Genome-wide analysis of c-MYC-regulated mRNAs and miRNAs, and c-MYC DNA binding by next-generation sequencing.

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, the validation of single c-MYC targets identified by NGS is described. Taken together, these approaches allow an accelerated and comprehensive analysis of c-MYC function in numerous cellular contexts which will further illuminate the role of this important oncogene.