Automethylation of lysine methyltransferase SETDB1 on H3K9-like motifs regulates interactions with chromodomain proteins and controls its functions

Histone H3 lysine 9 trimethylation is essential for heterochromatin formation and maintenance, genome stability, and silencing of transposable elements (TEs) in embryonic stem cells (ESCs). The H3K9-specific lysine methyltransferase SETDB1 is crucial for mammalian development, as it controls ESC pluripotency and viability by regulating gene expression and TE silencing. Here, we demonstrate that SETDB1 undergoes automethylation on two lysine residues located within H3K9-like motifs in its catalytic domain. Notably, these automethylated lysines are necessary for the normal growth and viability of ESCs. While SETDB1 automethylation does not affect its catalytic activity, it is crucial for its interaction with chromodomain-containing partners, including SUV39H1, CDYL, and Heterochromatin Protein 1 gamma (HP1gamma). The integrity of the two automethylated lysines is required for SETDB1 localization to its target sites and for effective silencing of both coding genes and TEs. Expression of an automethylation-deficient SETDB1 fails to properly establish H3K9me3 and disrupts HP1gamma recruitment at target loci. Collectively, our findings uncover a previously unknown mechanism regulating SETDB1 function, essential for maintaining the fitness of mouse ESCs. Highlights- SETDB1 undergoes automethylation on two lysines within its catalytic domain - The automethylation-deficient form of SETDB1 is enzymatically active, yet it compromises mESCs fitness - SETDB1 automethylation-deficient mutant impairs chromatin association and pan-genomic H3K9me3 landscape - SETDB1 automethylation regulates its interactions with many chromodomain-containing partners