The p53 tumor suppressor protein is regulated by multiple post-translational modifications, including lysine methylation. We previously found that monomethylation of p53 at lysine 382 (p53K382me1) by the protein lysine methyltransferase (PKMT) SET8/PR-Set7 represses p53 transactivation of target genes. However, the molecular mechanism linking p53K382 monomethylation to repression is not known. Here we show in biochemical and crystallographic studies the preferential recognition of p53K382me1 by the triple malignant brain tumor (MBT) repeats of the chromatin compaction factor L3MBTL1. We demonstrate that SET8-mediated methylation of p53 at Lys-382 promotes the interaction between L3MBTL1 and p53 in cells, and the chromatin occupancy of L3MBTL1 at p53 target promoters. In the absence of DNA damage, L3MBTL1 interacts with p53K382me1 and p53-target genes are repressed, whereas depletion of L3MBTL1 results in a p53-dependent increase in p21 and PUMA transcript levels. Activation of p53 by DNA damage is coupled to a decrease in p53K382me1 levels, abrogation of the L3MBTL1-p53 interaction, and disassociation of L3MBTL1 from p53-target promoters. Together, we identify L3MBTL1 as the second known methyl-p53 effector protein, and provide a molecular explanation for the mechanism by which p53K382me1 is transduced to regulate p53 activity.The reversible and dynamic methylation of proteins on the nitrogen side-chain of lysine residues can greatly increase the signaling potential of the modified factor (1, 2). Lysine residues can accept up to three methyl groups, forming mono-, di-, and trimethylated derivatives, with a unique activity frequently being coupled to the specific methylation state. The chemical addition of methyl moieties to histones is not believed to intrinsically affect chromatin structure. Rather, the principal mechanism by which histone lysine methylation is thought to manifest functionally occurs through regulation of modular protein-protein interactions (3-5). In this regard, the protein(s) that recognize a methylated lysine within a specific sequence context can define the functional outcome associated with that specific lysine methylation event. Thus, mechanistic insight into how lysine methylation influences a biological program requires knowledge of the proteins and domains that recognize and transduce this modification.In addition to histones, several other proteins such as the tumor suppressor p53 undergo lysine methylation, arguing that this modification may be a common mechanism for modulating protein-protein interactions and key cellular signaling pathways (6 -8). p53 plays a pivotal role in the regulation of cellular responses to various forms of genotoxic stresses, and its activity is coordinated by a complex network of post-translational modifications (9, 10). We previously demonstrated that the protein lysine methyltransferase (PKMT) 5 PR-Set7/SET8 monomethylates p53 exclusively at lysine 382 (p53K382me1), and that the placement of this modification negatively regulates p53 activity (11). Spec...