Epitranscriptomic control of erythropoiesis

项目摘要 我们最近发现，N6-甲基腺苷（m6 A）mRNA的调节在人类红细胞中起着重要作用， 细胞发育我们观察到编码m6 A mRNA甲基转移酶（MTase）复合物的基因， 包括胃L14、胃L3和WTAP，是促进红系细胞基因表达程序所必需的。 人红系白血病（HEL）细胞和人原代双能 造血祖细胞（HPC）。重要的是，我们发现m6 A mRNA标记促进>200的翻译。 基因，其中许多在红系基因表达和分化中起作用。两个钻石- 布莱克凡贫血和骨髓增生异常综合征与这些基因的突变有关。目的 这项资助的目的是阐明这种新的基因调控形式如何有助于正常和异常的 红细胞生成为此，在目的1中，我们检验了m6 A-mRNA标记促进选择性表达的假设。 翻译人类红细胞生成所需的基因。在目标2中，我们测试了m6 A标记的假设， 编码SET结构域组蛋白/蛋白甲基转移酶基因网络的mRNAs对红系造血细胞的生长至关重要。 谱系规范我们将创建一个人红细胞生成过程中所有m6 A mRNA标记事件的资源， 在检查m6 A甲基化对关键红细胞转录调节因子（例如，GATA 1，IKZF 1， KLF 2和ZFPM 1）以及特异性因子是否在谱系特异性标记事件中发挥作用。在目标3中， 定义成人红细胞生成和造血的m6 A-mRNA“甲基化组”，并询问m6 A 骨髓增生异常综合征（MDS）中mRNA甲基化改变。 我们对MDS的兴趣源于几个观察结果。首先，我们发现m6 A mRNA甲基化发生在70 在与MDS相关的104个基因中，包括10个最频繁突变的基因中的8个（例如，TET2、SF3B1、 ASXL 1、RUNX 1、DNMT 3A、ZRSR 2和STAG 2）。第二，人红系形成的阻断， 成年祖细胞通过抑制m6 A MTase复制MDS相关贫血的表型。第三，m6 A-mRNA- 多个MDS患者样本的测序初步研究提供了显著降低mRNA标记的证据， 对于剪接基因和红细胞生成的其它关键调节因子的网络（例如，U2 AF 2和KLF 1）。大约， 三分之一的MDS患者进展为急性髓性白血病（AML），预后不良。虽然多个 MDS涉及体细胞事件，包括染色体异常、线粒体（代谢） 功能障碍、促炎细胞因子表达异常和红细胞生成失调， 已经明确显示启动或驱动疾病进展。 这项研究将明确m6 A-mRNA甲基化在调节正常红细胞生成中的作用 并确定改变的m6 A-mRNA是否有助于MDS的贫血。

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.