Epitranscriptomic control of erythropoiesis

项目总结 我们最近发现，N6-甲基腺苷(M6A)的mRNA调节在人类红细胞中起着至关重要的作用 细胞发育。我们观察到编码m6A mRNA甲基转移酶(MTase)复合体的基因， 包括，METTL14，METTL3和WTAP，是促进红系基因表达程序所必需的 人红系白血病(HEL)细胞及其红系特性的初步研究 造血祖细胞(HPC)。关键的是，我们发现m6A mRNA标记促进了&gt；200的翻译 基因，其中许多在红系基因表达和分化中起作用。两颗钻石- Blackfan贫血和骨髓增生异常综合征与这些基因的突变有关。目的 这项拨款的目的是阐明这种新的基因调控形式如何有助于正常和不正常 红血球生成。为此，在目标1中，我们检验了m6A-mRNA标记促进选择性的假设 人类红细胞生成所需基因的翻译。在目标2中，我们检验了m6A标记的假设 编码组蛋白/蛋白甲基转移酶基因网络的mRNAs在红系中起关键作用 世系规范。我们将创建一个人类红细胞生成过程中所有M6A mRNA标记事件的资源， 在研究m6A甲基化对关键的红系转录调控因子(如GATA1，IKZF1， KLF2和ZFPM1)，以及特异性因素是否在谱系特异性标记事件中发挥作用。在目标3中，我们 定义m6A-mR NA“甲基组”对成人红细胞和造血的作用，并询问m6A是否 在骨髓增生异常综合征(MDS)中，mRNA甲基化发生改变。 我们对MDS的兴趣源于几个观察。首先，我们发现m6A基因的甲基化发生在 在104个与MDS相关的基因中，包括10个最频繁突变的基因中的8个(例如，TET2，SF3B1， ASXL1、RUNX1、DNMT3A、ZRSR2和STAG2)。第二，人类红系形成受阻 成体祖细胞通过抑制m6A MTase复制MDS相关贫血的表型。第三，m6A-mRNA- 对多个MDS患者样本进行的测序试点研究提供了显著降低mRNA标记的证据 用于剪接基因网络和其他红细胞生成的关键调控因子(例如，U2AF2和KLF1)。大约， 三分之一的MDS患者进展为急性髓系白血病(AML)，预后较差。尽管有多个 躯体事件与MDS有关，包括染色体异常、线粒体(代谢) 功能障碍，促炎细胞因子的异常表达，以及红细胞生成失调，都没有 明确地被证明是引发或推动疾病进展的因素。 拟议中的研究将确定m6a-mrna甲基化在调节正常红细胞生成中所起的作用。 并确定m6A-mRNA改变是否与MDS的贫血有关。

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.