Extended Data Fig. 2: Derivation of degron tagged METTL3 cells and their effect on Xist-mediated silencing.
From: m6A and the NEXT complex direct Xist RNA turnover and X-inactivation dynamics
a, Western blot showing the METTL3-FKBP12F36V protein level in two independent clones (C3 and H5) under ChrRNA-seq analysis conditions shown in Fig. 1d (top). TBP loading control. Western blot showing another clone of C-terminal METTL3 dTAG degron (M12D) generated in the Xist_Exon7-Bgl background exhibits sensitivity to dTAG-13 treatment at both 2 and 24 h (bottom). KAP1 loading control. b, Western blot showing the protein level for RBM15 and METTL14 for C3 and H5 clones under ChrRNA-seq analysis conditions shown in Fig. 1d. c, Western blot showing protein size and level for WT METTL3, FKBP12F36V tagged METTL3, and GFP-tagged WT or catalytic mutant METTL3. TBP loading control. The blot is representative of two biologically independent experiments. d, Bar plot showing the proportion of differentially expressed genes in Mettl3 KO mESCs23 (top) or 26 h dTAG-13 treated METTL3-FKBP12F36V mESCs (bottom) with m6A modification. The number of differentially up- or down-regulated genes are indicated. m6A peaks/sites are annotated from ChrMeRIP-seq data21 and miCLIP-seq2 data54. e, Allelic ratio difference between dTAG + Dox samples and Dox samples in Fig. 1d. Values from METTL3 N-terminal or C-terminal FKBP12F36V are averaged and shown as METTL3N (blue) and METTL3C (red) respectively. Green line on ChrX ideogram indicates location of Xist. Dashed line denotes 0, indicating no silencing difference. f-h, As in (e), but for gene group analysis. Boxplots showing the difference of allelic ratio towards three different gene categories defined previously: initial X-linked gene expression level17 (f), initial promoter chromatin state15 (g), gene silencing kinetics22 (h). FKBP12F36V indicated as dTAG. Centre lines indicate the median, box limits indicate the first and third quartiles, and whiskers indicate 1.5× IQR.
