Genome-Scale Transcriptional Analysis of Megakaryocytic Cell Cultures Reveals Insights into Lineage-Specific Differentiation

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Little is known about the transcriptional events underlying megakaryocytic (Mk) differentiation from hematopoietic stem cells. We sought to identify genes and pathways not previously associated with megakaryopoiesis and to evaluate the CHRF-288-11 (CHRF) megakaryoblastic cell line as a model system for investigating megakaryopoiesis. Using DNA microarrays, Q-RT-PCR, and protein-level assays, we compared the dynamic gene expression pattern of phorbol-ester-induced differentiation of CHRF cells to cytokine-induced Mk differentiation of human mobilized peripheral blood CD34+ cells. Transcriptional patterns of well-known Mk genes were similar between the two systems. CHRF cells constitutively express some early Mk genes including GATA-1. Several Rho and G-protein-coupled-receptor signaling pathway components were up-regulated, including genes not previously associated with Mk cells. Ontological analysis revealed up-regulation of defense-response genes, including both known and candidate platelet-derived contributors to inflammation. Up-regulation of interferon-responsive genes occurred in the cell line, but not in the primary cells, likely due to a genetic mutation in JAK2. The observed gene expression patterns in the CHRF and primary Mk cell cultures suggested that p53 activity increases during terminal Mk differentiation. In support of this, RNA-interference-mediated reduction of p53 expression in the CHRF cells led to greater polyploidization and reduced cell death. Mk cells derived from cultures initiated with p53/ mouse bone marrow also displayed greater polyploidization. Q-RT-PCR analysis identified seven genes that are part of the p53 regulon in Mk cells. The global gene expression data from primary Mk cell cultures were also leveraged to identify novel regulators of gene expression in Mk cells. We used ontological annotations and comparison with parallel granulocytic cultures to identify 199 transcription factors and co-regulators that were differentially expressed in Mk cells. These included both known Mk regulators and genes not previously associated with Mk cells. Protein expression and nuclear localization was confirmed in Mk cells for the candidate Mk transcription factors RFX5, MXD1, and FHL2. This analysis of megakaryopoiesis, which integrates dynamic gene expression data with protein abundance and activity assays, has identified a number of genes and pathways that may help govern megakaryopoiesis. This has significantly broadened our understanding of the molecular orchestration of Mk differentiation

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  • 08/06/2018
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