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PF4-Cre mediated deletion of SRF in murine megakaryocytes.

ABSTRACT: Srf is a MADS-box transcription factor that is critical for muscle differentiation. Its function in hematopoiesis has not yet been revealed. Mkl1, a cofactor of Srf, is part of the t(1;22) translocation in acute megakaryoblastic leukemia, and plays a critical role in megakaryopoiesis. In order to test the role of Srf in megakaryocyte development, we crossed Pf4-Cre mice, which express Cre recombinase in cells committed to the megakaryocytic lineage, to SrfF/F mice in which functional Srf is no longer expressed after Cre-mediated excision. Pf4-Cre/SrfF/F (KO) mice are born with normal mendelian frequency, but have significant macrothrombocytopenia with approximately 50% reduction in platelet count. In contrast, the BM has increased numbers and percentages of CD41+ megakaryocytes (WT: 0.41+/-0.06%; KO: 1.92+/-0.12%) with significantly reduced ploidy. KO mice show significantly increased megakaryocyte progenitors in the BM by both FACS analysis and CFU-Mk. Megakaryocytes lacking Srf have abnormal stress fiber and demarcation membrane formation and platelets lacking Srf have abnormal actin distribution. In vitro and in vivo assays reveal platelet function defects in KO mice. Critical actin cytoskeletal genes are downregulated in KO megakaryocytes. Thus, Srf is required for normal megakaryocyte maturation and platelet production, due at least in part, to regulation of cytoskeletal genes.

ORGANISM(S): Mus musculus

PROVIDER: GSE21859 | GEO | 2010/07/13

SECONDARY ACCESSION(S): PRJNA127039

REPOSITORIES: GEO

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PF4-Cre mediated deletion of SRF in murine megakaryocytes.

Project description:Srf is a MADS-box transcription factor that is critical for muscle differentiation. Its function in hematopoiesis has not yet been revealed. Mkl1, a cofactor of Srf, is part of the t(1;22) translocation in acute megakaryoblastic leukemia, and plays a critical role in megakaryopoiesis. In order to test the role of Srf in megakaryocyte development, we crossed Pf4-Cre mice, which express Cre recombinase in cells committed to the megakaryocytic lineage, to SrfF/F mice in which functional Srf is no longer expressed after Cre-mediated excision. Pf4-Cre/SrfF/F (KO) mice are born with normal mendelian frequency, but have significant macrothrombocytopenia with approximately 50% reduction in platelet count. In contrast, the BM has increased numbers and percentages of CD41+ megakaryocytes (WT: 0.41+/-0.06%; KO: 1.92+/-0.12%) with significantly reduced ploidy. KO mice show significantly increased megakaryocyte progenitors in the BM by both FACS analysis and CFU-Mk. Megakaryocytes lacking Srf have abnormal stress fiber and demarcation membrane formation and platelets lacking Srf have abnormal actin distribution. In vitro and in vivo assays reveal platelet function defects in KO mice. Critical actin cytoskeletal genes are downregulated in KO megakaryocytes. Thus, Srf is required for normal megakaryocyte maturation and platelet production, due at least in part, to regulation of cytoskeletal genes. C-kit+CD41+ megakaryocyte progenitors from PF4-Cre/SRF C57BL/6 SRF WT (3) and C57BL/6 SRF KO (3) mice were sorted by flow cytometry and cultured for three days in thrombopoietin.

2010-07-13 | E-GEOD-21859 | biostudies-arrayexpress

Label-free quantitative proteomics analysis of bone marrow cells of MK-LCM WT versus KO mice

Project description:High ploidy large cytoplasmic megakaryocytes (LCM) are critical negative regulators of hematopoietic stem cells (HSC) and are responsible for platelet formation. Using a mouse knockout model with normal megakaryocyte numbers but essentially devoid of LCM (MK-LCM KO), we demonstrated a pronounced increase in bone marrow HSC concurrent with endogenous mobilization and extramedullary hematopoiesis. When HSC isolated from a MK-LCM KO microenvironment were transplanted in lethally irradiated mice, the absence of LCM increased HSC in BM, blood and spleen. Severe thrombocytopenia was observed in animals with diminished LCM, although there was no change in megakaryocyte ploidy distribution. In contrast, WT HSC-generated LCM regulated a normal HSC pool and prevented thrombocytopenia. The present label-free quantitative LC-MSMS data was used to determine proteins that are differentially expressed in bone marrow cells of MK-LCM WT versus MK-LCM KO mice.

