UC Riverside

Deregulated MYC expression is associated with most types of cancer in humans. MYC deregulation may occur via alterations of the MYC gene itself or via aberrant activation of upstream signaling pathways that activate MYC gene expression and/or increase MYC protein stability via post-translational modifications. Here, I show that MYC is endogenously acetylated at lysine (K) residues K149, K158, and K323 through the characterization of three corresponding novel acetyl-c-MYC antibodies. Having established the existence of endogenously acetylated MYC in cancer cells, this strongly implies a link between such modifications and the regulation of MYC biological functions. Therefore, I test for the importance of MYC K residues acetylated by p300/CBP and GCN5 by analyzing the functional properties of three acetylation-defective MYC K to arginine (R) mutants (e.g. K149R, K158R and K323R). The assays I use highlight MYC’s regulation of cellular proliferation, metabolism, apoptosis and oncogenic transformation in a well-established Rat1a fibroblast system that constitutively overexpresses MYC wild type (WT) and the MYC mutants. With this system I show that MYC K149R and K158R mutants are defective in oncogenic transformation, as indicated by the reduction of anchorage-independent growth of cells in soft agar and reduced tumor formation in athymic nude mice. I also demonstrate that the MYC K158R mutant (a site that would be preferentially modified by p300) is less proliferative and forms fewer foci after cells reach confluence, maintains stronger cellular adhesions compared to MYC WT and the other MYC mutants, and is more apoptotic than MYC WT under conditions of serum starvation. In comparison, the MYC K323R mutant (a site that would be preferentially modified by GCN5) does not exhibit any of these defects, but does appear to affect cellular metabolism, as cells expressing this mutant hastily acidify the culture medium. Finally, by means of RNA sequencing, I show that different sets of MYC-regulated genes are affected (both up- or down-regulated) by the K to R MYC mutants. This data strongly implies that MYC is capable of differential regulation of discrete subsets of genes, via gene-selective mechanisms involving K residues that are acetylated by p300 or GCN5.