Nuclear m6A reader YTHDC1 regulates the scaffold function of LINE1 RNA in mouse ESCs and early embryos [ICM ChIP-seq]

N6-methyladenosine (m6A) on chromosome-associated regulatory RNAs (carRNAs), including repeat RNAs, plays important roles in tuning the chromatin state and transcription, but the intrinsic mechanism remains unclear. Here, we report that YTHDC1 plays indispensable roles in the embryonic stem cell (ESC) self-renewal and differentiation potency, which highly depends on its m6A-binding ability. Ythdc1 is required for sufficient rRNA synthesis and repression of the 2-cell transcriptional program in ESCs, which recapitulates the transcriptome regulation by the LINE1 scaffold. Detailed analyses revealed that YTHDC1 recognizes m6A on LINE1 RNAs in the nucleus and regulates the formation of the LINE1-NCL partnership and the chromatin recruitment of KAP1. Moreover, the establishment of H3K9me3 on LINE1 scaffold-regulated regions, including 2-cell retrotransposons, is interrupted in Ythdc1-depleted ESCs and inner cell mass (ICM) cells, which consequently increases the transcriptional activities. Our study reveals a role of m6A in regulating the RNA scaffold, providing a new model for the RNA-chromatin cross-talk. rRNA-free total RNA-seq was performed for Ythdc1 f/f (control), Ythdc1 cKO, W378A-YTHDC1 rescue, WT-YTHDC1 rescue, WT (control for Mettl3 KO), Mettl3 KO, control shRNA-expressed and Kap1 shRNA-expressed mouse ES cell lines with 2-3 replicates for each line. Nuclear RIP-seq for YTHDC1 was performed using control ES cells with 2 replicates for IP and 1 reaction for input. Cross-Linked ChIP-seq of KAP1 and H3K9me3 was perfomed for Ythdc1 f/f , Ythdc1 cKO, W378A-YTHDC1 rescue and WT-YTHDC1 rescue ES cells with 2 replicates for each IP and 1 reaction for each input. Low-input RNA-seq was performed for control and Ythdc1 KO ICM cells with 2 replicates each. Ultra-low-input native ChIP-seq of H3K9me3 was performed for control and Ythdc1 KO ICM cells with 2 replicates for each IP and 1 reaction for each input.