KAT–independent gene regulation by Tip60 promotes ESC self-renewal but not pluripotency. Mus musculus

Although histone-modifying enzymes are assumed to function in a manner dependent on their enzymatic activities, this assumption remains untested for many factors. Here we show the Tip60 (Kat5) lysine acetyltransferase (KAT), which is essential for embryonic stem cell (ESC) self-renewal and pre-implantation development, performs these functions independently of its KAT activity. Unlike ESCs depleted of Tip60, KAT–deficient ESCs exhibited minimal alterations in gene expression, chromatin accessibility at Tip60 binding sites, and self-renewal, thus demonstrating a critical KAT–independent role of Tip60 in ESC maintenance. In contrast, KAT–deficient ESCs exhibited impaired differentiation into mesoderm and endoderm, demonstrating a second, KAT–dependent function in differentiation. Consistent with this phenotype, KAT–deficient mouse embryos exhibited post-implantation developmental defects. These findings establish separable KAT–dependent and KAT–independent functions of Tip60 in ESCs and during embryonic development, raising the possibility of undiscovered catalysis-independent functions of additional KATs. Overall design: The data represent several experiments: (1) RNA-seq experiments examining gene expression changes in Tip60 catalytic inactive, Ep400 catalytic inactive, double mutants, or knockdowns (RNAi) in ESCs relative to controls, (2) RNA-seq experiments examining changes in gene expression of wild type controls and Tip60 catalytic inactive mutants during ESC differentiation, and (3) ATAC-seq experiments examining chromatin accessibility in control, Tip60 catalytic inactive, and Tip60 knockdown ESCs. The data represent ChIPseq experiment examining changes in H4 acetylation and H2AZ occupany in Tip60 catalytic inactive and Ep400 catalytic inactive mutant ESCs respectively, relative to controls.