Nat10 regulates the homeostasis of pluripotency and 2C state in mESCs by N4-acetylcytidine mRNA modification [ChIP-seq]

Mouse embryonic stem cells (mESCs) were isolated from the inner cell mass (ICM), which can prevent an excessive number of cells from transitioning to the 2-cell state and retain only 1%–5% of cells transiently expressing 2C genes such as Zscan4 and ERVs, and 95%–99% of normally expressing pluripotent transcription factors such as Oct4 and Nanog. The homeostasis state is always regulated by epigenetics especially heterochromatin modifications. However, it is still unclear what a specific factor can regulate this homeostasis state. Here we report that N-acetyltransferase 10 (Nat10), the writer for N4-acetylcytidine (ac4C) modification of mRNA, can serve as a new heterochromatin associated protein which can regulate the homeostasis of 2C genes and the expression of pluripotency genes in mESCs through a dual role. Mechanistically, Nat10 deficiency decreased ac4C modification on Oct4 and Esrrb mRNAs, resulting in decreased pluripotency. At the same time, ac4C modification on the heterochromatin component Kap1 mRNA is also reduced, which indirectly leads to a decrease in the binding of the H3K9me3 histone modification to the 2C gene, thereby removing the repression of the 2C genes and allowing the 2C genes to be expressed at high levels. These findings elucidate a novel role of Nat10 in mESCs and reveal new mechanisms linking the 2C program and pluripotency to ac4C modifications in mESCs. About 2×107 of Nat10 CON and Nat10 KD mESCs were first fixed with 1% paraformaldehyde, and then lysed and sonicated to achieve the majority of DNA fragments at 100–500bp. DNA fragments were then enriched by immunoprecipitation with 5 μg H3K9me3 antibody (ab8898, Abcam) and Dynabeads M280 (Life Technologies), rocked at 4 ℃ overnight. The immunoprecipitated material was eluted from the beads by heating for 30 min at 65 ℃. Then, samples were incubated at 65 ℃ for overnight to reverse crosslinking. The samples were then extracted with phenol: chloroform: isoamyl alcohol (25: 24: 1, pH > 7.8) followed by chloroform, ethanol precipitated in the presence of glycogen, and re-suspended in ddH2O. ChIP enriched DNA was used for library construction by Novogene Corporation. Each library was subsequently sequenced, resulting in ~20 million reads (125 bp).