2023-05-10 | PXD040423 | Pride

Cell-autonomous function of Runx1 transcriptionally regulates megakaryocytic maturation in mice (expression)

Project description:RUNX1 transcription factor (TF) is a key regulator of megakaryocytic development and when mutated is associated with familial platelet disorder and predisposition to acute myeloid leukemia (FPD-AML). We used mice lacking Runx1 specifically in megakaryocytes (MKs) to characterize the Runx1-mediated transcriptional program during advanced stages of MK differentiation. Gene expression and chromatin-immunoprecipitation-sequencing (ChIP-seq) of Runx1 andÂ p300Â identified functional Runx1-bound MK enhancers. Runx1/p300 co-bound regions showed significant enrichment in genes important for MK and platelet homeostasis. Runx1-bound regions were highly enriched in RUNX and ETS motifs and to a lesser extent in GATA motif.Â The data providesÂ the first example of genome-wide Runx1/p300 occupancy in maturating FL-MK,Â unravels the Runx1-regulated program controlling MK maturationÂ in vivoÂ andÂ identifies itsÂ bona fideÂ regulated genes. It advances our understandingÂ of the molecular events that upon mutations in RUNX1 lead to theÂ predisposition to familial platelet disorders and FPD-AML. Gene expression profiles of mature megakaryocytes taken from fetal livers of megakaryocyte-specific Runx1 knockout mice, using Runx1F/F/Pf4-Cre mice versus control (WT) mice.

2013-03-22 | E-GEOD-45373 | biostudies-arrayexpress

Mus musculus

Project description:PF4-Cre mediated deletion of SRF in murine megakaryocytes.

| PRJNA127039 | ENA

Shared roles for Scl and Lyl1 in murine platelet production and function

Project description:The Stem Cell Leukemia (Scl or Tal1) protein forms part of a multimeric transcription factor complex required for normal megakaryopoiesis. However, unlike other members of this complex such as Gata1, Fli1 and Runx1, mutations of Scl have not been observed as a cause of inherited thrombocytopenia. We postulated that functional redundancy with its closely related family member, Lymphoblastic Leukemia 1 (Lyl1) might explain this observation. To determine if Lyl1 can substitute for Scl in megakaryopoiesis, we examined the platelet phenotype of mice lacking one or both factors in megakaryocytes. Conditional Scl knockout mice crossed with transgenic mice expressing Cre recombinase under the control of the mouse platelet factor 4 (Pf4) promoter generated megakaryocytes with markedly reduced but not absent Scl. These Pf4SclcKO mice had mild thrombocytopenia and subtle defects in platelet aggregation. However, Pf4SclcKO mice generated on a Lyl1-null background (double knockout, DKO mice) had severe macrothrombocytopenia, abnormal megakaryocyte morphology, defective pro-platelet formation and markedly impaired platelet aggregation. DKO megakaryocytes, but not single knockouts, had reduced expression of Gata1, Fli1, Nfe2 and many other genes that cause inherited thrombocytopenia. These gene expression changes were significantly associated with shared Scl and Lyl1 E-box binding sites that were also enriched for Gata1, Ets and Runx1 motifs. Thus, Scl and Lyl1 share functional roles in platelet production and function by regulating expression of partner proteins including Gata1 and Fli1. We propose that this functional redundancy provides one explanation for the absence of Scl and Lyl1 mutations as a cause of inherited thrombocytopenia.

2020-12-21 | GSE125025 | GEO

A Critical Role of RUNX1 in Governing Megakaryocyte-Primed Hematopoietic Stem Cell Differentiation [RNA-seq]

Project description:RUNX1 is crucial for multiple stages of hematopoiesis and its mutation can cause familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML). We aim to study the role of RUNX1 in megakaryocyte-biased HSCs differentiation to megakaryocytes. Here, by using Runx1F/FMx1-Cre mouse model, we sorted CD41pos HSCs and CD41neg HSCs in both RUNX1 WT and KO, and compared their gene expression profiles.

