MicroRNAs and hematopoietic differentiation

描述(申请人提供)：了解人类从胎儿到成人的血红蛋白转换是很重要的，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方面具有明确的作用。其他人的工作发现了三种转录因子，它们是MYB、BCL11A和KLF1，它们调控着3个珠蛋白基因的表达。这笔赠款建立在并扩展了我们最近的发现，即两个高度保守和共转录的miRNAs-miR-15a，miR-16a-在伽马珠蛋白基因调控中的重要作用。在人类13三体中，胎儿血红蛋白(HBF)转换和持续时间延迟，新生儿胚胎血红蛋白升高。我们将关键区域定位在染色体13q14带上，并确定miR-15a和16-1的表达增强是HBF水平升高的首选候选基因。事实上，我们发现，在原代人类红系祖细胞中，这些microRNAs表达增加50%会导致胎儿和胚胎血红蛋白基因表达增加。我们发现Myb是在红系细胞中调节3珠蛋白表达的miR-15a和16-1的一个保守的mRNA靶点。在这里，我们将首先通过下调纯化培养的红系祖细胞中miR-15a-16-1的表达，并通过分析miR15a-16-1基因缺失的小鼠的胎儿和成人红细胞生成情况，确定哪个红系祖细胞是miR-15a-16-1的主要红系造血靶细胞。同时，我们将研究miR-15-16适度(50%)过表达对培养的人和小鼠红系祖细胞的影响，以及我们将产生的miR-15-16在所有造血细胞或仅在红系细胞中过表达~50%的小鼠的影响。我们将确定miR-15a-16基因敲除和适度过表达对细胞周期和细胞增殖、BFU-E祖细胞自我更新和红系分化的影响，重点研究主要红系蛋白的表达，包括成人、胎儿和胚胎珠蛋白。我们还将确定在红细胞生成的不同阶段，miR-15-16靶向的mRNAs网络。具体地说，我们将确定miR-15a-16-1在人和小鼠细胞中过表达和敲除(mRNA-seq)引起的mRNA水平和翻译的全球变化，并通过分析核糖体占有率的变化(核糖体足迹分析)。然后，我们将通过几种计算方法分析组合的实验数据，以构建一个miRNA-mRNA相互作用网络，最终改变包括3珠蛋白在内的关键人类和小鼠红系蛋白的表达。利用报告基因分析，我们将确认关键的人和小鼠红系mRNAs作为miR-15-16的靶标，然后通过适当的敲除或过表达实验来确定关键mRNAs作为靶标的功能，重点是人类珠蛋白基因的调控。因此，我们的首要目标是确定更多调控3珠蛋白基因表达的基因。

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.