Increased lipid transport and de novo synthesis lead to dynamic lipidome remodeling and regulation of lipid-dependent kinases during megakaryopoiesis and proplatelet formation
Ontology highlight
ABSTRACT:
INSTRUMENT(S): timsTOF Pro
ORGANISM(S): Mus Musculus (mouse)
TISSUE(S): Megakaryocyte Differentiation, Megakaryocyte
SUBMITTER: 
Christopher Gerner
LAB HEAD: Christopher Gerner
PROVIDER: PXD037622 | Pride | 2023-08-22
REPOSITORIES: Pride
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Project description:The transcription factor c-Myb plays a key role in human primary CD34+ hematopoietic progenitor cells (HPCs) lineage choice, by enhancing erythropoiesis at the expense of megakaryopoiesis. We previously demonstrated that c-Myb affects erythroid versus megakaryocyte lineage decision in part by transactivating KLF1 and LMO2 expression. To further unravel the molecular mechanisms through which c-myb affects lineage fate decision, we profiled the miRNA and mRNA changes in myb-silenced CD34+ HPCs. The integrative analysis of miRNA/mRNA expression changes upon c-myb silencing in human CD34+ HPCs highlighted a set of 19 miRNA with 150 anticorrelated putative target mRNAs. Among the miRNAs downregulated in myb-silenced progenitors with the highest number of predicted target mRNAs, we selected hsa-miR-486-3p based on the in vitro effects of its overexpression on HPCs commitment. Indeed, morphological and flow cytometric analyses after liquid culture showed that hsa-miR-486-3p overexpression in HPCs enhanced erythroid and granulocyte differentiation while restraining megakaryocyte and macrophage differentiation. Moreover, collagen-based clonogenic assay demonstrated a strong impairement megakaryocyte commitment upon hsa-miR-486-3p overexpression in CD34+ cells. Moreover, in order to identify the mRNA target through which hsa-miR-486-3p affects lineage fate decision, we profiled the mRNA changes in mimic transfected CD34+ HPC by means of Affymetrix GeneAtlas U219 strip array. Gene expression profiling of hsa-miR-486-3p overexpressing CD34+ cells enabled us to identify a set of 8 genes downregulated and computationally predicted, putative hsa-miR-486-3p targets. Among them, we selected c-maf transcript as upregulated upon myb silencing. Worth of note, c-maf silencing in CD34+ progenitor cells was able to reverse the affects of myb silencing on erythroid versus megakaryocyte lineage choice. Integrative miRNA/mRNA analysis highlighted a set of miRNAs and anticorrelated putative target mRNAs modulated upon myb silencing, therefore potential players in myb-driven HPCs lineage choice. Among them, we demonstrated the hsa-miR-486-3p/c-maf pair as partially contributing to the effects of myb on HPCs commitment. Therefore, our data collectively identified myb-driven hsa-miR-486-3p upregulation and subsequent c-maf downregulation as a new molecular mechanism through which cMyb favours erythropoiesis while restraining megakaryopoiesis. Gene expression profile (GEP) was performed on total RNA derived from three independent experiments at 24h after the last nucleofection.
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Project description:The fetal to adult switch in mouse hematopoietic stem cell (HSC) behavior is characterized by entry into quiescence and alterations in lineage output. Here we identify the ability of HSCs to undergo emergency megakaryopoiesis following acute inflammatory insult as an outcome of this developmental switch. Single cell accessibility mapping of fetal and adult HSCs resolves a heterogeneous chromatin landscape consistent with a two-step megakaryocyte lineage priming process. Importantly, we demonstrate that the accumulation of megakaryocyte primed HSCs is under the control of the developmentally restricted Lin28b RNA binding protein. Persistent Lin28b protein expression in postnatal HSCs delays the formation of a primed HSC pool and limits emergency megakaryopoiesis. Taken together, we identify Lin28b as a negative regulator of a megakaryocyte primed HSC state that is enforced at the chromatin level during unperturbed organismal development. These findings highlight the ontogenically timed onset of inflammatory responsiveness, with important implications for the delicate balance between host protection and tissue damage during neonatal life.
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Project description:The fetal to adult switch in mouse hematopoietic stem cell (HSC) behavior is characterized by entry into quiescence and alterations in lineage output. Here we identify the ability of HSCs to undergo emergency megakaryopoiesis following acute inflammatory insult as an outcome of this developmental switch. Single cell accessibility mapping of fetal and adult HSCs resolves a heterogeneous chromatin landscape consistent with a two-step megakaryocyte lineage priming process. Importantly, we demonstrate that the accumulation of megakaryocyte primed HSCs is under the control of the developmentally restricted Lin28b RNA binding protein. Persistent Lin28b protein expression in postnatal HSCs delays the formation of a primed HSC pool and limits emergency megakaryopoiesis. Taken together, we identify Lin28b as a negative regulator of a megakaryocyte primed HSC state that is enforced at the chromatin level during unperturbed organismal development. These findings highlight the ontogenically timed onset of inflammatory responsiveness, with important implications for the delicate balance between host protection and tissue damage during neonatal life.
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