Dataset Information

Screening for genes that regulate the differentiation of human megakaryocytic lineage cells.

ABSTRACT: Different combinations of transcription factors (TFs) function at each stage of hematopoiesis, leading to distinct expression patterns of lineage-specific genes. The identification of such regulators and their functions in hematopoiesis remain largely unresolved. In this study, we utilized screening approaches to study the transcriptional regulators of megakaryocyte progenitor (MkP) generation, a key step before platelet production. Promising candidate genes were generated from a microarray platform gene expression commons and individually manipulated in human hematopoietic stem and progenitor cells (HSPCs). Deletion of some of the candidate genes (the hit genes) by CRISPR/Cas9 led to decreased MkP generation during HSPC differentiation, while more MkPs were produced when some hit genes were overexpressed in HSPCs. We then demonstrated that overexpression of these genes can increase the frequency of mature megakaryocytic colonies by functional colony forming unit-megakaryocyte (CFU-Mk) assay and the release of platelets after in vitro maturation. Finally, we showed that the histone deacetylase inhibitors could also increase MkP differentiation, possibly by regulating some of the newly identified TFs. Therefore, identification of such regulators will advance the understanding of basic mechanisms of HSPC differentiation and conceivably enable the generation and maturation of megakaryocytes and platelets in vitro.

SUBMITTER: Zhu F

PROVIDER: S-EPMC6176640 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Screening for genes that regulate the differentiation of human megakaryocytic lineage cells.

Zhu Fangfang F Feng Mingye M Sinha Rahul R Seita Jun J Mori Yasuo Y Weissman Irving L IL

Proceedings of the National Academy of Sciences of the United States of America 20180827 40

Different combinations of transcription factors (TFs) function at each stage of hematopoiesis, leading to distinct expression patterns of lineage-specific genes. The identification of such regulators and their functions in hematopoiesis remain largely unresolved. In this study, we utilized screening approaches to study the transcriptional regulators of megakaryocyte progenitor (MkP) generation, a key step before platelet production. Promising candidate genes were generated from a microarray plat ...[more]

PMID: 30150396

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Project description:Programmed cell death-2 (PDCD2) protein is enriched in embryonic, hematopoietic, and neural stem cells, however, its function in stem/progenitor cell differentiation is unclear. We investigated the effects of PDCD2 knockdown on the development and differentiation of hematopoietic progenitor cells (HPC). CD34(+) cells derived from normal human bone marrow and K562 leukemic cells were effectively transduced with short-hairpin RNA to knockdown PDCD2. Colony-forming assays were used to investigate the effects of PDCD2 loss on HPC clonogenic potential and on 12-O-tetradecanoyl-phorbol-13-acetate-and arabinofuranosylcytosine-induced terminal differentiation. In CD34(+) clonogenic progenitors, PDCD2 knockdown decreased the total number of colony-forming units, increased the number of colony-forming units-granulocyte-erythroid-macrophage-megakaryocyte and burst-forming unit-erythroid primitive colonies, and decreased the number of burst-forming unit-erythroid mature colonies. Similar results were observed in K562 cells, suggesting that PDCD2 is important for HPC differentiation and/or survival, and for erythroid lineage commitment. Furthermore, 12-O-tetradecanoyl-phorbol-13-acetate-induced megakaryocytic differentiation and proliferation of K562 cells was not affected by PDCD2 knockdown. In contrast, arabinofuranosylcytosine-induced erythroid differentiation of K562 cells was significantly reduced with PDCD2 knockdown, with no effect on cell proliferation. The effects of PDCD2 knockdown were attributed to a cell cycle arrest at G(0)/G(1), along with increased messenger RNA expression of early progenitor factors c-MYB and GATA-2, and decreased expression of erythroid factors GATA-1, EpoR, and ?-globin. We conclude that PDCD2 loss of function(s) impedes erythroid differentiation by inducing cell cycle arrest and increasing expression of early hematopoietic progenitor factors. These findings suggest that PDCD2 has a novel regulatory role in human hematopoiesis and is essential for erythroid development.

Dataset Information

Screening for genes that regulate the differentiation of human megakaryocytic lineage cells.

Publications

Screening for genes that regulate the differentiation of human megakaryocytic lineage cells.

Similar Datasets

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