(a) Heat map analysis based on RNA-seq analysis in
Mettl14 KO vs. nondeleted control NSCs. (b,c) Gene ontology
(GO) analysis of genes down- and up-regulated in Mettl14 KO vs.
nondeleted control E14.5 NSCs. GO analysis were performed by DAVID.
Differentially expressed genes had an adjusted P < 0.01
and a 2-fold or greater expression difference. Among differentially expressed
genes, 1099 are up-regulated and 1487 are down -regulated. Numbers of gene
counts and exact P values for each GO term are listed in Supplementary Fig. 4a.
(e) Representative western blots of acid-extracted histones from
E14.5 NSCs using antibodies recognizing H3K4–1me, H3K4–3me,
H3K27–3me, H3K9–3me, H3K27-ac, H3K9-ac, pan-acetyl- H3, uH2AK119,
uH2BK120, and H3S28 phosphorylation. The band sizes range from 17 to 23 KD as
expected for modified histones. For uncropped images, see Supplementary Fig. 6a. (f)
Quantitation of western blots from E14.5 and E17.5 NSCs, one-way ANOVA (WT:
n = 8, KO: n = 12, and Het:
n = 8 independent NSCs cultures; H3K4me1,
P = 0.1123, F (2, 25) = 2.39; H3K4me3,
P = 1.06442E-09, F (2, 25) = 52.77;
H3K9me3, P = 0.2096, F (2, 25) = 1.664;
H3K27me3, P = 0.00013, F (2, 25) = 13.07;
H3K9ac, P = 0.1461, F (2, 25) = 2.08; H3K27ac,
P = 4.796E-06, F (2, 25) = 20.8; H3ac,
P = 0.3676, F (2, 25) = 1.042; H2AK119Ubi,
P = 0.3592, F (2, 25) = 1.067; H2BK120Ubi,
P = 0.1192, F (2, 25) = 2.319; H3S28pho,
P = 0.5347, F (2, 25) = 0.642) followed by
Bonferroni’ spost hoc test (H3K4me1, WT vs. KO,
P = 0.2376, 95% C.I. = - 0.4713 to 0.09065, WT vs. Het,
P = 0.9999, 95% C.I. = −0.2629 to 0.3527; H3K4me3,
WT vs. KO, P = 1.157E-08, 95% C.I. = −0.5518 to
−0.3128, WT vs. Het, P = 0.9999, 95% C.I. =
−0.134 to 0.1278; H3K9me3, WT vs. KO, P = 0.4574, 95%
C.I. = −0.3314 to 0.1054, WT vs. Het, P = 0.9999, 95%
C.I. = −0.1942 to 0.2842; H3K27me3, WT vs. KO, P =
0.0008, 95% C.I. = −1.131 to −0.2956, WT vs. Het,
P = 0.9999, 95% C.I. = −0.3891 to 0.5256; H3K9ac, WT
vs. KO, P = 0.321, 95% C.I. = −0.4577 to 0.1121, WT vs.
Het, P = 0.1141, 95% C.I. = −0.5732 to 0.05098; H3K27ac,
WT vs. KO, P = 1.769E-05, 95% C.I. = −1.591 to
−0.6358, WT vs. Het, P = 0.9999, 95% C.I. =
−0.5908 to 0.4556; H3ac, WT vs. KO, P = 0.6463, 95% C.I.
= −0.4945 to 0.2007, WT vs. Het, P = 0.9999, 95% C.I. =
−0.3309 to 0.4307; H2AK119Ubi, WT vs. KO, P = 0. 5288,
95% C.I. = −0.1242 to 0.3523, WT vs. Het, P = 0.9999,
95% C.I. = −0.2759 to 0.2459; H2BK120Ubi, WT vs. KO, P =
0.6171, 95% C.I. = −0.2165 to 0.5511, WT vs. Het, P =
0.6457, 95% C.I. = - 0.5982 to 0.2426; H3S28pho, WT vs. KO, P =
0.9999, 95% C.I. = −0.2407 to 0.2961, WT vs. Het, P =
0.8731, 95% C.I. = −0.3915 to 0.1965). (f) Cell growth analysis based on
an MTT assay of NSCs treated with vehicle/DMSO or the MLL1 inhibitor MM-102, the
CBP/P300 inhibitor C646, or the Ezh2 inhibitor GSK343. Shown is the absorbance
ratio of KO to non-deleted controls at each drug dose. One-way ANOVA
(n = 3 independent experiments for all experimental groups;
GSK343, P = 4.232E-05, F (3, 8) = 38.47; C646,
P = 0.0003, F (3, 8) = 23.43; MM-102,
P = 0.0025, F (3, 8) = 11.91) followed by
Bonferroni’s post hoc test (GSK343, c vs. 1.25,
P = 0.0035, 95% C.I. = −0.2477 to −0.05943, c
vs. 2.5, P = 0.0002, 95% C.I. = −0.3265 to
−0.1383, c vs. 5, P = 1.979E-05, 95% C.I. =
−0.4169 to - 0.2287; C646, c vs. 1.25, P = 0.0036, 95%
C.I. = −0.1158 to −0.02744, c vs. 2.5, P =
0.0236, 95% C.I. = −0.09574 to −0.007344, c vs. 5,
P = 0.000103, 95% C.I. = −0.1654 to −0.07702;
MM-102, c vs. 0.0625, P = 0.0507, 95% C.I. = −8.591E-05
to 0.06086, c vs. 1.25, P = 0.9999, 95% C.I. = - 0.03858 to
0.02237, c vs. 2.5, P = 0.0615, 95% C.I. = −0.05958 to
0.001368). Graphs represent the mean ± SD. Dots represent data from
individual data points. ns = nonsignificant. * P < 0.05,
** P < 0.01, *** P < 0.001, ****
P < 0.0001.