The c-Myb transcription factor is highly expressed in immature hematopoietic cells and down-regulated during
differentiation. c-myb is essential for the hematopoietic development, as c-myb-/- mice die at E15 due to failure of fetal
hepatic erythropoiesis. To gain further insights into the role of c-myb during the hematopoietic lineage commitment, we
studied the effects of c-Myb silencing in human CD34+ hematopoietic stem/progenitor cells. c-Myb silencing in CD34+
cells was performed by transfection of siRNAs using the Amaxa Nucleofector® Technology. In order to keep c-Myb
expression silenced for all the commitment phase of CD34+ cells, each sample was nucleofected 3 times, once a day.
Moreover, to exclude non-specific effects of siRNA nucleofection, for each experiment, together with the sample
transfected with the siRNAs targeting c-Myb, one sample electroporated without siRNAs and one transfected with a
non-targeting siRNA were performed. c-Myb silencing effects on CD34+ cells differentiation ability were studied by
methylcellulose and collagen-based clonogenic assays and by morphological and immunophenotypic analyses after
liquid culture. Furthermore, we investigated by microarray analysis the changes in gene expression induced by c-Myb
silencing. Methylcellulose assay revealed a remarkable increase of the percentage of monocyte (CFU-M) colonies and a
decrease of the erythroid ones (BFU-E) in c-Myb-silenced CD34+ cells. Moreover, collagen-based clonogenic assay
demonstrated that c-Myb silencing strongly enhances the megakaryocyte commitment of CD34+ cells. In agreement
with these data, flow cytometric analysis showed an increase in mono-macrophage and megakaryocyte fractions in cmyb-silenced
cells, while the erythroid population was strongly decreased. Morphological evaluation of May
Grunwald-Giemsa stained cytospins further supported the conclusion that c-myb silencing forces the CD34+ cells
commitment towards the macrophage and megakaryocyte lineages at the expense of the erythroid one. Gene expression
profiling of c-Myb silenced CD34+ cells enabled us to identify new 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. Functional rescue experiments showed that the
retroviral vector-mediated overexpression of KLF1 and LMO2 transcription factors in c-Myb silenced cells is able to
rescue, at least in part, the impaired erythroid differentiation. 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. In particular, we identified c-Myb-driven KLF1 and LMO2 transactivation
as the molecular mechanism through which c-Myb regulates erythroid versus megakaryocyte lineage fate decision
