c-myb supports erythropoiesis through the transactivation of KLF1 and LMO2 expression
ABSTRACT The c-myb transcription factor is highly expressed in immature hematopoietic cells and down-regulated during differentiation. To define its role during the hematopoietic lineage commitment, we silenced c-myb in human CD34(+) hematopoietic stem/progenitor cells. Noteworthy, c-myb silencing increased the commitment capacity toward the macrophage and megakaryocyte lineages, whereas erythroid differentiation was impaired, as demonstrated by clonogenic assay, morphologic and immunophenotypic data. Gene expression profiling and computational analysis of promoter regions of genes modulated in c-myb-silenced CD34(+) cells identified the transcription factors Kruppel-Like Factor 1 (KLF1) and LIM Domain Only 2 (LMO2) as putative targets, which can account for c-myb knockdown effects. Indeed, chromatin immunoprecipitation and luciferase reporter assay demonstrated that c-myb binds to KLF1 and LMO2 promoters and transactivates their expression. Consistently, the retroviral vector-mediated overexpression of either KLF1 or LMO2 partially rescued the defect in erythropoiesis caused by c-myb silencing, whereas only KLF1 was also able to repress the megakaryocyte differentiation enhanced in Myb-silenced CD34(+) cells. Our data collectively demonstrate that c-myb plays a pivotal role in human primary hematopoietic stem/progenitor cells lineage commitment, by enhancing erythropoiesis at the expense of megakaryocyte diffentiation. Indeed, we identified KLF1 and LMO2 transactivation as the molecular mechanism underlying Myb-driven erythroid versus megakaryocyte cell fate decision.
SourceAvailable from: Carmela Mannarelli[Show abstract] [Hide abstract]
ABSTRACT: Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by megakaryocyte hyperplasia, bone marrow fibrosis, and abnormal stem cell trafficking. PMF may be associated with somatic mutations in JAK2, MPL, or CALR. Previous studies have shown that abnormal megakaryocytes play a central role in the pathophysiology of PMF. In this work, we studied both gene and microRNA (miRNA) expression profiles in CD34+ cells from PMF patients. We identified several biomarkers and putative molecular targets such as FGR, LCN2, and OLFM4. By means of miRNA-gene expression integrative analysis, we found different regulatory networks involved in the dysregulation of transcriptional control and chromatin remodeling. In particular, we identified a network gathering several oncomiRs (e.g., miR-155-5p) and targeted genes whose abnormal function has been previously associated to myeloid neoplasms, including JARID2, NR4A3, CDC42, and HMGB3. Since the validation of miRNA-target interactions unveiled JARID2/miR-155-5p as the strongest relationship in the network, we studied the function of this axis in normal and PMF CD34+ cells. We showed that JARID2 downregulation mediated by miR 155 5p overexpression leads to increased in vitro formation of CD41+ megakaryocyte precursors. These findings suggest that overexpression of miR-155-5p and the resulting downregulation of JARID2 may contribute to megakaryocyte hyperplasia in PMF.Blood 08/2014; 124(13). DOI:10.1182/blood-2013-12-544197 · 9.78 Impact Factor
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ABSTRACT: Currently, bone marrow transplantation is the only curative treatment for β-thalassemia and sickle cell disease. In rare cases, sustained and full fetal hemoglobin production was observed in patients after failure of bone marrow transplantation. This rendered the patients transfusion-free, despite genetic disease and transplant rejec- tion. The mechanisms underlying this phenomenon remain unexplored. We have studied a trio (father-mother-child) in which the affected child became transfusion-independent after rejection of an allogeneic bone marrow graft. Remarkably, we found that his non-thalassemic mother also expressed unusually high levels of γ-globin. High HbF in one of the parents may therefore be of prognostic value in these rare cases. Genotyping of the HBB locus and the HbF quantitative trait loci HBS1L-MYB, KLF1 and BCL11A, and protein expression analysis of KLF1 and BCL11A, failed to explain the increased HbF levels, indicating that an as yet unidentified HbF modifier locus may be involved. We hypothesize that epigenetic events brought about by the transplantation procedure allow therapeutic levels of HbF expression in the child. Potential implications of our observations for reactivation of γ-globin expression and interpretation of the French globin gene therapy case are discussed.
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ABSTRACT: The human β-globin locus is comprised of embryonic, fetal and adult globin genes that are expressed in a developmental stage-specific manner. Mutations in the globin locus give rise to the β-globinopathies, β-thalassemia and sickle cell disease, that begin to manifest symptoms around the time of birth. Although the fetal globin genes are autonomously silenced in adult stage erythroid cells, mutations lying both within and outside of the locus lead to natural variations in the level of fetal globin gene expression, and some of these significantly ameliorate the clinical symptoms of the β-globinopathies. Multiple reports have now identified several transcription factors that are involved in fetal globin gene repression in definitive (adult) stage erythroid cells (the TR2/TR4 heterodimer, c-Myb, KLFs, BCL11A, and SOX6). To carry out their repression functions, chromatin modifying enzymes (such as DNA methyltransferase, histone deacetylases and lysine-specific histone demethylase 1) are additionally involved as a consequence of forming large macromolecular complexes with the DNA-binding subunits of these cellular machines. This review focuses on the molecular mechanisms underlying fetal globin gene silencing and possible near future molecularly-targeted therapies for treating the β-globinopathies.Molecular and Cellular Biology 07/2014; 34(19). DOI:10.1128/MCB.00714-14 · 5.04 Impact Factor