Abstract
Hematopoietic development involves the coordinated activity of differentiation and cell cycle regulators. In current models of mammalian cell cycle control, E2f activators (E2f1, E2f2, and E2f3) are portrayed as the ultimate transcriptional effectors that commit cells to enter and progress through S phase. Using conditional gene knock-out strategies, we show that E2f1-3 are not required for the proliferation of early myeloid progenitors. Rather, these E2fs are critical for cell survival and proliferation at two distinct steps of myeloid development. First, E2f1-3 are required as transcriptional repressors for the survival of CD11b(+) myeloid progenitors, and then they are required as activators for the proliferation of CD11b(+) macrophages. In bone marrow macrophages, we show that E2f1-3 respond to CSF1-Myc mitogenic signals and serve to activate E2f target genes and promote their proliferation. Together, these findings expose dual functions for E2f1-3 at distinct stages of myeloid development in vivo, first as repressors in cell survival and then as activators in cell proliferation. In summary, this work places E2f1-3 in a specific signaling cascade that is critical for myeloid development in vivo.
Highlights
Molecular events regulating hematopoiesis involve coordinated expression of components that regulate the cell cycle [1]
We show that E2f1–3 respond to CSF1-Myc mitogenic signals and serve to activate E2f target genes and promote their proliferation
E2f1–3 Are Essential for Myeloid Cell Survival and Development—To explore the potential role for the E2f1–3 transcription factors in hematopoietic development, we first examined their expression in the bone marrow of wild type mice
Summary
Mice—E2f1Ϫ/Ϫ, E2f2Ϫ/Ϫ, and c-Mycf/f mice were a gift from Michael Greenberg, Stuart Orkins, and Andreas Trumpp, respectively. To determine the expression of E2fs in different hematopoietic lineages within the bone marrow, cells from bone marrow were isolated and stained with lineage-specific antibodies (Cd11b, Ter119 and B220) and sorted using FACS. Cells were stimulated by the addition of RPMI supplemented with 50 ng/ml recombinant human CSF-1 and harvested at the indicated time points. Cells were stimulated to proliferate by the addition of DMEM supplemented with 15% FBS or 100 ng/ml recombinant human CSF-1 and harvested at the indicated time points. For BrdU incorporation assays, serumor CSF-1-stimulated cells were incubated with 50 M BrdU for the indicated time and subsequently fixed with methanol and acetic acid (1:1). Promoter Luciferase Assays—NIH-3T3 cells expressing the wild type CSF-1R, mutant CSR-IR [809] receptors, and c-Mycf/f cells were transfected with the E2f3a and E2f3b luciferase expression vectors, together with Renilla as an internal control, as described previously [32]. Cells were harvested at various times after stimulation, and luciferase activity was measured using a luminometer
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