Dataset Information

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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE245115 | GEO | 2023/12/24

REPOSITORIES: GEO

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Project description:Background Tip60 (KAT5) is the histone acetyltransferase (HAT) of the mammalian Tip60/NuA4 complex. While Tip60 is important for early mouse development and mouse embryonic stem cell (mESC) pluripotency, the function of Tip60 as reflected in a genome-wide context is not yet well understood. Results Gel filtration of nuclear mESCs extracts indicate incorporation of Tip60 into large molecular complexes and exclude the existence of large quantities of âfreeâ Tip60 within the nuclei of ESCs. Thus, monitoring of Tip60 binding to the genome should reflect the behaviour of Tip60-containing complexes. The genome-wide mapping of Tip60 binding in mESCs by chromatin immunoprecipitation (ChIP) coupled with high-throughput sequencing (ChIP-seq) shows that the Tip60 complex is present at promoter regions of predominantly active genes that are bound by RNA polymerase II (Pol II) and contain the H3K4me3 histone mark. The coactivator HAT complexes, Tip60- and Mof (KAT8)-containing (NSL and MSL), show a global overlap at promoters, whereas distinct binding profiles at enhancers suggest different regulatory functions of each essential HAT complex. Interestingly, Tip60 enrichment peaks at about 200 bp downstream of the transcription start sites suggesting a function for the Tip60 complexes in addition to histone acetylation. The comparison of genome-wide binding profiles of Tip60 and c-Myc, a somatic cell reprogramming factor that binds predominantly to active genes in mESCs, demonstrate that Tip60 and c-Myc co-bind at 50â60 % of their binding sites. We also show that the Tip60 complex binds to a subset of bivalent developmental genes and defines a set of mESC-specific enhancer as well as super-enhancer regions. Conclusions Our study suggests that the Tip60 complex functions as a global transcriptional co-activator at most active Pol II promoters, co-regulates the ESC-specific c-Myc network, important for ESC self-renewal and cell metabolism and acts at a subset of active distal regulatory elements, or super enhancers, in mESCs. Genome- wide binding of Tip60 co-activator complexes