MicroRNAs and hematopoietic differentiation

描述（由申请人提供）：了解人类胎儿到成人血红蛋白的转换是很重要的，特别是考虑到胎儿血红蛋白（HbF）水平升高在改善镰状细胞病和2-地中海贫血的临床症状中的明确作用。其他人的工作确定了三种调节珠蛋白基因表达的转录因子，MYB， BCL11A和KLF1。这项资助建立并扩展了我们最近关于两个高度保守和共转录的mirna - miR-15a， miR-16a -在γ球蛋白基因调控中的重要作用的发现。在人类13三体中，胎儿血红蛋白（HbF）的转换和持续延迟以及新生儿胚胎血红蛋白的升高。我们将关键区域定位在染色体带13q14上，并确定miR-15a和16-1的表达增强是HbF水平升高的首选候选。事实上，我们发现这些microrna在原代人红系祖细胞中的表达增加50%，导致胎儿和胚胎血红蛋白基因表达升高。我们发现Myb在红细胞中是miR-15a和16-1的一个保守mRNA靶点，调控3 -球蛋白的表达。在这里，我们将首先通过在纯化培养的红系祖细胞中敲低miR-15a-16-1的表达，并通过分析miR15a-16-1位点缺失的小鼠的胎儿和成年红细胞生成情况，确定哪个红系祖细胞是miR-15a-16-1的主要促红细胞生成目标。同时，我们将研究miR-15- 16适度（50%）过表达对培养的人类和小鼠红细胞祖细胞的影响，以及我们将产生的小鼠中，miR-15- 16在所有造血细胞或仅在红细胞中过表达约50%。我们将确定miR-15a-16-敲低和适度过表达对细胞周期和细胞增殖、BFU-E祖细胞自我更新以及红细胞分化的影响，重点关注包括人类成人、胎儿和胚胎球蛋白在内的主要红细胞蛋白的表达。我们还将确定miR-15- 16在红细胞生成不同阶段靶向的mrna网络。具体来说，我们将确定人类和小鼠细胞中miR-15a-16-1过表达和敲低诱导的mRNA水平和翻译的全局变化（mRNA- seq）以及核糖体占用变化的分析（核糖体足迹测定）。然后，我们将通过几种计算方法分析综合实验数据，以构建miRNA- mRNA相互作用的网络，最终改变包括3珠蛋白在内的关键人类和小鼠红细胞蛋白的表达。通过报告基因检测，我们将验证关键的人类和小鼠红系mrna作为miR-15- 16的靶标，然后通过适当的敲低或过表达实验确定关键mrna作为靶标的功能，重点研究人类珠蛋白基因的调控。因此，我们的首要目标是确定调节珠蛋白基因表达的其他基因。

DESCRIPTION (provided by applicant): Understanding the fetal to adult hemoglobin switch in humans is important, especially given the clear role that elevated levels of fetal hemoglobin (HbF) have in ameliorating the clinical symptoms of sickle cell disease and 2-thalassemia. Work by others identified three transcription factors that regulate 3 globin gene expression, MYB, BCL11A, and KLF1. This grant builds on and extends our recent discoveries concerning the important roles of two highly conserved and cotranscribed miRNAs - miR-15a, miR-16a - in gamma globin gene regulation. In human trisomy 13 there is delayed switching and persistence of fetal hemoglobin (HbF) and elevation of embryonic hemoglobin in newborns. We localized the critical region to chromosomal band 13q14 and identified enhanced expression of miR-15a and 16-1 as top candidates for the elevated HbF levels. Indeed, we showed that a 50% increased expression of these microRNAs in primary human erythroid progenitor cells resulted in elevated fetal and embryonic hemoglobin gene expression. We showed that Myb is one conserved mRNA target of miR-15a and 16-1 in erythroid cells that regulate 3 globin expression. Here we will first determine which erythroid progenitor cell is the primary erythropoietic target of miR-15a- 16-1 by knocking down miR-15a-16-1 expression in purified cultured erythroid progenitors and by analyzing fetal and adult erythropoiesis in mice deleted for the miR15a-16-1 locus. In parallel we will study the effects of modest (50%) overexpression of miR-15- 16 in cultured human and murine erythroid progenitors and also in mice we will generate in which miR-15- 16 is overexpressed by ~50% in all hematopoietic cells or only in erythroid cells. We will determine effects of miR-15a-16- knockdown and modest overexpression on cell cycle and cell proliferation, self renewal of BFU-E progenitors, and on erythroid differentiation, focusing on expression of major erythroid proteins including the human adult, fetal, and embryonic globins. We will also determine the network of mRNAs targeted by miR-15- 16 at different stages of erythropoiesis. Specifically, we will determine the global changes in mRNA levels and translation induced by miR-15a-16-1 overexpression and knockdown in human and murine cells (mRNA- seq) and by analysis of changes in ribosome occupancy (iribosome-footprinting assay). We will then analyze the combined experimental data by several computational approaches to construct a network of miRNA- mRNA interactions that ultimately modify expression of key human and murine erythroid proteins including 3 globin. Using reporter gene assays we will validate key human and murine erythroid mRNAs as targets of miR-15- 16 and then determine the function of key mRNAs as targets by appropriate knockdown or overexpression experiments, focusing on human globin gene regulation. Our overriding goal is thus to identify additional genes that regulate 3 globin gene expression.