Epitranscriptomic control of erythropoiesis

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PROJECT SUMMARY We recently found that N6-methyladenosine (m6A) mRNA regulation plays an essential role in human red blood cell development. We observed that genes coding for the m6A mRNA methyltransferase (MTase) complex, including, METTL14, METTL3, and WTAP, are required for promoting erythroid gene expression programs in human erythroid leukemia (HEL) cells and for the erythroid lineage specification in human primary, bipotent hematopoietic progenitor cells (HPCs). Critically, we find that m6A mRNA marks promote the translation of >200 genes, many of which play a role in erythroid lineage gene expression, and differentiation. Both Diamond- Blackfan anemia, and Myelodysplastic Syndromes are associated with mutations in these genes. The purpose of this grant is to elucidate how this novel form of gene regulation contributes to normal and abnormal erythropoiesis. To this end, in Aim 1, we test the hypothesis that m6A-mRNA marking promotes selective translation of genes required for human erythropoiesis. In Aim 2, we test the hypothesis that m6A marking of mRNAs coding for a network of SET domain histone/protein methyltransferase genes is critical for erythroid lineage specification. We will create a resource of all m6A mRNA marking events during human erythropoiesis, while examining the effect of m6A methylation on key erythroid transcriptional regulators (e.g., GATA1, IKZF1, KLF2, and ZFPM1) and whether specificity factors play roles in lineage-specific marking events. In Aim 3, we define the m6A-mRNA "methylome" for human adult erythropoiesis and hematopoiesis and ask whether m6A mRNA methylation is altered in myelodysplastic syndrome (MDS). Our interest in MDS stems from several observations. First, we found that m6A mRNA methylation occurs in 70 out of 104 genes associated with MDS, including 8 of the 10 most frequently mutated genes (e.g., TET2, SF3B1, ASXL1, RUNX1, DNMT3A, ZRSR2, and STAG2). Second, the blockage of erythroid lineage formation in human adult progenitors by inhibition of m6A MTase copies the phenotype of MDS-related anemias. Third, m6A-mRNA- sequencing pilot studies of multiple MDS patient samples provided evidence of significantly lower mRNA marking for networks of splicing genes and other key regulators of erythropoiesis (e.g., U2AF2 and KLF1). Approximately, one-third of patients with MDS progress to acute myeloid leukemia (AML), with poor outcomes. Although multiple somatic events have been implicated in MDS, including chromosomal abnormalities, mitochondrial (metabolic) dysfunction, abnormal expression of pro-inflammatory cytokines, and dysregulated erythropoiesis, none have been shown unequivocally to initiate or drive disease progression. The proposed studies will define the role that m6A-mRNA methylation plays in regulating normal erythropoiesis and determine whether altered m6A-mRNA contributes to the anemia of MDS.