Perna Amalia, Alberi Lavinia Auber
Department of Medicine, University of Fribourg, Fribourg, Switzerland.
Swiss Integrative Center for Human Health, Fribourg, Switzerland.
Front Cell Neurosci. 2019 Mar 19;13:95. doi: 10.3389/fncel.2019.00095. eCollection 2019.
Chromatin immunoprecipitation (ChIP) is an assay developed in order to define the dynamic nature of transcription processes. This method has been widely employed to identify methylated and acetylated DNA sequences in a variety of organs both in animals and humans. Nevertheless, this technique is significantly less employed to study transcriptional targets of specific nuclear signaling factors (TFs) and the data published so far have mainly used cell culture material and have been hardly reproduced in tissue. As nuclear signaling underlies important adaptive and maladaptive responses in chronic conditions such as cancer and neurodegeneration, there is a need for streamlining the upfront workflow of TF-ChIP for subsequent target sequencing. Based on the typical low concentration of the signaling transcriptional complex and the complexity/length of the ChIP Seq protocol, the field of cellular signaling has been confronted with a major roadblock in identifying clinically relevant targets of pathological and physiological signaling pathways. The present protocol offers a standardized procedure for detecting signaling targets in any whole tissue or specific dissected regions. The advantages of the protocol compared to the existing published methods are: (1) the small amount of starting material; appropriate for tissue subregions; (2) the optimization of DNA fragmentation from whole tissue; (3) suitability for sparsely populated tissues (i.e., brain); (4) the specificity of the TF-targeting readout; and (5) high DNA quality for sequencing or hybridization. The present protocol is highly detailed and can be reproduced using both fresh and fresh-frozen tissue. This is particularly relevant in the clinical setting, where specimen integrity is often the limiting step and where transcriptional target profiling is therapeutically relevant. The method is centered on Notch signaling but can be applied to a variety of nuclear signaling pathways as long as specific antibodies are available for pull down. Taken the superior yield/readout of this procedure, ChIP may finally provide relevant information about dynamic downstream gene changes for use in both basic research and clinical applications.
染色质免疫沉淀法（ChIP）是为了定义转录过程的动态性质而开发的一种分析方法。该方法已被广泛用于鉴定动物和人类各种器官中的甲基化和乙酰化DNA序列。然而，该技术在研究特定核信号因子（TFs）的转录靶点方面应用较少，迄今为止发表的数据主要使用细胞培养材料，且在组织中难以重现。由于核信号是癌症和神经退行性变等慢性疾病中重要的适应性和 maladaptive 反应的基础，因此需要简化 TF-ChIP 的前期工作流程以便后续进行靶点测序。基于信号转录复合物的典型低浓度以及 ChIP Seq 方案的复杂性/长度，细胞信号领域在识别病理和生理信号通路的临床相关靶点方面面临着一个主要障碍。本方案提供了一种用于检测任何全组织或特定解剖区域中信号靶点的标准化程序。与现有已发表方法相比，该方案的优点包括：（1）起始材料量少；适用于组织亚区域；（2）对全组织DNA片段化的优化；（3）适用于细胞稀少的组织（如脑）；（4）TF靶向读数的特异性；以及（5）用于测序或杂交的DNA质量高。本方案非常详细，使用新鲜组织和新鲜冷冻组织均可重现。这在临床环境中尤为重要，因为样本完整性往往是限制步骤，而转录靶点分析在治疗上具有相关性。该方法以Notch信号为中心，但只要有用于下拉的特异性抗体，就可应用于多种核信号通路。鉴于此程序具有优异的产量/读数，ChIP最终可能为基础研究和临床应用提供有关动态下游基因变化的相关信息。
