Extended Data Fig. 7: Mechanism of H3K36me3-dependent m6A modification.
From: Histone H3 trimethylation at lysine 36 guides m6A RNA modification co-transcriptionally
a, Binding of METTL3 (top) and METTL14 (bottom) to target mRNAs was determined by CLIP–qPCR assays. b, qRT–PCR data showing that knockdown of SETD2 in HepG2 and HeLa cells did not downregulate the expression of m6A MTC genes. c, qRT–PCR data showing that overexpression of SETD2 in A498 cells did not significantly change the expression of m6A MTC genes. d, qRT–PCR showing that overexpression of KDM4A in HEK293T cells did not affect the expression of individual m6A MTC genes. Gene expression in b–d was normalized to GAPDH mRNA. e, Expression of individual MTC proteins in cells after shRNA silencing (shSETD2 sh#1 and sh#2), SETD2 overexpression (OE; wild type or mutants R1625C and ΔSRI), or KDM4A overexpression was determined by western blot analysis. f, Interaction of METTL3 and METTL14 in SETD2-knockdown or control cells with forced expression of HA–METTL3. g, Western blot showing forced expression of METTL3, METTL14 and WTAP in HeLa cells. h, Co-immunoprecipitation of HA-tagged individual m6A MTC proteins in HeLa cell lysates with or without pretreatment with DNase (DN; 100 U ml−1) or RNase (RN; 2 μg ml−1) for 1 h at room temperature. i, Co- immunoprecipitation of HA-tagged METTL14 showed no interaction between METTL14 and mono- or di-methylation of H3K36 (H3K36me1 and H3K36me2) in HeLa cells. j, Co-immunoprecipitation of endogenous METTL3 or METTL14 in HeLa cells. The interaction between METTL3 or METTL14 with H3K36me3 was detected by western blot analysis. k, Co-immunoprecipitation of endogenous H3K36me3 in HepG2 cells without or with shRNA knockdown of individual m6A MTC genes. l, The m6A/A ratio was quantified by LC–MS/MS in HepG2 cells after knockdown of SETD2 and/or METTL14. Data are mean ± s.d. of two (b, l) or three (a, c, d) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001; two-tailed Student’s t-test. Images in e–k are representative of three independent experiments.
