Microarray of Human primary megakaryocyte from CALR mutated patient
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Isabelle Plo
PROVIDER: E-MTAB-9048 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Ba/F3 mouse cell lines overexpressing or not different types of CALRm (type 1 (del52, del34, del46) and type 2 (del19, ins5)) were starved 6 hours from cytokines and collected. RNA were submitted to microarrays.
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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.
2021-11-02 | E-MTAB-8609 | biostudies-arrayexpress
Project description:Dnm2fl/fl Pf4-Cre (Dnm2Plt-/-) mice lacking the endocytic GTPase dynamin 2 (DNM2) in platelets and megakaryocytes (MKs) develop hallmarks of myelofibrosis. At the cellular level, the tyrosine kinase JAK2 is constitutively active but decreased in expression in Dnm2Plt-/- platelets. Additionally, Dnm2Plt-/- platelets cannot endocytose the thrombopoietin (TPO) receptor Mpl, leading to elevated circulating TPO levels. Here, we assessed whether the hyperproliferative phenotype of Dnm2Plt-/- mice was due to JAK2 constitutive activation or elevated circulating TPO levels. In unstimulated Dnm2Plt-/- platelets, STAT3 and to a lower extent STAT5 were phosphorylated, but their phosphorylation was slowed and diminished upon TPO stimulation. We further crossed Dnm2Plt-/- mice in the Mpl-/- background to generate Mpl-/- Dnm2Plt-/- mice lacking Mpl ubiquitously and DNM2 in platelets and MKs. Mpl-/- Dnm2Plt-/- platelets had severely reduced JAK2 and STAT3 but normal STAT5 expression. Mpl-/- Dnm2Plt-/- mice had severely reduced bone marrow MK and hematopoietic stem and progenitor cell numbers. Additionally, Mpl-/- Dnm2Plt-/- mice had severe erythroblast maturation defects, decreased expression of hemoglobin and heme homeostasis genes, increased expression of ribosome biogenesis and protein translation genes, and developed anemia with grossly elevated plasma erythropoietin levels, leading to early fatality by postnatal day 25. Mpl-/- Dnm2Plt+/+ mice had impaired EB development at three weeks of age, which normalized with adulthood. Together, the data shows that DNM2-dependent Mpl-mediated endocytosis in platelets and MKs is required for steady-state hematopoiesis and provides novel insights into a developmentally controlled role for Mpl in normal erythropoiesis, regulating hemoglobin and heme production.
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Project description:Myelofibrosis (MF) is caused by genetic abnormalities involving the thrombopoietin (TPO)/MPL/JAK2 axis. Furthermore MF patients have elevated serum TPO levels. MF is also associated with reduced GATA1 content in MK suggesting that this abnormality represents a phenotypic modifier. In 2014, Dr. Crispino suggested that in MF abnormal TPO signaling induces a ribosomal deficiency hampering GATA1 mRNA translation in MK. Support for MK GATA1 deficiency as “phenotypic modifier” in MF was provided by the observation that mice carrying the Gata1low mutation reducing Gata1 transcription in MK develop myelofibrosis. Since reduced RBC half-life subject these mice to continuous “erythroid stress”, we investigated the TPO/Mpl axis in this model. In Gata1low and wild-type mice, TPO mRNA was expressed by bone marrow (BM), spleen and liver. The greatest expression (by 300-fold) was detected in liver. Gata1low livers expressed TPO mRNA levels 6-fold greater than wild-type livers. TPO protein was detected in BM, spleen, liver and peritoneum washes and plasma. The greatest levels where detected in plasma. Gata1low plasma contained TPO levels 2-fold lower than wild-type plasma, but 2-times greater than plasma from bleed wild-type mice and Mplnull mice with similar thrombocytopenia, suggesting that TPO is overproduced in Gata1low mice. JAK2 and STAT5 were easily detected in Gata1low BM bur barely detectable in wild-type BM, suggesting that in the former MPL is prompt to signaling activation. Furthermore, Gata1low LSK expressed levels of Mpl