小鼠Gata1 mRNA 3’端非翻译区在正常和应激造血中的作用及其调控机制研究

Gata1，an essential transcription factor whose level varies during erythroid development, is precisely regulated. However, how the level of Gata1 is controlled has not been fully understood. MicroRNAs (miRNAs) are small noncoding RNAs that can regulate the expression of specific target genes and play important roles in hematopoiesis. Our microarray data reveals that in the early erythroid developmental stages, the expression levels of 11 miRNAs are much higher compared to the levels of miR-144/451, the most abundantly expressed miRNAs in late stage red blood cells. Further bioinformatics analysis indicates that two of those miRNAs (miR-709 and miR-706) can potentially bind to multiple sites of the 3’ untranslated region (3’UTR) of Gata1 mRNA. Therefore, we knocked out part of the Gata1 3’UTR sequence that contains potential miR-709 and miR-706 binding sites. Our preliminary data indicate that Gata1 3’UTR knockout mice (both heterozygous and homozygous) are anemic and their Gata1 levels are higher than that in the normal littermates. We thus hypothesize that Gata1 3’UTR is an important functional element for Gata1 expression and loss of binding between Gata1-3’UTR and miR-709/miR-706 increases the level of Gata1, which leads to disrupted erythropoiesis. This proposal describes plans for the extensive study on 1) the role of Gata1 3’UTR in erythropoiesis under both baseline and stress conditions; 2) the role of miR-709 and miR-706 in erythropoiesis; and 3) the direct regulatory relationship between miR-709/miR-706 and Gata1 expression. We will analyze Gata1 3’UTR knockout mice we have generated as an in vivo model and utilize in vitro systems including fetal liver-derived hematopoietic stem cell culture to examine what the function of Gata1-3’UTR and miR-709/miR-706 are, and whether both miR-709 and miR-706 affect erythroid development through the inhibition of Gata1. Our goal is to map a new molecular pathway through which Gata1 is regulated in normal and stress erythropoiesis and to demonstrate a proof of principle that miRNAs can target major transcription factors for homeostasis during development.

红系生成必需的转录因子Gata1表达水平受精密调控但机制未明。miRNA可抑制特定靶基因表达，对造血起重要作用。我们芯片显示miR-709和miR-706表达水平在红系发育早期明显增高，且这些miRNA在小鼠Gata1的3’端非翻译区（3’UTR）存在多个潜在结合位点，我们敲除含这两个miRNA潜在结合位点的Gata1-3’UTR片段后发现敲除鼠呈贫血症状且Gata1水平增加。推测：Gata1-3’UTR是一功能元件，与miR-709和miR-706失去结合后Gata1表达增加可致造血障碍。本课题计划以Gata1-3’UTR突变鼠作体内模型，给合造血干细胞培养等体外系统确定：正常和应激造血时Gata1-3’UTR功能；miR-709和miR-706对造血的影响；miR-709和miR-706与Gata1的关系。旨在找出调节红系生成新途径并证明一普遍规律：转录因子表达可受miRNA有效抑制。

红系生成必需的转录因子Gata1表达水平受精密调控。miRNAs可抑制特定靶基因表达，对造血起重要作用。我们的实验结果显示，miR-709和miR-706可以直接调控小鼠Gata1的3’端非翻译区（3’UTR）并产生功能改变，具体表现为：1. 我们敲除含miR-709和miR-706潜在结合位点的Gata1-3’UTR片段后发现敲除鼠呈贫血症状；2. 我们对野生型小鼠和Gata1-3’UTR敲除小鼠（KO）同时注射5-FU（fluorouracil，抗代谢肿瘤化疗药），模拟肿瘤化疗毒副作用引起的因红细胞前体细胞被大量杀伤所致的急性贫血，结果发现在这种应激造血中，Gata1-3’UTR敲除反而可以改善贫血。这些结果说明Gata1-3’UTR是一个功能元件，与miR-709和miR-706失去结合后Gata1表达增加可致造血障碍。