Fig. 1: Acute depletion of METTL3 results in accelerated XCI.
From: m6A and the NEXT complex direct Xist RNA turnover and X-inactivation dynamics
a, Schematic outline of N-terminal FKBP12F36V tagging of METTL3. b, Western blot showing the FKBP12F36V–METTL3 fusion protein and METTL14 protein level for two independent clones (G4 and H1) after 2 or 24 h of dTAG-13 treatment. SETDB1 was used as a loading control. Short (middle) and long (bottom) exposure were also explored. c, Schematic outline detailing cell line specification and experimental design. d, Box plot showing the allelic ratio of X-linked genes (n = 396) from ChrRNA-seq analysis for each sample and condition indicated above and below, respectively. Two independent lines tagged with an N-terminal degron (G4 and H1 clone) and C-terminal FKBP12F36V (C3 and H5 clone) were included for this analysis, alongside untagged WT cells (C7H8 clone). The y axis denotes an allelic ratio ranging from 0 to 1. Two red dashed lines indicate the allelic ratio in ES cells (NoDox) and WT cells induced for 1 day with Dox. P values were calculated using a two-sided paired t-test. e, PCA using allelic ratio of X-linked genes (n = 374) for samples in d, along with time-course WT (C7H8) samples. f, Box plot depicting the allelic ratio of X-linked genes (n = 410) from ChrRNA-seq analysis for the complementation assay where either GFP–METTL3 (WT_P2B3 clone) or GFP–METTL3-D395A (D395A_1F clone) was expressed from the Rosa26 locus in a C-terminal METTL3 dTAG degron cell line (H5). Both WT_P2B3 and D395A_1F clones retain both Xcast and X129. The red dashed line indicates the allelic ratio at 0.5. Samples and conditions are indicated above and below, respectively. P values were calculated using a two-sided paired t-test. Two biological replicates were averaged. In box plots (d,f), center lines indicate the median, box limits indicate the first and third quartiles and whiskers indicate 1.5× the interquartile range (IQR).
