Loss of G6b-B results in defective TPO signaling and blocks megakaryocyte-lineage differentiation
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE155735 | GEO | 2023/04/03
REPOSITORIES: GEO
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Project description:EZH2, the enzymatic component of PRC2, has been identified as a key factor in hematopoiesis. EZH2 loss of function mutations have been found in myeloproliferative neoplasms, more particularly in myelofibrosis, but the precise function of EZH2 in megakaryopoiesis is not fully delineated. Here, we show that EZH2 inhibition by small molecules and shRNA induces MK commitment by accelerating lineage marker acquisition without change in proliferation. Later in differentiation, EZH2 inhibition blocks proliferation and endomitosis and decreases proplatelet formation. EZH2 inhibitors similarly reduce polyploidization and proplatelet formation of JAK2V617F MK. In transcriptome profiling, the defect in proplatelet formation was associated with an aberrant actin cytoskeleton regulation pathway, whereas polyploidization was associated with an inhibition of expression for a set of genes involved in DNA replication and repair, and an upregulation of CDK inhibitors, more particularly CDKN1A and CDKN2D. The knockdown of CDKN1A and/or CDKN2D could partially rescue the percentage of polyploid MKs. However only CDKN1A was regulated by H3K27me3 suggesting that EZH2 controls MK polyploidization through a direct regulation of CDKN1A and indirectly of CDKN2D.
2021-03-31 | E-MTAB-8611 | biostudies-arrayexpress
Project description:Megakaryocyte (MK) differentiation is well described in morphologic terms but its molecular counterparts and the basis for platelet release are incompletely understood. We profiled mRNA expression in populations of primary mouse MKs representing successive differentiation stages. Genes associated with DNA replication are highly expressed in young MKs, in parallel with endomitosis. Intermediate stages are characterized by disproportionate expression of genes associated with the cytoskeleton, cell migration and G-protein signaling, whereas terminally mature MKs accumulate hemostatic factors, including many membrane proteins. We used these expression profiles to extract a reliable panel of molecular markers for MKs of early, intermediate or advanced differentiation, and establish its value using mouse models of defective thrombopoiesis resulting from absence of GATA-1, NF-E2 or tubulinß1. Computational analysis of the promoters of late-expressed MK genes identified new candidate targets for NF-E2, a critical transcriptional regulator of platelet release. One such gene encodes the kinase adaptor protein LIMS1/PINCH1, which is highly expressed in MKs and platelets and significantly reduced in NF-E2-deficient cells. Transactivation studies and chromatin immunoprecipitation implicate Lims1 as a direct target of NF-E2 regulation. Attribution of stagespecific genes, in combination with various applications, thus constitutes a powerful way to study MK differentiation and platelet biogenesis Experiment Overall Design: MK progenitors expand in mouse bone marrow or fetal liver cell preparations cultured with thrombopoietin, and mature over 5-6 days in vitro. After 3 days of culture, a significant fraction of cells shows features of committed MK progenitors, and numerous terminally mature, proplatelet-forming MKs appear by day 5. Because isolation of cell populations that correspond to sequential stages in MK differentiation is hindered by the lack of synchrony in primary MK cultures, we applied flow cytometry to harvest sub-populations that are substantially enriched for MKs with defined properties. High surface expression of the lineage marker CD41 identified MKs, whereas forward-scatter (FSC) properties distinguished cells on the basis of size. We collected thrombopoietic culture suspensions from days 3, 4 and 6, and sorted populations by flow cytometry. RNAs were prepared from sorted cells to probe Affymetrix MOE430A mouse oligonucleotide arrays.
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Project description:The hematopoietic system gives rise to a heterogeneous population of terminally differentiated cells that reside in the bone marrow (BM) to fulfill their roles in immunity, blood clotting and tissue oxygenation. Hematopoietic stem and progenitor cells are at the apex of a hierarchically organized maturation cascade constantly replenishing the pool of differentiated cells to maintain blood cell homeostasis. The bone marrow microenvironment is functionally compartmentalized by heterogeneous niche cells that provide physical and soluble signals to spatio-temporally organize hematopoiesis. Megakaryocytes (MKs) are niche cells of hematopoietic origin that support hematopoietic stem cell (HSCs) homeostasis and generate circulating platelets. Platelet production involves the release of MK fragments while their cell body is entirely consumed in a process termed thrombopoiesis. Consequently, replenishment of fragmented MKs from MK progenitors (termed megakaryopoiesis) is required to ensure sufficient platelet production, but also to maintain MK homeostasis within the HSC niche. Here, we used intravital imaging of the megakaryocytic lineage to identify the spatio-temporal patterns of megakaryopoiesis during steady state and thrombocytopenia. We show that MK consumption during thrombopoiesis is compensated by immediate and local proliferation of MK progenitors. MK homeostasis is controlled by plasmacytoid dendritic cells (pDCs) that scan the BM and secrete Interferon alpha (INFa) upon detection of exhausted megakaryocytes to trigger megakaryopoiesis. We identified local pDC-derived INFa release as a physiological inflammatory stimulus of the bone marrow niche that synchronizes megakaryopoiesis and thrombopoiesis to maintain homeostasis of MKs and platelets in steady state and under stress. Viral infection with SARS-CoV-2 can manipulate pDC-driven MK proliferation leading to inappropriate megakaryopoiesis, which has been associated with thrombotic complications during COVID-19.
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Project description:Megakaryocyte (MK) differentiation is well described in morphologic terms but its molecular counterparts and the basis for platelet release are incompletely understood. We profiled mRNA expression in populations of primary mouse MKs representing successive differentiation stages. Genes associated with DNA replication are highly expressed in young MKs, in parallel with endomitosis. Intermediate stages are characterized by disproportionate expression of genes associated with the cytoskeleton, cell migration and G-protein signaling, whereas terminally mature MKs accumulate hemostatic factors, including many membrane proteins. We used these expression profiles to extract a reliable panel of molecular markers for MKs of early, intermediate or advanced differentiation, and establish its value using mouse models of defective thrombopoiesis resulting from absence of GATA-1, NF-E2 or tubulinß1. Computational analysis of the promoters of late-expressed MK genes identified new candidate targets for NF-E2, a critical transcriptional regulator of platelet release. One such gene encodes the kinase adaptor protein LIMS1/PINCH1, which is highly expressed in MKs and platelets and significantly reduced in NF-E2-deficient cells. Transactivation studies and chromatin immunoprecipitation implicate Lims1 as a direct target of NF-E2 regulation. Attribution of stagespecific genes, in combination with various applications, thus constitutes a powerful way to study MK differentiation and platelet biogenesis Keywords: expression profiling differentiation megakaryocyte
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