Covalent modification of histones by protein lysine methyltransferases (PKMTs) influences diverse biological and disease processes. Greater than fifty PKMTs are predicted to be present in the human proteome; however, the catalytic activity and substrate specificity for the majority of these enzymes is unknown. Here, we perform a biochemical screen of over forty human candidate PKMTs and demonstrate that SETD6 (SET domain containing 6) monomethylates chromatin-associated RelA at lysine 310 (RelAK310me1). RelA, a subunit of NF-KB, a pivotal transcriptional regulator of inflammation, is repressed when methylated by SETD6. SETD6 attenuates RelA-driven gene expression and oncogenic cellular programs including inflammatory responses in primary immune cells. Depletion of SETD6 results in the secretion of more than 20 cytokines in primary mouse BMDMs and primary human Monocyte-derived dendritic cells. Furthermore, we identify the ankryin repeat of GLP (G9a-like protein) as a recognition motif for RelAK310me1. SETD6-dependent binding of GLP to RelAK310me1 promotes a repressed chromatin state at RelAK310me1-occupied genes through GLP-mediated H3K9 methylation. This repressive pathway is overridden by NF-KB-activation linked PKCzeta-mediated phosphorylation of RelA at serine 311 (RelAS311ph), which blocks GLP binding to RelAK310me1 and drives target gene expression. Together, our findings identify SETD6 as a novel PKMT and regulator of NF-KB signaling, and demonstrate a new paradigm for how integrated crosstalk between modifications on transcription factors and histones modulates key physiologic and pathologic programs.
