Experimental and Molecular Pathology, Institute of Pathology, Ludwig-Maximilians-Universität München, Munich, Germany.
German Cancer Consortium (DKTK), Munich, Germany.
Methods Mol Biol. 2021;2318:119-160. doi: 10.1007/978-1-0716-1476-1_7.
The c-MYC oncogene is activated in ~50% of all tumors and its product, the c-MYC transcription factor, regulates numerous processes, which contribute to tumor initiation and progression. Therefore, the genome-wide characterization of c-MYC targets and their role in different tumor entities is a recurrent theme in cancer research. Recently, next-generation sequencing (NGS) has become a powerful tool to analyze mRNA and miRNA expression, as well as DNA binding of proteins in a genome-wide manner with an extremely high resolution and coverage. Since the c-MYC transcription factor regulates mRNA and miRNA expression by binding to specific DNA elements in the vicinity of promoters, NGS can be used to generate integrated representations of c-MYC-mediated regulations of gene transcription and chromatin modifications. Here, we provide protocols and examples of NGS-based analyses of c-MYC-regulated mRNA and miRNA expression, as well as of DNA binding by c-MYC. Furthermore, we describe the validation of single c-MYC targets identified by NGS . Taken together, these approaches allow an accelerated and comprehensive analysis of c-MYC function in numerous cellular contexts. Ultimately, these analyses will further illuminate the role of this important oncogene.
c-MYC 癌基因在大约 50%的所有肿瘤中被激活，其产物 c-MYC 转录因子调节许多过程，这些过程有助于肿瘤的发生和发展。因此，对 c-MYC 靶点的全基因组特征及其在不同肿瘤实体中的作用是癌症研究中的一个反复出现的主题。最近，下一代测序（NGS）已成为一种强大的工具，可在全基因组范围内以极高的分辨率和覆盖率分析 mRNA 和 miRNA 的表达以及蛋白质的 DNA 结合。由于 c-MYC 转录因子通过结合启动子附近的特定 DNA 元件来调节 mRNA 和 miRNA 的表达，因此 NGS 可用于生成 c-MYC 介导的基因转录和染色质修饰调控的综合表现。在这里，我们提供了基于 NGS 的 c-MYC 调节的 mRNA 和 miRNA 表达以及 c-MYC 的 DNA 结合分析的方案和实例。此外，我们还描述了通过 NGS 鉴定的单个 c-MYC 靶点的验证。总之，这些方法允许在许多细胞环境中加速和全面地分析 c-MYC 的功能。最终，这些分析将进一步阐明这个重要癌基因的作用。
