Abstract
Many of the regulatory features governing erythrocyte specification, maturation, and associated disorders remain enigmatic. To identify new regulators of erythropoiesis, we utilize a functional genomic screen for genes affecting expression of the erythroid marker CD235a/GYPA. Among validating hits are genes coding for the N6-methyladenosine (m6A) mRNA methyltransferase (MTase) complex, including, METTL14, METTL3, and WTAP. We demonstrate that m6A MTase activity promotes erythroid gene expression programs through selective translation of ~300 m6A marked mRNAs, including those coding for SETD histone methyltransferases, ribosomal components, and polyA RNA binding proteins. Remarkably, loss of m6A marks results in dramatic loss of H3K4me3 marks across key erythroid-specific KLF1 transcriptional targets (e.g., Heme biosynthesis genes). Further, each m6A MTase subunit and a subset of their mRNAs targets are required for human erythroid specification in primary bone-marrow derived progenitors. Thus, m6A mRNA marks promote the translation of a network of genes required for human erythropoiesis.
Highlights
Many of the regulatory features governing erythrocyte specification, maturation, and associated disorders remain enigmatic
Since technical limitations precluded the use of human hematopoietic stem and progenitor cells (HSPCs) for large scale functional genomic screens, we took advantage of Human erythroleukemia (HEL) cells as a surrogate model, based on their expression of key markers and transcription factors associated with erythropoiesis[28]
Transduction of HEL cells with lentiviral vectors expressing sgRNAs targeting GYPA, GATA1, or LMO2 resulted in significant reduction of CD235a expression (Supplementary Fig. 1a) and, for sgGATA1 and sgLMO2, significant changes in gene expression of erythroid gene targets as well as erythroid progenitor stagespecific gene expression programs (Supplementary Fig. 1b, c)
Summary
Many of the regulatory features governing erythrocyte specification, maturation, and associated disorders remain enigmatic. M6A-marked mRNAs in HEL cells were enriched for regulators of hematopoiesis and erythropoiesis (e.g., GATA1, FLI1, KLF1, and MPL) (Fig. 2c) and genes with causal roles in erythroid-related diseases (Fig. 2d, e).
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