Regulation and function of the microRNA-144/451 cluster during megakaryocytic/erythroid differentiation

转录因子和microrna是胚胎发生过程中造血干细胞出现的主要调节因子，并调节造血谱系（如骨髓生成）的分化。转录因子通过结合基因调控序列中的特定位点来调节基因表达，并招募具有表观遗传功能的辅因子复合物。转录因子是microrna的主要靶标，经常产生调控环，驱动和稳定特定的基因表达程序。由突变、染色体易位或转录因子和microrna的表观遗传改变引起的基因表达失调是人类白血病发展的主要原因。我们的初步数据显示microRNA簇miR144/451的表达直接受到Tal1、GATA1和RUNX1转录因子的调控。miR144/451在红细胞分化期间表达上调，在巨核细胞分化期间表达下调。我们发现与miR144/451调控元件结合的转录因子在分化过程中发生了变化。此外，我们发现白血病融合蛋白RUNX1/ETO与miR144/451启动子结合并下调miR144/451的表达。在这个项目中，我们想研究miR144/451在巨核细胞/红细胞分化过程中的转录调控，重点研究miR144/451调控元件的表观遗传变化。此外，我们想分析RUNX1/ETO对miR144/451表达的影响，以评估RUNX1/ETO对红细胞分化的抑制作用是否部分通过miR144/451介导。在这些实验中，我们将使用原代人CD34+细胞检测miR144/451对巨核细胞/红细胞分化的影响。为了研究miR144/451影响分化的机制，我们想分析miR144是否靶向Tal1和/或RUNX1的3- utr，并通过这种方式创建一个调控回路。此外，我们希望使用基于silac的质谱法在蛋白质组范围内鉴定单个microrna miR144和miR451的靶标。该研究将破译由miR144/451和转录因子创建的网络，该网络调节巨核细胞/红细胞分岔的基因表达程序。

Transcription factors and microRNAs are central regulators of hematopoietic stem cell emergence during embryogenesis and regulate differentiation of hematopoietic lineages such as myelopoiesis. Transcription factors regulate gene expression by binding to specific sites in regulatory sequences of genes and recruit cofactor complexes with epigenetic functions. Transcription factors are a main target of microRNAs and often create regulatory loops, which drive and stabilize specific gene expression programs. Deregulation of gene expression caused by mutations, chromosomal translocations or epigenetic alterations of transcription factors and microRNAs are a major cause for the development of leukaemia in humans. Our preliminary data show that expression the microRNA cluster miR144/451 is directly regulated by the transcription factors Tal1, GATA1 and RUNX1. Expression of miR144/451 is upregulated during erythroid and down regulated during megakaryocytic differentiation. We found that the transcription factor binding to regulatory elements of miR144/451 changes during differentiation. Furthermore, we found that the leukemic fusion protein RUNX1/ETO binds to the miR144/451 promoter and down regulates miR144/451 expression.In this project we want to examine the transcriptional regulation of miR144/451 during megakaryocytic/erythroid differentiation with a focus on epigenetic changes at regulatory elements of miR144/451. Furthermore, we want to analyse the influence of RUNX1/ETO on miR144/451 expression to evaluate if the repressive effect of RUNX1/ETO on erythroid differentiation is partly mediated through miR144/451. In relation to these experiments we will examine the influence of miR144/451 on megakaryocytic/erythroid differentiation using primary human CD34+ cells. To examine the mechanism how miR144/451 influences differentiation we want to analyse if miR144 targets the 3-UTRs of Tal1 and/or RUNX1 and this way creates a regulatory loop. Furthermore, we want to use SILAC-based mass spectrometry to identify targets of the single microRNAs, miR144 and miR451, in a proteome wide manner. The proposed investigations will decipher the network created by miR144/451 and transcription factors, which regulates the gene expression programs at the megakaryocytic/erythoid bifurcation.