Intermediate Steps In Erythroid, Megakaryocytic and Myeloid Lineage Specification

Abstract Abstract 4778 Normal human CD34 positive precursor cells can be expanded in vitro and converted with high efficiency into maturing erythroid cells or myeloid cells by treating the cells with erythropoietin (EPO) or with G-CSF, respectively. The maturation of megakaryocytic cells can also be induced from human CD34 positive precursor cells by treatment with thrombopoietin (TPO) with lower efficiency. The induction time varies from 9 to 12 days depending on the lineages. Total RNAs were extracted every other day during the induction period for the purpose of gene profiling at intermediate steps of the differentiation. The samples were analyzed with the “Whole Transcriptome Shotgun Sequencing (RNA-seq)” method. Comparison of the gene expression among these three lineages at different stages of differentiation shows complex changes of a substantial number of genes in categories including transcription factors, cytokines etc. Basically, many genes change similarly in both erythroid and megakaryocytic lineage differentiation, the number of this shared genes are much less when comparing myeloid cells with one of these lineages. Initially during erythropoietin treatment, a large number of megakaryocyte mRNAs are upregulated to levels comparable to those of the erythroid specific genes, or to those seen in precursor cells treated with thrombopoietin. After several days of erythropoietin treatment, most of the megakryocyte related genes are down regulated, while there is continued upregulation of erythroid genes. Unlike many other megakaryocytic genes, the level of mRNA for the thrombopoietin receptor MPL is rapidly down-regulated suggesting that erythroid lineage commitment occurs prior to silencing of many megakaryocytic genes. Comparison of the linage specific genes identified groups of cell surface proteins which may be applied as new markers for sorting various cell populations. Some of these include DARC, LEPR, TFRC, BMPR2, TFR2, AXL, EPHB4, IL15RA, TNFRSF19, FZD5, ULBP1, CELSR2, RYK, MRGPRE, CRY1 which are specific for erythroid lineage; FPR1, EVI2A, CD14, and others totaling 50 proteins for myeloid lineage and more than 80 highly expressed surface markers such as GP1BA, F2RL3, GP1BB, ADRA2A, GABRE unique for megakaryocytic lineage. In parallel, proteomic analysis was carried out with an Isotope coded affinity tag (ICAT)-based protein profiling and Multiplexed Isobaric Tagging Technology (iTRAQ™). Statistical analysis shows good correlation between the relative changes of RNA and protein expression levels during the erythropoiesis, although there were large discrepancies in the relative levels of mRNA and protein at any one time point. Only those genes with very low or very high RNA values show the poor correlations. CTCF and its associated factor Rad21 are involved in the establishment of enhancer boundary elements and chromosomal conformation. We have begun genome wide analysis of these factors during erythroid and myeloid cell growth and differentiation. In this project, we also aimed to investigate the modulation mechanisms of CTCF during the CD34 positive cells differentiation into different lineages. Taking the advantage of the ChIP-Sequencing (ChIP-Seq) technology, we found that progressive changes in the sites of binding of the DNA binding protein CTCF occurs, and are not limited to the regions embedding erythroid genes, suggesting that there is a lineage specific global reorganization of chromatin. In addition to transcriptional controls, there are prominent post-transcriptional effects regulating the expression of key transcription factors including CTCF. Through this study, the regulation mechanisms of the chromatin remodeling, signal pathways and other, molecular events during hematopoiesis will be described in detail. Disclosures: No relevant conflicts of interest to declare.