Extended Data Fig. 3: The YTHDC2/LTR7 axis prevents the neuronal fate of hESC via LINC-ROR.
a, Gene Ontology (GO) analysis of the genes differentially expressed in YTHDC2 KO versus control hESCs. b, GO analysis of the genes whose promoters are located within 20kb of YTHDC2 ChIP-seq peaks. c, YTHDC2 KO promotes neuronal fate of hESC-derived neuronal cells and embryoid bodies (EBs). The protein levels of the indicated neuronal markers were analyzed by Western blot. n = 3 biologically independent experiments. d, RT-qPCR analysis of gene expression of EBs derived from wild-type control versus YTHDC2 KO hESCs. The marker genes for neuronal, endoderm, mesoderm, and trophectoderm lineages were analyzed. e–h, Epigenetic silencing of LTR7 loci by dCas9-KRAB/CARGO (CRISPRi) lead to: increased histone H3K9me3 and decreased H3K27ac modifications on LTR7 loci, determined by ChIP-seq(e, f); reduction of HERV-H transcripts, determined by RNA-seq (g) and RT-qPCR (h). i-k, LTR7 CRISPRi did not affect hESCs morphology (i) or the expression of pluripotency markers at RNA (j) and protein (k) levels. l, LTR7 CRISPRi promotes neuronal fate of hESC-derived neuronal cells and embryoid bodies (EBs). The protein levels of the indicated neuronal markers were analyzed by Western blot as indicated. n = 3 biologically independent experiments. m, LINC-ROR expression is substantially decreased in YTHDC2 KO compared to the wild-type control (Ctrl) cells, determined by RT-qPCR. n, RT-qPCR analysis reveals a notable reduction of LINC-ROR in hESCs upon epigenetic silencing of LTR7s. o, RT-qPCR analysis showing efficient knockdown (KD) of LINC-ROR by two gRNAs targeting LINC-ROR sequence. p, CasRx-mediated KD of LINC-ROR did not alter the expression of pluripotency markers in hESCs, determined by RT-qPCR analysis. Data represent mean ± s.e.m. from three (h, j, m-p) or four (d)independent experiments. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. P values were calculated by two-tailed Student′s test in (d, h, j, m-p). Exact p values are in Source Data 1.
