Nature 359:295C300

Nature 359:295C300. E2F2 for knockout (KO) embryos are anemic (6,C8), a defect that can be suppressed by a functionally normal placenta (9, 10). In addition, inactivation of specifically in hematopoietic stem cells (HSC) or the erythroid lineage leads to mild anemia and mild splenomegaly Dinoprost tromethamine (11,C14). Interestingly, while the role of Rb in the control of postnatal erythropoiesis is cell autonomous (12, 13), Rb appears to elicit both cell-autonomous and non-cell-autonomous signals to maintain normal erythropoiesis during embryogenesis (10, 15, 16). These data suggest that Rb may make different contributions to embryonic erythropoiesis and postnatal erythropoiesis. It is largely accepted that Rb exerts its function mainly through its interactions with the E2F family of transcription factors (4, 5, 17,C20). In mammalian cells, there are eight genes (to locus encoding two isoforms, E2F3a and E2F3b (4, 5, 17,C20). Based on their structural domains and Dinoprost tromethamine their impact on gene transcription, E2Fs can be broadly divided into two groups (18). The activator group, consisting of E2F1, E2F2, and E2F3, transcriptionally activates E2F target genes during the G1/S transition of the cell cycle when they are released from Rb binding and inhibition. On the other hand, members of the repressor group transcriptionally repress E2F target genes in quiescent or terminally differentiated cells. Based on their structural domains, the repressor group can be further divided into two subclasses, canonical repressors (E2F4, E2F5, and E2F6) and atypical repressors (E2F7 and E2F8). While transcriptional repression mediated by E2F4 and E2F5 depends on their binding to the Rb pocket protein and the other two pocket proteins, p107 and p130, E2F6-, E2F7-, and E2F8-mediated PJS repression is thought to be pocket protein independent, as none of them contain the consensus pocket-protein-binding domain. Although E2F6 has been shown to exert its repressor function through a polycomb repressor complex (21), it is unclear how E2F7 and E2F8 impose transcriptional repression. Consistent with the intimate interactions between Rb and Rb-pocket-protein-binding E2Fs (i.e., E2F1 to E2F5), numerous studies using mouse models have shown that E2Fs, particularly activator E2Fs, are important mediators for Rb function in the nervous system, lenses, placentae, and fetal livers (FL) (16, 22,C29). However, whether non-pocket-protein-binding E2Fs, namely, E2F6, E2F7, and E2F8, Dinoprost tromethamine can also mediate Rb function is largely unknown. We recently uncovered a surprising functional interaction between Rb and E2F8 in the erythroid lineage (12). Specifically, while the inactivation of or in HSC or the erythroid lineage led to mild erythropoietic defects, the concomitant inactivation of both genes synergized to trigger severe anemia, which is characterized by profound ineffective erythropoiesis and mild hemolysis. Here we report that the concomitant ablation of and in HSC or the erythroid lineage led to a partial differentiation block at a critical stage of erythroid terminal differentiation where cells are programmed to permanently exit the cell cycle. Importantly, we also show that the loss of triggered a series of cell cycle defects that have been previously unappreciated, including stressed DNA replication and prolonged cell cycle progression. Interestingly, these defects Dinoprost tromethamine were exacerbated by the concomitant loss of but were rescued by the inactivation of bromodeoxyuridine (BrdU) incorporation assay, BrdU (Sigma) was administered through i.p. injection at a concentration of 150 g/g of body weight. Mice were sacrificed after 45 min. Single-cell suspensions prepared from BM cells were stained for erythroid staging as described above, followed by intracellular marker staining with BrdU antibodies using a BrdU-fluorescein isothiocyanate (FITC) kit (BD Biosciences) according Dinoprost tromethamine to the manufacturer’s recommendations. For H2AX and phospho-histone 3 (PH3) staining, after staining for erythroid staging, cells were fixed, permeabilized, and stained with antibodies against H2AX.

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