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

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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.