Nat10 regulates the homeostasis of pluripotency and 2C state in mESCs by N4-acetylcytidine mRNA modification [RNA-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. mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEB Next First Strand Synthesis Reaction Buffer (5×). First strand cDNA was synthesized using formed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 30 ends of DNA fragments, NEB Next Adaptors with hairpin loop structure were ligated to prepare for hybridization. To select cDNA fragments of preferentially 150–200 bp in length, the library fragments were purified with AMPure XP random hexamer primer and M-MLV Reverse Transcriptase (RNase H). Second strand cDNA synthesis was subsequently per-system (Beckman Coulter, Beverly, USA). Then 3 μL USER Enzyme (NEB, USA) was used with size-selected and cDNA adaptor ligated at 37 ℃ for 15 min followed by 5 min at 95 ℃ prior to PCR. PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index Primer. At last, PCR products were purified using AMPure XP system and library quality assessed on the Agilent Bioanalyzer 2100 system. Cluster of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform.