Role of the Tudor-knot domain of the TIP60 lysine acetyltransferase [ChIP-seq]

The histone lysine acetyltransferase TIP60 is the main enzyme that catalyzes histone H4 acetylation in cells. Domains on TIP60 regulate its enzymatic activity from different aspects. Here we use a CRISPR-Cas9 tiling screen to scan for essential domains on TIP60 protein and found that the Tudor-knot domain is essential for cell survival and intercellular H4 acetylation. We performed in-vitro biochemical assays and demonstrated Tudor-knot domain is not a histone reader. And deficiency of the Tudor-knot domain has mild effects on TIP60 intracellular localization, as well as the TIP60 complex’s constitution. But Tudor-knot deficiency significantly reduces TIP60 HAT activity both in vivo and in vitro. By comparing the catalytic efficiency of nucleosome substrate and histone octamer substrate, as well as TIP60 protein alone or TIP60 complex, we found the nucleosomal structure and other TIP60 complex components are required for Tudor-knot relative HAT activity regulation. We propose that the Tudor-knot domain function to increase nucleosome accessibility. Finally, we show that the Tudor-knot domain is required for TIP60-dependent transcription regulation. Altogether, our study reveals a mechanism that the Tudor-knot domain that regulates TIP60-dependent transcription through the regulation of TIP60 substrate catalytic efficiency. We performed ChIP-seq experiments of histone H4ac and HA-KAT5 in H1299 cell lines that overexpress HA-KAT5-FL, HA-KAT5-Y47A, and HA-KAT5-dN. Histone H4 pan-acetyl (Active Motif, catalog 39925, lot 12517004), and HA tag (clone C29F4) (Cell Signaling Technology, catalog 3724S, lot 8) were used.