mRNA 3-times greater than wild-type cells but expressed cell-surface levels of MPL 2-times lower than wild-type cells and similar to those on LSK from TPO-treated wild-type mice, suggesting that MPL is down-modulated in Gata1low LSK. The Crispino’s hypothesis that in MF activation of TPO/MPL/JAK2 induces a ribosomal deficiency hampering GATA1 mRNA translation and the realization that this axis is activated in Gata1low mice made us question the original hypothesis that reduced content of GATA1 in Gata1low MK results from deletion of lineage-specific enhancers. Microarray analyses indeed identified that Gata1low BM express a discordant ribosome signature including reduced expression of RPS24 and RPS36A, two genes mutated in Diamond Blackfan Anemia, a disease characterized by inefficient GATA1 mRNA translation. Electron microscopy identified that the cytoplasm of Gata1low MK contained poorly developed endoplasmic reticulum with rare polysomes. In conclusion, these results validate the Gata1low model as a MF model by indicating that these mice express an activated TPO/MPL axis and an abnormal ribosomal signature which may reduce efficiency of Gata1 mRNA translation.
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Project description:Myelofibrosis (MF) is caused by genetic abnormalities involving the thrombopoietin (TPO)/MPL/JAK2 axis. Furthermore MF patients have elevated serum TPO levels. MF is also associated with reduced GATA1 content in MK suggesting that this abnormality represents a phenotypic modifier. In 2014, Dr. Crispino suggested that in MF abnormal TPO signaling induces a ribosomal deficiency hampering GATA1 mRNA translation in MK. Support for MK GATA1 deficiency as “phenotypic modifier” in MF was provided by the observation that mice carrying the Gata1low mutation reducing Gata1 transcription in MK develop myelofibrosis. Since reduced RBC half-life subject these mice to continuous “erythroid stress”, we investigated the TPO/Mpl axis in this model. In Gata1low and wild-type mice, TPO mRNA was expressed by bone marrow (BM), spleen and liver. The greatest expression (by 300-fold) was detected in liver. Gata1low livers expressed TPO mRNA levels 6-fold greater than wild-type livers. TPO protein was detected in BM, spleen, liver and peritoneum washes and plasma. The greatest levels where detected in plasma. Gata1low plasma contained TPO levels 2-fold lower than wild-type plasma, but 2-times greater than plasma from bleed wild-type mice and Mplnull mice with similar thrombocytopenia, suggesting that TPO is overproduced in Gata1low mice. JAK2 and STAT5 were easily detected in Gata1low BM bur barely detectable in wild-type BM, suggesting that in the former MPL is prompt to signaling activation. Furthermore, Gata1low LSK expressed levels of Mpl mRNA 3-times greater than wild-type cells but expressed cell-surface levels of MPL 2-times lower than wild-type cells and similar to those on LSK from TPO-treated wild-type mice, suggesting that MPL is down-modulated in Gata1low LSK. The Crispino’s hypothesis that in MF activation of TPO/MPL/JAK2 induces a ribosomal deficiency hampering GATA1 mRNA translation and the realization that this axis is activated in Gata1low mice made us question the original hypothesis that reduced content of GATA1 in Gata1low MK results from deletion of lineage-specific enhancers. Microarray analyses indeed identified that Gata1low BM express a discordant ribosome signature including reduced expression of RPS24 and RPS36A, two genes mutated in Diamond Blackfan Anemia, a disease characterized by inefficient GATA1 mRNA translation. Electron microscopy identified that the cytoplasm of Gata1low MK contained poorly developed endoplasmic reticulum with rare polysomes. In conclusion, these results validate the Gata1low model as a MF model by indicating that these mice express an activated TPO/MPL axis and an abnormal ribosomal signature which may reduce efficiency of Gata1 mRNA translation.
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