Fig. 5.

Promoter histone H3K4me3 marks are lost in the absence of m⁶A methyltransferase activity. a An H3K4me3 relative read distribution plot around the transcription start site (TSS) of methylated genes in CRISPR NTC, WTAP-KO, METTL3-KO, and METTL14-KO HEL cells. The plot shows a marked reduction in H3K4me3 marks following loss of WTAP and METTL14 with a more modest effect with loss of METTL3. b Bedgraphs of normalized H3K4me3 CUT&RUN data for select genes with reduced methylation following m⁶A loss. c An upset plot of the H3K4me3 peaks found in CRISPR NTC, WTAP-KO, METTL3-KO, and METTL14-KO HEL cells. The pattern of H3K4me3 loss is consistent with loss of KLF1 transcriptional regulation. Genes with reduced H3K4me3 following loss of any of the three m⁶A MTase components are enriched for genes downregulated following Klf1 KO. d A Venn diagram showing enrichment for KLF1 CHIP-seq target genes⁶³ among the H3K4me3 peaks lost following WTAP-KO or METTL14-KO, suggesting m⁶A-mediated epigenetic regulation of the KLF1 transcriptional program. e KLF1 CHIP-qPCR validation of several genes with or without altered H3K4me3 levels following METTL3-KO and WTAP-KO, (n = 4, mean ± SEM). f A diagram of the iron procurement, heme synthesis and transport, and hemoglobin assembly in erythroid cells, highlighting regulation by KLF1 and altered H3K4me3 marking following m⁶A loss. *: KLF1 CHIP-seq target genes⁶³; Blue: reduced H3K4me3 marking and transcript expression following m⁶A-MTase inhibition; Green: reduced H3K4me3 marking or transcript expression following m⁶A-MTase inhibition. These results highlight inhibition of multiple pathways involved in hemoglobin synthesis following loss of m⁶A possibly through down regulation of the KLF1 transcriptional program. g The proposed model for the role of m⁶A in translational regulation of erythroid gene expression and erythropoiesis