2023-01-20 | GSE211935 | GEO

A Critical Role of RUNX1 in Governing Megakaryocyte-Primed Hematopoietic Stem Cell Differentiation [CUT&RUN Chip-seq]

Project description:RUNX1 is crucial for multiple stages of hematopoiesis and its mutation can cause familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML). We aim to study the role of RUNX1 in megakaryocyte-biased HSCs differentiation to megakaryocytes. Here, by using Runx1F/FMx1-Cre mouse model ,we sorted CD41pos HSCs and CD41neg HSCs in both RUNX1 WT and KO, and tested the RUNX1 direct binding targets in these cells genome.

2023-01-20 | GSE212001 | GEO

RNA-Seq Analysis of Gfi1b KO Megakaryocytes

Project description:Megakaryocytes isolated from Gfi1b flox/flox mice carrying PF4-Cre or not, and from Gfi1b flox/flox mice carrying ROSA-Cre-ERT with or without tamoxifen injection were analyzed for differential expression by RNA-Seq

2017-01-21 | GSE71310 | GEO

Transcription profiling by array of mouse bone marrow lineage- c-Kit+ Sca-1+ cells in which floxed Mpl alleles were specifically and homozygously deleted in megakaryocytes and platelets

Project description:Thrombopoietin (TPO) acting via its receptor Mpl is the major cytokine regulator of platelet number. To precisely define the role of specific hematopoietic cells in TPO dependent hematopoiesis, we generated mice that express the Mpl receptor normally on stem/progenitor cells but lack expression on megakaryocytes and platelets (MplPF4cre/PF4cre). MplPF4cre/PF4cre mice displayed profound megakaryocytosis and thrombocytosis with a remarkable expansion of megakaryocyte-committed and multipotential progenitor cells, the latter displaying biological responses and a gene expression signature indicative of chronic TPO over-stimulation as the underlying causative mechanism, despite a normal circulating TPO level. Thus, TPO signaling in megakaryocytes is dispensable for platelet production; its key role in control of platelet number is via generation and stimulation of the bipotential megakaryocyte precursors. Nevertheless, Mpl expression on megakaryocytes and platelets is essential to prevent megakaryocytosis and myeloproliferation by restricting the amount of TPO available to stimulate the production of megakaryocytes from the progenitor cell pool.

2014-06-10 | E-MTAB-2389 | biostudies-arrayexpress

Sp1/Sp3 transcription factors regulate hallmarks of megakaryocyte maturation, and platelet formation and function

Project description:Sp1 and Sp3 belong to the Specificity proteins (Sp)/Krüppel-like transcription factor family. They are closely related, ubiquitously expressed and recognize G-rich DNA motifs. They are thought to regulate generic processes such as cell cycle and growth control, metabolic pathways and apoptosis. Ablation of Sp1 or Sp3 in mice is lethal, and combined haploinsufficiency results in hematopoietic defects during the fetal stages. Here, we show that in adult mice conditional ablation of either Sp1 or Sp3 has minimal impact on hematopoiesis, while the simultaneous loss of Sp1 and Sp3 results in severe macrothrombocytopenia and platelet dysfunction. We employed flow cytometry, cell culture and electron microscopy and show that although megakaryocyte numbers are normal in bone marrow and spleen, they display a less compact demarcation membrane system and a striking inability to form proplatelets. Through megakaryocyte transcriptomics and platelet proteomics we identified several cytoskeleton-related proteins and downstream effector kinases, including Mylk, that were downregulated upon Sp1/Sp3 depletion, providing an explanation for the observed defects in megakaryopoiesis. We show that Mylk is required for proplatelet formation and stabilization and for ITAM-receptor mediated platelet aggregation. Our data highlights the specific vs generic role of these ubiquitous transcription factors in the highly specialized megakaryocytic lineage. Megakaryocyte mRNA profiles of Sp1fl/fl::Sp3fl/fl (WTlox) and Pf4-Cre::Sp1fl/fl::Sp3fl/fl (dKO) mice were generated by deep sequencing, in triplicate.

2014-12-18 | E-GEOD-58707 | biostudies-arrayexpress

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