FTO mediates LINE1 m6A demethylation and chromatin regulation in mESCs and mouse tissues
N6-methyladenosine (m6A) is the most abundant internal modification in mammalian messenger RNA (mRNA). It is installed by writer proteins and can be reversed by erasers. FTO was the first RNA demethylase shown to catalyze oxidative demethylation of m6A in RNA. Despite extensive studies, the main physiological substrates of FTO and the related functional pathways remain elusive in many systems, in particular during early mammalian development. Here, we show that FTO mediates the m6A demethylation of chromosome-associated repeat RNAs in mouse embryonic stem cells (mESCs), especially the long-interspersed element-1 family (LINE1) RNA, thereby affecting their abundances to regulate chromatin state. Overall design: ChIP profiles of histone marks and transcription factors in WT and Fto knockout mESCs were generated by deep sequencing, in duplicate using Illumina NovaSeq 6000. Nuclear RNA lifetime and nascent RNA synthesis of WT and Fto knockout mESCs were generated by deep sequencing, in triplicate, using Illumina NovaSeq 6000 machine.Embryos at GV, MII and morula stages were transferred to 500 ?l Trizol Reagent and then mixed well with 100 ?l chloroform added. The mixture was transferred Qiagen MaXtract High Density tubes (129046). After centrifuge, upper and aqueous phase was removed. RNA was precipitated by adding equal volume isopropanol and washing by 75% ethanol. Then purified RNA were analyzed by RT-qPCR or subjected to library generation using SMARTer Stranded Total RNASeq Kit (Takara Bio) following manufacturer?s protocol. Libraries were sequenced on an Illumina NovaSeq 6000 platform in paired-end read mode with 150 bp per read. ATAC-Seq was performed on wild-type and FTO knockout mESCs.