Extended Data Fig. 9: Silencing of SETD2 demethylates H3K36me3 and m6A of pluripotency factors and inhibits the in vitro differentiation of mouse ES cells.
From: Histone H3 trimethylation at lysine 36 guides m6A RNA modification co-transcriptionally
a, qRT-PCR showing downregulation of Setd2, but not m6A MTC genes (Mettl3, Mettl14 or Wtap), in Dox-induced SETD2-knockdown mouse ES cells. b, Western blot showing reduction of SETD2 and decrease of H3K36me3 in Dox-induced SETD2-knockdown mouse ES cells after 48 h of Dox treatment. c, Immunofluorescence showing expression of stage-specific embryonic antigen-1 (SSEA1; left) and OCT4 (right) in control (−Dox) and SETD2-knockdown (+Dox) mouse ES cells after 3 days of LIF withdrawal. Scale bars, 10 μm. d, Changes in mRNA expression of endoderm differentiation markers during in vitro differentiation of mouse ES cells. e, shRNA knockdown of Mettl14 by mouse ES cells as determined by qRT–PCR. f, shRNA silencing of METTL14 promotes the expression of pluripotency markers in mouse ES cells. HSP90 serves as a loading control. g, shRNA silencing of METTL14 delayed emerging of endoderm markers in mouse ES cells as determined by qRT-PCR. h, Numbers of H3K36me3 peaks identified in mouse ES cells with or without Dox-induced SETD2 knockdown by ChIP–seq. i, ChIP–seq revealed global hypomethylation of H3K36me3 in mouse ES cells with doxycycline treatment as shown by histogram distribution and box plots. For the box plot, top whisker denotes the ninety-fifth percentile (shCtrl = 2.519, shSETD2 = 1.081), top of the box denotes the seventy-fifth percentile (shCtrl = 1.281, shSETD2 = 0.527), horizontal lines denotes the median (shCtrl = 0.886, shSETD2 = 0.312), bottom of the box is the twenty-fifth percentile (shCtrl = 0.454, shSETD2 = 0.155), and bottom whisker is the fifth percentile (shCtrl = 0.000, shSETD2 = 0.000). P value was calculated using two-sided Wilcoxon and Mann–Whitney test. j, Dot blot (left) and quantification (right, data are mean ± s.d.) revealed reduction of cellular m6A in mouse ES cells by doxycycline-induced knockdown of SETD2. k, m6A-seq revealed global hypomethylation of m6A in mouse ES cells after doxycycline treatment as shown by histogram distribution and box plots. For the box plot, top whisker is the ninety-fifth percentile (shCtrl = 18.500, shSETD2 = 10.200), top of the box is the seventy-fifth percentile (shCtrl = 9.530, shSETD2 = 5.360), horizontal line is the median (shCtrl = 5.480, shSETD2 = 3.700), bottom of the box is the twenty-fifth percentile (shCtrl = 3.530, shSETD2 = 2.110), and bottom whisker is fifth percentile (shCtrl = 2.000, shSETD2 = 0.000). P value was calculated using two-sided Wilcoxon and Mann–Whitney test. l, HOMER motif analysis revealed conserved motifs enriched in hypomethylated peaks. P value was calculated by HOMER algorithm. m, The enrichment of hypomethylated m6A peak distribution. The enrichment fold was determined by the proportion of m6A peaks normalized by the length of the region. n, Gene set enrichment analysis (GSEA)31 of downregulated genes after differentiation (day (D) 0 to D6) and upregulated genes after SETD2 silencing (−Dox to +Dox). o, Determination of H3K36me3 abundance in pluripotency factors by ChIP–qPCR. p, mRNA levels of pluripotency factors in mouse ES cells as examined by qRT-PCR. Data are mean ± s.d. of two (a) or three (d, e, g, j, o and p) independent experiment. *P < 0.05, **P < 0.01, ***P < 0.001; two-tailed Student’s t-test. Images in b, f and j are representative of three independent experiments.
