Bianchi E, Bulgarelli J, Ruberti S, Rontauroli S, Sacchi G, Norfo R, Pennucci V, Zini R, Salati S, Prudente Z, Ferrari S, Manfredini R
Department of Life Sciences, Center for Regenerative Medicine 'Stefano Ferrari', University of Modena and Reggio Emilia, Modena, Italy.
Department of Life Sciences, Center for Genome Research, University of Modena and Reggio Emilia, Modena, Italy.
Cell Death Differ. 2015 Dec;22(12):1906-21. doi: 10.1038/cdd.2015.30. Epub 2015 Apr 10.
The transcription factor MYB has a key role in hematopoietic progenitor cells (HPCs) lineage choice, by enhancing erythropoiesis at the expense of megakaryopoiesis. We previously demonstrated that MYB controls erythroid versus megakaryocyte lineage decision by transactivating KLF1 and LMO2 expression. To further unravel the molecular mechanisms through which MYB affects lineage fate decision, we performed the integrative analysis of miRNA and mRNA changes in MYB-silenced human primary CD34+ HPCs. Among the miRNAs with the highest number of predicted targets, we focused our studies on hsa-miR-486-3p by demonstrating that MYB controls miR-486-3p expression through the transactivation of its host gene, ankyrin-1 (ANK1) and that miR-486-3p affects HPCs commitment. Indeed, overexpression and knockdown experiments demonstrated that miR-486-3p supports the erythropoiesis while restraining the megakaryopoiesis. Of note, miR-486-3p also favors granulocyte differentiation while repressing the macrophage differentiation. To shed some light on the molecular mechanisms through which miR-486-3p affects HPCs lineage commitment, we profiled the gene expression changes upon miR-486-3p overexpression in CD34+ cells. Among the genes downregulated in miR-486-3p-overexpressing HPCs and computationally predicted to be miR-486-3p targets, we identified MAF as a miR-486-3p target by 3'UTR luciferase reporter assay. Noteworthy, MAF overexpression was able to partially reverse the effects of miR-486-3p overexpression on erythroid versus megakaryocyte lineage choice. Moreover, the MYB/MAF co-silencing constrained the skewing of erythroid versus megakaryocyte lineage commitment in MYB-silenced CD34+ cells, by restraining the expansion of megakaryocyte lineage while partially rescuing the impairment of erythropoiesis. Therefore, our data collectively demonstrate that MYB favors erythropoiesis and restrains megakaryopoiesis through the transactivation of miR-486-3p expression and the subsequent downregulation of MAF. As a whole, our study uncovers the MYB/miR-486-3p/MAF axis as a new mechanism underlying the MYB-driven control of erythroid versus megakaryocyte lineage fate decision.
转录因子MYB在造血祖细胞（HPC）谱系选择中起关键作用，它通过增强红细胞生成而以巨核细胞生成为代价。我们之前证明，MYB通过反式激活KLF1和LMO2的表达来控制红系与巨核细胞谱系的决定。为了进一步阐明MYB影响谱系命运决定的分子机制，我们对MYB沉默的人原代CD34 + HPC中的miRNA和mRNA变化进行了综合分析。在预测靶标数量最多的miRNA中，我们通过证明MYB通过反式激活其宿主基因锚蛋白-1（ANK1）来控制miR-486-3p的表达，且miR-486-3p影响HPC的定向分化，从而将研究重点聚焦于hsa-miR-486-3p。事实上，过表达和敲低实验表明，miR-486-3p支持红细胞生成，同时抑制巨核细胞生成。值得注意的是，miR-486-3p还促进粒细胞分化，同时抑制巨噬细胞分化。为了阐明miR-486-3p影响HPC谱系定向分化的分子机制，我们分析了CD34 +细胞中miR-486-3p过表达后的基因表达变化。在miR-486-3p过表达的HPC中下调且经计算预测为miR-486-3p靶标的基因中，我们通过3'UTR荧光素酶报告基因检测确定MAF是miR-486-3p的靶标。值得注意的是，MAF过表达能够部分逆转miR-486-3p过表达对红系与巨核细胞谱系选择的影响。此外，MYB/MAF共沉默通过抑制巨核细胞谱系的扩增，同时部分挽救红细胞生成的损伤，从而限制了MYB沉默的CD34 +细胞中红系与巨核细胞谱系定向分化的偏向。因此，我们的数据共同表明，MYB通过反式激活miR-486-3p的表达以及随后下调MAF来促进红细胞生成并抑制巨核细胞生成。总体而言，我们的研究揭示了MYB/miR-486-3p/MAF轴是MYB驱动的红系与巨核细胞谱系命运决定控制的一种新机制。
