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中心调节因子，并调节造血谱系如骨髓生成的分化。转录因子通过与基因调控序列中的特定位点结合来调控基因表达，并募集具有表观遗传功能的辅因子复合物。转录因子是microRNA的主要靶点，通常会产生调控环，从而驱动和稳定特定的基因表达程序。由转录因子和microRNA的突变、染色体易位或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。我们的初步数据表明，microRNA簇miR 144/451的表达直接受转录因子Tal 1，GATA 1和RUNX 1的调控。miR 144/451的表达在红系细胞中上调，在巨核细胞分化中下调。我们发现，在分化过程中，与miR 144/451的调节元件结合的转录因子发生变化。 此外，我们还发现白血病融合蛋白RUNX 1/ETO与miR 144/451启动子结合，下调miR 144/451的表达，本研究旨在探讨miR 144/451在巨核细胞/红细胞分化过程中的转录调控，重点关注miR 144/451调控元件的表观遗传变化。此外，我们想分析RUNX 1/ETO对miR 144/451表达的影响，以评估RUNX 1/ETO对红系分化的抑制作用是否部分通过miR 144/451介导。关于这些实验，我们将使用原代人CD 34+细胞检查miR 144/451对巨核细胞/红细胞分化的影响。为了研究miR 144/451如何影响分化的机制，我们想要分析miR 144是否靶向Tal 1和/或RUNX 1的3-UTR，并且这种方式创建了调节环。此外，我们希望使用基于SILAC的质谱法以蛋白质组范围的方式鉴定单个microRNA，miR 144和miR 451的靶标。拟议的研究将破译由miR 144/451和转录因子创建的网络，该网络调节巨核细胞/类巨核细胞分叉处的基因表达程序。

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.