Extended Data Fig. 8: YTHDC1 stabilizes METTL3 on heterochromatin.
From: METTL3 regulates heterochromatin in mouse embryonic stem cells
a–c, Box plots (a), aggregation plots (b), and UCSC snapshot (c) showing METTL3 levels on IAPEz-int in YTHDC1WT, Ythdc1 KO and YTHDC1W429A cell lines. ****P < 0.0001 (exact P values from left to right: 2.3 × 10−176, 1.2 × 10−195), two-sided paired t-test. d–f, Box plots (d), aggregation plots (e), and UCSC snapshot (f) showing H3K9me3 levels on IAPEz-int in YTHDC1WT, Ythdc1 KO and YTHDC1W429A cell lines. ****P < 0.0001 (exact P values from left to right: 7.1 × 10−63, 4.8 × 10−279), two-sided paired t-test. g–j, Heat maps (g), box plots (h), aggregation plots (i), and UCSC snapshots (j) showing H4K20me3 levels on IAPEz-int in YTHDC1WT, Ythdc1 KO and YTHDC1W429A cell lines. ****P < 0.0001 (exact P values from left to right: 8.8 × 10−30, 7.8 × 10−114), two-sided paired t-test. k, Western blots showing protein levels of METTL3 and YTHDC1 in control, Mettl3 KO, Ythdc1 KD and Mettl3 KO + Ythdc1 KD cell lines. l, ChIP–qPCR showing H3K9me3 enrichment level on IAPEz elements in control, Mettl3 KO, Ythdc1 KD and Mettl3 KO + Ythdc1 KD cell lines. *P < 0.05, **P < 0.01; two-sided t-test. Exact P values are provided in the Source Data. m, Western blots showing Cas9 protein levels upon Dox treatment in Mettl3 KO cell lines. n, ChIP–qPCR of H3K9me3 (left) and Cas9 (right) on IAP and control regions in Mettl3 KO cell lines expressing dCas9–YTHDC1. The mean of three biological replicates ± s.d. is shown. *P < 0.05, **P < 0.01; two-sided t-test. Exact P values are provided in the Source Data. o, Western blots showing Cas9 and METTL3 protein levels upon Dox treatment in Mettl3 KO + METTL3APPA cell lines. p, ChIP–qPCR of H3K9me3 (left) and Cas9 (right) on IAP and control in Mettl3 KO + METTL3APPA cell lines expressing dCas9–YTHDC1. The mean of three biological replicates ± s.d. is shown. Exact P values are provided in the Source Data. q, Co-immunoprecipitation coupled with western blots showing interactions of YTHDC1 with reintroduced METTL3 (wild-type or catalytically mutated) in Mettl3 KO cells with or without triptolide treatment. Uniquely mapped ChIP–seq reads were used in a, c, d, f–h, j. Uniquely + randomly mapped ChIP–seq reads were used in b, e, i. Heat maps were ranked according to METTL3 density in parental cells in descending order in g. For the box plots in a, d, h, the middle line and lower and upper hinges of the box plot correspond to the median and the first and third quartiles, respectively. The whiskers extend from the hinges to no further than 1.5 × IQR from the hinge. Outlying points are plotted individually. Blots are representative of two independent experiments in k, m, o, q. For blots source data, see Supplementary Fig. 1. MTA and MaSat used in ChIP–qPCR are examples of repetitive elements not bound by METTL3.
