METTL14 is a chromatin regulator independent of its RNA N6-methyladenosine methyltransferase activity

Abstract METTL3 and METTL14 are two components that form the core heterodimer of the main RNA m6A methyltransferase complex (MTC) that installs m6A. Surprisingly, depletion of METTL3 or METTL14 displayed distinct effects on stemness maintenance of mouse embryonic stem cell (mESC). While comparable global hypo-methylation in RNA m6A was observed in Mettl3 or Mettl14 knockout mESCs, respectively. Mettl14 knockout led to a globally decreased nascent RNA synthesis, whereas Mettl3 depletion resulted in transcription upregulation, suggesting that METTL14 might possess an m6A-independent role in gene regulation. We found that METTL14 colocalizes with the repressive H3K27me3 modification. Mechanistically, METTL14, but not METTL3, binds H3K27me3 and recruits KDM6B to induce H3K27me3 demethylation independent of METTL3. Depletion of METTL14 thus led to a global increase in H3K27me3 level along with a global gene suppression. The effects of METTL14 on regulation of H3K27me3 is essential for the transition from self-renewal to differentiation of mESCs. This work reveals a regulatory mechanism on heterochromatin by METTL14 in a manner distinct from METTL3 and independently of m6A, and critically impacts transcriptional regulation, stemness maintenance, and differentiation of mESCs.


Figure S2 .
Figure S2.METTL3 and METTL14 acted similarly on m 6 A-mediated regulation.(A-B) m 6 A levels on mRNA (A) and caRNAs (B) in Mettl3 Control and Mettl3 CKO, Mettl14 Control

Figure S4 .
Figure S4.METTL3 and METTL14 displayed distinct chromatin binding preferences.(A) Western blot assays showing the specificity and IP efficiency of the METTL14 antibodies we used.(B) Average profiles of METTL14 at gene level (from 5KB upstream of TSS to 5KB downstream of TTS) in Mettl14 Control or Mettl14 CKO mESCs (top panel).Heatmap of METTL14 genomic binding at peaks center and their flanking regions in Mettl14 Control or Mettl14 CKO mESCs (bottom panel).CUT&RUN signal has been normalized to E. coli Spike-in DNAs.(C) Average profiles of various histone modification on METTL3 and METTL14 CUT&RUN peaks in wild type mESCs.(D) Bar graphs showing the overlapping ratios (Jaccard index) of METTL14 CUT&RUN, METTL3 CUT&RUN and METTL3 ChIP-seq peaks with different histone modifications (GSE36114 and GSE126243) in wild type mESCs.(E) Bar graphs showing the overlapping ratios (Jaccard index) of METTL14-specific sites, METTL3specific sites and METTL3/METTL14-m 6 A sites with different histone modifications in wild type mESCs.(F-G) The correlations of METTL14 CUT&RUN signal between two biological replicates in Mettl3 Control and Mettl3 CKO mESCs (F), and METTL3 CUT&RUN signal between two biological replicates in Mettl14 Control and Mettl14 CKO mESCs (G).(H) Average profile of METTL3 CUT&RUN signal at gene level in Mettl14 Control and Mettl14 CKO mESCs.(I) Profiles of METTL14 and METTL3 CUT&RUN signals on genes that are significantly down-regulated (down-DEGs) upon Mettl14 knockout in mESCs.

Figure S5 .
Figure S5.METTL14-occupied genomic regions marked with H3K27me3 are less enriched of m 6 A than the non-marked ones.(A) Heatmap of H3K27me3 modification at four identified clusters in mESCs cultured under different cell culture conditions: "LIF+Serum" condition refers to the presence of LIF in the medium containing Serum, and "LIF+2i" condition refers to serumfree medium containing 2i. (B-C) IGV visualization (B) and bar plot for quantification (C) showing H3K27me3 levels on Tfap2c, Hic1 and NKx2-2 genes in Mettl14 Control and Mettl14 CKO mESCs.(D) Boxplot of caRNA abundance that derived from METTL14 bound DNA loci.METTL14 peaks were categorized into H3K27me3 modified (H3K27me3 (+)) and unmodified (H3K27me3 (-)) groups.(E) Bar graph showing the percentage of METTL14 peaks overlapped with caRNA m 6 A peaks.METTL14 peaks were categorized into H3K27me3 modified (H3K27me3 (+)) and unmodified (H3K27me3 (-)) groups.

Figure S6 .
Figure S6.METTL14 directly regulates H3K27me3 deposition in mESCs.(A) Average profiles of H3K27me3 modification at gene level (from 2.5KB upstream of TSS to 2.5KB downstream of TTS) in Mettl14 Control and Mettl14 CKO mESCs, respectively.(B) Western blot (left panel) with quantification (right panel) of H3K27me3 in Mettl14 Control and Mettl14 CKO mESCs.n=3 per condition.P values were calculated using Two-tailed Student's t-test.(C) Heatmap showing H3K27me3 CUT&RUN signal (left panel) and H3K27me3 signal changes (right panel) around the peak centers (+/-5KB, left panel) upon Mettl14 CKO in mESCs.CUT&RUN signal are normalized according to SNAP-CUTANA_K-MetStat_Panel_Analysis pipeline.(D) Heatmap showing H3K27me3 binding and binding changes upon Mettl14 knockout in mESCs at the four clusters of METTL3 and METTL14 peak centers and the flanking 2.5KB regions.(E) H3K27me3 modifications on METTL3 (left panel) or METTL14 (right panel) CUT& RUN peak centers and the flanking 5KB in Mettl14 Control and Mettl14 CKO mESCs, respectively.(F) Cumulative distributions of H3K27me3 modified gene expression fold-changes

Figure S7 .
Figure S7.METTL14 regulates H3K27me3 changes during neuronal differentiation of mESCs.(A) Heatmap showing H3K27me3 modification level changes during mESCs differentiation (day 6 vs. day 0) on four clusters of METTL3 and METTL14 CUT&RUN peak centers and the flanking 2.5KB regions.(B-C) Scatter plot showing the correlation between METTL14 CUT&RUN signal and H3K27me3 level changes comparing NPCs vs. ESCs (B), and Mettl14 KO vs. wildtype (WT) NPC (C).Sites ranked by METTL14 CUT&RUN signal were grouped and averaged into 100 data point.(D) Boxplot of gene expression level fold-changes (log 2 FC) upon Mettl14 knockout (left panel); after GSK343 treatment in Mettl14 knockout mEB (middle panel); or the rescue degree (right panel).Rescue degree was calculated as log 2 FC from (middle panel) subtracted the log 2 FC from (left panel).Genes were categorized into two subgroups according to whether they were modified by m 6 A or H3K27me3.P values were calculated by a nonparametric Wilcoxon-Mann-Whitney test.(E) Alkaline phosphatase staining of Mettl14 Control, Mettl14 CKO, and Mettl14 CKO mESCs treated with GSK343 under two different concentrations.