Extended Data Fig. 8: METTL14 binds to H3K36me3 in vitro and in vivo.
From: Histone H3 trimethylation at lysine 36 guides m6A RNA modification co-transcriptionally
a, Schematic of the indirect or direct models of H3K36me3 recruiting MTC. Left, the ‘adaptor’ model refers to an indirect interaction of m6A MTC with H3K36me3 through known H3K36me3-binding proteins (adaptors). Right, the ‘reading and writing’ (R/W) model proposes that the m6A writer complex functions as reader of H3K36me3 (green dot) and is recruited to chromatin to catalyse m6A methylations (yellow dots) in newly synthesized RNAs. b, Potential association of METTL3 or METTL14 with known H3K36me3 readers was examined by co-immunoprecipitation in HeLa cells with forced expression of HA-tagged METTL3 or METTL14. c, Western blot showing knockdown efficiency of MSH2 siRNA (si-MSH2). si-NC denotes non-targeting control siRNA. d, Western blot showing that MSH2 siRNA did not affect the interaction between METTL14 and H3K36me3. e, The direct binding of Flag-tagged recombinant human METTL3 or METTL14 proteins with histone H3 peptides with (me3+) or without (me3−) K36me3 modifications was examined by in vitro pull-down assays. f, Gel-shift assay of H3K9me3 or unmethylated histone H3 with recombinant human METTL3 or METTL14 in native SDS–PAGE gel. g, Top, schematic of full-length (FL) METTL14 and its truncations. Bottom, co-immunoprecipitation coupled with western blot showing the interaction of ectopically expressed full-length or truncated METTL14 with H3K36me3 in HeLa cells. h, Co-immunoprecipitation coupled with western blot showing the interaction of ectopically expressed truncated (Δ186–456 or Δ117–456) METTL14 with H3K36me3 in HepG2 cells. i, Co-immunoprecipitation coupled with western blot showing the interaction of ectopically expressed full-length or truncated (Δ138–143 or Δ153–161) METTL14 with H3K36me3 in HepG2 cells. j, Dot blot (right) and quantification (left, data are mean± s.d.) of m6A abundance in METTL14-inducible knockout cells (sgMETTL14) transduced with different METTL14 variants. Data are mean ± s.d. k, Pearson correlation coefficients of METTL14 ChIP–seq peaks with genomic H3K27me3 features in non-overlapping, non-repetitive windows of different sizes along the genome. l, Percentages of various RNA species containing METTL14-binding sites detected by PAR-CLIP sequencing analysis. m, The proportion (pie chart, top) and enrichment (histogram, bottom) of METTL14 RNA-binding sites distribution in gene body regions, identified by PAR-CLIP sequencing. n, HOMER motif analysis of T-to-C mutations or truncations identified by METTL14 PAR-CLIP sequencing. P value was calculated by HOMER algorithm. o, Co-immunoprecipitation showing that METTL14 bound to Ser2-phosphorylated (pSer2) Pol II in HeLa cells. p, Co-localization of METTL14 with H3K36me3 (top) or Pol II (pSer2) (bottom) in the nuclei of HepG2 cells. Scale bars, 10 μm. q, Dot blot (right) and quantification (left; data are mean ± s.d.) showing enrichment of m6A in chromatin-bound RNAs compared to that in RNAs from other cell fractions. r, Western blot showing treatment with the Pol II inhibitor DRB (100 µM for 3 h) did not affect H3K36me3 levels in HeLa cells. Dimethylsulfoxide (DMSO) was used as a vehicle control. s, t, Dot blot (right) and quantification (left, data are mean ± s.d.) showing the m6A abundance in total RNA (s) or poly(A) RNA (t) was reduced after DRB treatment (100 µM for 3 h) in HeLa cells. u, Co-immunoprecipitation showing association of METTL14 with H3K36me3, METTL3 or Pol II CTD, with or without DRB treatment. v, Distribution of METTL14-binding sites on chromatin in DRB-treated cells. Images in b–j, o and q–u are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001; two-tailed student’s t-test.
