MicroRNAs and hematopoietic differentiation

描述（由申请人提供）：了解人类胎儿至成人血红蛋白的转换非常重要，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-地中海贫血临床症状方面的明确作用。其他人的工作确定了三种调节3珠蛋白基因表达的转录因子，MYB，BCL 11 A和KLF 1。这项资助建立在我们最近发现的两种高度保守和共转录的miRNAs - miR-15 a，miR-16 a-在γ珠蛋白基因调控中的重要作用的基础上，并扩展了这一发现。在人类13三体中，存在胎儿血红蛋白（HbF）的延迟转换和持续存在以及新生儿中胚胎血红蛋白的升高。我们将关键区域定位于染色体带13 q14，并将miR-15 a和16-1的表达增强确定为HbF水平升高的最佳候选者。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是调节3珠蛋白表达的红系细胞中miR-15 a和16-1的一个保守的mRNA靶标。 在此，我们将首先通过敲低纯化培养的红系祖细胞中的miR-15 a-16 -1表达并通过分析miR 15 a-16 -1基因座缺失的小鼠中的胎儿和成人红细胞生成来确定哪种红系祖细胞是miR-15 a-16 - 1的主要红细胞生成靶标。平行地，我们将研究miR-15- 16在培养的人和鼠红系祖细胞中适度（50%）过表达的影响，并且还在我们将产生的小鼠中，其中miR-15- 16在所有造血细胞中或仅在红系细胞中过表达约50%。我们将确定miR-15 a-16敲低和适度过表达对细胞周期和细胞增殖、BFU-E祖细胞自我更新和红系分化的影响，重点关注主要红系蛋白的表达，包括人成人、胎儿和胚胎球蛋白。 我们还将确定miR-15- 16在红细胞生成的不同阶段靶向的mRNA网络。具体而言，我们将确定在人和鼠细胞中由miR-15 a-16-1过表达和敲低诱导的mRNA水平和翻译的总体变化（mRNA-seq），并通过分析核糖体占有率的变化（虹膜体足迹测定）。然后，我们将通过几种计算方法来分析组合的实验数据，以构建最终修饰关键的人类和小鼠红系蛋白（包括3珠蛋白）表达的miRNA- mRNA相互作用网络。使用报告基因测定，我们将验证关键的人类和小鼠红细胞mRNA作为miR-15- 16的靶点，然后通过适当的敲低或过表达实验确定关键mRNA作为靶点的功能，重点是人类珠蛋白基因调控。因此，我们的首要目标是确定额外的基因，调节3珠蛋白基因的表达。

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.