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      A Key Commitment Step in Erythropoiesis Is Synchronized with the Cell Cycle Clock through Mutual Inhibition between PU.1 and S-Phase Progression


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          During red blood cell development, differentiation and cell cycle progression are intimately and uniquely linked through interdependent mechanisms involving the erythroid transcriptional suppressor PU.1 and the cyclin-dependent kinase inhibitor p57 KIP2.


          Hematopoietic progenitors undergo differentiation while navigating several cell division cycles, but it is unknown whether these two processes are coupled. We addressed this question by studying erythropoiesis in mouse fetal liver in vivo. We found that the initial upregulation of cell surface CD71 identifies developmentally matched erythroblasts that are tightly synchronized in S-phase. We show that DNA replication within this but not subsequent cycles is required for a differentiation switch comprising rapid and simultaneous committal transitions whose precise timing was previously unknown. These include the onset of erythropoietin dependence, activation of the erythroid master transcriptional regulator GATA-1, and a switch to an active chromatin conformation at the β-globin locus. Specifically, S-phase progression is required for the formation of DNase I hypersensitive sites and for DNA demethylation at this locus. Mechanistically, we show that S-phase progression during this key committal step is dependent on downregulation of the cyclin-dependent kinase p57 KIP2 and in turn causes the downregulation of PU.1, an antagonist of GATA-1 function. These findings therefore highlight a novel role for a cyclin-dependent kinase inhibitor in differentiation, distinct to their known function in cell cycle exit. Furthermore, we show that a novel, mutual inhibition between PU.1 expression and S-phase progression provides a “synchromesh” mechanism that “locks” the erythroid differentiation program to the cell cycle clock, ensuring precise coordination of critical differentiation events.

          Author Summary

          Hematopoietic progenitors that give rise to mature blood cell types execute simultaneous programs of differentiation and proliferation. One well-established link between the cell cycle and differentiation programs takes place at the end of terminal differentiation, when cell cycle exit is brought about by the induction of cyclin -dependent kinase inhibitors. It is unknown, however, whether the cell cycle and differentiation programs are coordinated prior to cell cycle exit. Here, we identify a novel and unique link between the cell cycle clock and the erythroid (red blood cell) differentiation program that takes place several cell division cycles prior to cell cycle exit. It differs from the established link in several respects. First, it takes place at the onset, rather than at the end, of erythroid terminal differentiation, preceding the chromatin changes that enable induction of red cell genes. Second, it is initiated by the suppression, rather than the induction, of a cyclin -dependent kinase inhibitor. It therefore causes the cell to enter S-phase, rather than exit the cell cycle. Specifically, we found that there is an absolute interdependence between S-phase progression at this time in differentiation, and a key commitment step in which, within a short few hours, cells become dependent on the hormone erythropoietin, undergo activating changes in chromatin of red cell genes, and activate GATA-1, the erythroid master transcriptional regulator. Arresting S-phase progression at this time prevents execution of this commitment step and subsequent induction of red cell genes; conversely, arresting differentiation prevents S-phase progression. However, once cells have undergone this key commitment step, there is no longer an interdependence between S-phase progression and the induction of erythroid genes. We identified two regulators that control a “synchromesh” mechanism ensuring the precise locking of the cell cycle clock to the erythroid differentiation program during this key commitment step.

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          Most cited references57

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          Multilineage gene expression precedes commitment in the hemopoietic system.

          We have tested the hypothesis that multipotential hemopoietic stem and progenitor cells prime several different lineage-affiliated programs of gene activity prior to unilineage commitment and differentiation. Using single cell RT-PCR we show that erythroid (beta-globin) and myeloid (myeloperoxidase) gene expression programs can be initiated by the same cell prior to exclusive commitment to the erythroid or granulocytic lineages. Furthermore, the multipotential state is characterized by the coexpression of several lineage-affiliated cytokine receptors. These data support a model of hemopoietic lineage specification in which unilineage commitment is prefaced by a "promiscuous" phase of multilineage locus activation.
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            Nucleosome destabilization in the epigenetic regulation of gene expression.

            Assembly, mobilization and disassembly of nucleosomes can influence the regulation of gene expression and other processes that act on eukaryotic DNA. Distinct nucleosome-assembly pathways deposit dimeric subunits behind the replication fork or at sites of active processes that mobilize pre-existing nucleosomes. Replication-coupled nucleosome assembly appears to be the default process that maintains silent chromatin, counteracted by active processes that destabilize nucleosomes. Nucleosome stability is regulated by the combined effects of nucleosome-positioning sequences, histone chaperones, ATP-dependent nucleosome remodellers, post-translational modifications and histone variants. Recent studies suggest that histone turnover helps to maintain continuous access to sequence-specific DNA-binding proteins that regulate epigenetic inheritance, providing a dynamic alternative to histone-marking models for the propagation of active chromatin.
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              Discovering hematopoietic mechanisms through genome-wide analysis of GATA factor chromatin occupancy.

              GATA factors interact with simple DNA motifs (WGATAR) to regulate critical processes, including hematopoiesis, but very few WGATAR motifs are occupied in genomes. Given the rudimentary knowledge of mechanisms underlying this restriction and how GATA factors establish genetic networks, we used ChIP-seq to define GATA-1 and GATA-2 occupancy genome-wide in erythroid cells. Coupled with genetic complementation analysis and transcriptional profiling, these studies revealed a rich collection of targets containing a characteristic binding motif of greater complexity than WGATAR. GATA factors occupied loci encoding multiple components of the Scl/TAL1 complex, a master regulator of hematopoiesis and leukemogenic target. Mechanistic analyses provided evidence for crossregulatory and autoregulatory interactions among components of this complex, including GATA-2 induction of the hematopoietic corepressor ETO-2 and an ETO-2-negative autoregulatory loop. These results establish fundamental principles underlying GATA factor mechanisms in chromatin and illustrate a complex network of considerable importance for the control of hematopoiesis.

                Author and article information

                Role: Academic Editor
                PLoS Biol
                PLoS Biology
                Public Library of Science (San Francisco, USA )
                September 2010
                September 2010
                21 September 2010
                : 8
                : 9
                : e1000484
                [1 ]Department of Pediatrics and Department of Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America
                [2 ]Department of Reproduction and Development, Erasmus MC, University Medical Center, Rotterdam, The Netherlands
                Baylor College of Medicine, United States of America
                Author notes

                The author(s) have made the following declarations about their contributions: Conceived and designed the experiments: RP JRS QS YL KH MS. Performed the experiments: RP JRS QS YL KH MS. Analyzed the data: RP JRS QS YL KH MS. Wrote the paper: RP MS. Contributed to figure design: MK. Contributed to the timing of replication experiments: JG.

                Pop et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                : 3 March 2010
                : 4 August 2010
                Page count
                Pages: 19
                Research Article
                Developmental Biology
                Developmental Biology/Cell Differentiation
                Developmental Biology/Developmental Molecular Mechanisms

                Life sciences
                Life sciences


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