The erythroid phenotype is acquired through gene induction and morphological maturation in the space of 3 to 5 'differentiation divisions'. We recently uncovered a fundamental organizational feature of fetal liver erythropoiesis, whereby a novel S phase-dependent switch controls the transition from self-renewal to differentiation divisions (Pop et al., PLoS Biology 2010). Further, this switch also triggers an unusual process of genome-wide DNA demethylation, the first known example of such a process in somatic cells (Shearstone et al., Science 2011). The S phase-dependent switch takes place at the transition from flow-cytometric "subset 0" (S0, Lin-CD71med/low) to "subset 1" (S1, Lin-CD71high) in the murine fetal liver. It comprises several erythroid commitment events, including the onset of Epo dependence, activation of the erythroid master transcriptional regulator GATA-1, and an activating chromatin reconfiguration at the beta-globin locus-control region. These events take place synchronously, during early S phase of the last generation of colony-forming-unit erythroid (CFU-e) progenitors, and are dependent on S phase progression. The S phase-dependent switch at the S0/S1 transition represents a novel, pivotal interaction between the cell cycle and differentiation programs, distinct from the well-established interaction between terminal maturation and cell-cycle exit. The mechanisms underlying the switch and the process of global DNA demethylation that it triggers are largely unknown. We identified PU.1, a transcriptional repressor of erythropoiesis, as a central regulator of this switch. We propose that PU.1 coordinates the synchronous cell cycle and differentiation events at the S0/S1 transition, through novel, antagonistic interactions between PU.1 and S phase progression. Here we propose that the tumor suppressor protein pRb, and its family members, p107 and p130, mediate the antagonistic interactions between PU.1 and S phase, and hence act as gatekeepers of the S0/S1 erythroid commitment switch. We will investigate this hypothesis with the following two aims: 1) Determine whether the S phase-dependent switch at the S0/S1 transition is dysregulated in mice deleted for one, two or three Rb family proteins or in PU.1-null mice. We will determine whether PU.1 is able to exert its dual inhibitory functions on S phase and on erythroid differentiation in mice conditionally-deleted for one, two or three of the Rb family proteins pRb, p107 or p130. Further, we will determine whether the S0/S1 transition is accelerated or becomes S phase-independent in mice deleted for PU.1 or for Rb family proteins. 2) Determine whether PU.1 and/or Rb family members interact with DNMTs and regulate global DNA methylation at the S0/S1 transition. We will ask whether global or erythroid-specific DNA demethylation takes place prematurely in S0 cells of mice deleted for PU.1 or for Rb family proteins, and whether DNMTs are directly associated with PU.1 at gene promoters. This work has the potential to uncover fundamental mechanisms of Rb and PU.1 regulation of cell cycle and differentiation, relevant to leukemia and to anemia.
During the formation of blood cells, undifferentiated blood cell progenitors undergo two processes apparently in parallel: cell division cycles, giving rise to a large number of differentiated progeny;and a differentiation program, in which changes in signaling proteins, chromatin regulators and transcription factors bring about the expression of genes that are specific to only one blood cell type. It is not known how the differentiation program and the cell division program are coordinated during this process. Very recently, our laboratory has discovered a specific step in red cell formation, in which the cell division cycle and the differentiation programs are linked: neither can proceed without the other. Our hypothesis is that this is a key step in differentiation;and that it is likely to be dysregulationin carcinogenesis, where the coordination between cell division and differentiation is disrupted. The aim of this proposal is to investigate the molecular mechanism of this novel link between cell division and differentiation.