ChIP-Seq with reference exogenous genome (ChIP-Rx) of wild-type MYC and mutant MYC in U2OS-MYC-KO cells

MYC binds to thousands of gene regulatory elements in the genome such as promoters and enhancers and exerts an amplification effect on the expression of most genes, regardless of the E-box motif existence. In this study, we discovered that MYC is an RNA-binding protein with a high affinity to guanosine-rich RNAs. RNAs binding to MYC enhance its chromatin occupancy. This process helps on MYC-mediated regulation of gene expression. Mechanistically, Lys355, Arg356, and Arg357 in the basic region of MYC are essential for RNA binding. Alanine mutation of these three amino acids abolishes MYC RNA-binding function but causes minimal damage to MYC/MAX DNA-binding capacity. Only loss of MYC RNA-binding function, while maintaining DNA-binding function, significantly decreases MYC chromatin occupancy in vivo. Our study reveals a new dimension to MYC-mediated gene regulation by demonstrating that RNA-binding activity plays an important role in MYC’s function. Chromatin immunoprecipitation assays followed by sequencing (ChIP-Seq) with reference exogenous genome (ChIP-Rx) of doxycycline (Dox) inducible FLAG-tagged wild type MYC and mutant MYC in U2OS-MYC-KO cells. NIH3T3 cells (mouse) were used as the reference exogenous genome. Two biological replicates were sequenced for each lysate.