺序芯片的协议。这些协议允许调查心脏基因转录的表观遗传调控。
Introduction
心脏是要形成的第一器官，并成为在胚胎功能。的心脏是从来自第一和第二胚胎心脏字段1出现许多细胞谱系建造。从受精后的囊胚阶段到心脏形，胚胎细胞具有从而使许多细胞命运的决定。基因转录是在时间和空间依赖性管制，是胚胎发育过程中underlies细胞命运决定的关键生物过程。这样的方法是由基因组，包括增强子和心组成的基因的启动子中的绑定调节区特异性转录因子介导的。的DNA是围绕经受修饰例如乙酰化，甲基化，泛素化和/或磷酸化组蛋白包裹。组蛋白修饰导致取决于压抑，活化或蓄势基因转录在其上的组蛋白的赖氨酸残基被修饰2。
jove_content“>染色质免疫沉淀（芯片）已经成立年前3，是目前最广泛使用的技术，以确定任何修饰组蛋白或转录因子4。随着组蛋白或转录因子的免疫目标，结合的DNA可或者通过聚合酶链式反应（PCR）扩增或测序。芯片有技术上克服更具挑战性凝胶阻滞测定法5。然而芯片不意味着直接的转录因子的DNA，凝胶阻滞分析的优点的结合。在另一方面，沉淀结合DNA测序已开通的基因调节的新的全基因组的角度。胚胎干细胞（ES细胞）的胚状体中的概括（ 即 ，细胞聚集），或在二维培养心脏发育6的早期步骤，并在原则上足够的材料芯片提供。此外，人ES细胞分别表示Ca人细胞模型rdiogenesis虽然他们的心源性潜力取决于它们的表观遗传签名7。在开发的后期阶段，小鼠胚胎组织允许调查测定细胞特性所需的特定后生景观。然而，基因组在一个时间和细胞类型特异性方式8转录。基因转录的表观遗传调控具有局部区域内进行研究。在此，我们描述接着用胚胎干细胞，胚状体和心肌特异性胚胎地区PCR或测序沉淀，顺序芯片的协议。这些协议允许调查心脏基因转录的表观遗传调控。
Protocol
Representative Results
Discussion
表观遗传学已成为发育生物学研究的一个重要领域。如何遗传程序在胚胎细胞被激活以允许细胞胚胎谱系中获取的特定身份已经长时间为发育生物学家的一个关键问题。
芯片具有在过去几年内被广泛使用，并结合到DNA测序按照测序分辨率的改善。这已成为以在基因组中广泛依赖性进行调查细胞或特定的胚胎和成年组织的后生景观的强大技术。芯片分析缓冲和超声条件得到极?…
Declarações
The authors have nothing to disclose.
Acknowledgements
The authors acknowledge funding agencies, the IMI StemBANCC European community programme, the leducq Foundation (SHAPEHEART) and the Agence Nationale de la Recherche (Genopath)
Materials
| Formaldehyde | Sigma | F8775 | Cell Fixation |
| Glycine | Sigma | G8898 | Cross-link stop |
| Aprotinin | Fluka | 10820 | Proteases inhibitor |
| Leupeptin hemisulfate | Sigma | L2882 | Proteases inhibitor |
| PMSF | Sigma | P7626 | Proteases inhibitor |
| Protein A magnetic beads | Life technologies | 10001D | Immunoprecipitation |
| SPRI magnetic beads | Thermo Scientific | 15002-01 | DNA purification |
| Proteinase K | Life technologies | 25530-015 | Protein digestion |
| DNA BR standard | Life technologies | Q32850 | Calibration range |
| Syber green | Molecular Probes | S-11484 | DNA quantification |
| TE buffer | Invitrogen | P7589 | DNA quantification |
| PBX 1X | Life technologies | 14190-094 | Washing |
| DNase RNase free water | Life technologies | 10977-035 | Dilution |
| Axygen tube | Axygen | MCT-175-C | ChIP purifiction |
| Antibody | Company | Reference | ChIP concentration |
| H3K27ac | Abcam | ab4729 | 3 µg for ESC and EBs, 1 µg for tissues |
| H3K4me1 | Diagenode | C15410194 (pAb-194-050) | 3 µg for ESC and EBs, 1 µg for tissues |
| H3K36me3 | Diagenode | C15410058 (pAb-058-050) | 3 µg for ESC and EBs, 1 µg for tissues |
| H3K9me2 | Diagenode | C15410060 (pAb-060-050) | 3 µg for ESC and EBs, 1 µg for tissues |
| H3K4me3 | Diagenode | C15410030 (pAb-030-050) | 3 µg for ESC and EBs, 1 µg for tissues |
| H3K27me3 | Diagenode | C15410069 (pAb-069-050) | 3 µg for ESC and EBs, 1 µg for tissues |
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