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How are Red Cells Formed?
We study mammalian red cell formation (erythropoiesis) as an accessible experimental system in which to address fundamental questions in biology and disease. Deficits in red cell formation are common worldwide, manifesting as anemia, myelodysplasia or myeloproliferative disease. The study of erythroid progenitors contributes to our understanding of these conditions. It also reveals fundamental mechanisms in lineage commitment and differentiation, relevant to oncogenesis and regenerative medicine
Flow-cytometric analysis of erythroid progenitors
We pioneered the use of cell-surface markers in the staging of red cell progenitors in murine blood-forming tissue (Figure 1; Socolovsky et al., Blood 2001; Zhang et al., Blood 2003; Liu et al., Blood 2006; Pop et al., PLoS Biology 2010; Koulnis et al., JoVE, 2011). This approach allows us to study minimally-perturbed primary erythroid progenitors directly within the context of their physiological niche in vivo.
Figure 1: Identification of erythroid progenitors and precursors within mouse fetal liver or other hematopoietic tissue using cell surface markers CD71 and Ter119. The cells can be isolated and analyzed by flow cytometry.
Figure 2: Fetal liver erythroid cells are subdivided into subsets S0 to S5 containing increasingly mature erythroblasts, based on their CD71/Ter119 expression.
An S-phase –dependent developmental switch activates erythroid transcription
We found that several key committment events in early erythroid progenitors are synchronized within a single, S phase-dependent developmental switch, that takes place within S phase of a single cell cycle, at the transition from the S0 to the S1 subset (Figure 2, 3). They include a switch in chromatin configuration, activation of the erythroid master transcriptional regulator GATA-1 and the onset of dependence on erythropoietin (Epo), the principal hormonal regulator of erythropoiesis. This epigenetic switch is a new example of only a handful of known S phase-dependent developmental switches in metazoa. We are investigating the precise role of S phase in these commitment events.
Figure 3: An S –phase dependent epigenetic switch at the transition from S0 to S1: Several commitment events are synchronized in a single S phase, including dramatic changes in transcription, cell cycle regulation and chromatin.
Genome-wide loss in DNA methylation during erythroid differentiation
The erythroid S phase-dependent switch (Figure 3) is associated with a dramatic increase in the rate of DNA synthesis, apparently exceeding the DNA methylation capacity of the cells and resulting in genome-wide DNA demethylation (Figure 4,5). This global loss in methylation is required for the rapid induction of a subset of erythroid genes that are massively induced during erythropoiesis, including genes required for hemoglobin synthesis. This finding provides the first instance of a genome-wide loss in DNA methylation in normal (non-cancer) somatic cells. Previously, global demethylation was thought to be confined to the pre-implantation embryo and to primordial germ cells. We are investigating the mechanisms responsible for the global loss in DNA methylation, which may aid in understanding global demethylation in other contexts, including cancer and early development.
Figure 4: Red cell precursors are unusual amongst somatic (body) cells in that they undergo genome-wide loss in DNA methylation, a process that was previously thought to be confined to pluripotent cells in the early ermbryo or in the germline. Solid black circles indicates methylated CpG dinucleotides in DNA, empty circles incidate demethylated CpGs.
Figure 5: Genome-wide loss in DNA methylation. Each datapoint represents DNA methylation level in a 5kB window, sliding across the genome. Genomic DNA from increasingly mature erythroblasts (subsets S0 to S4/5) was analyzed using Reduced Representation Bisulfite Sequencing (RRBS).
This work is described in the following publications:
1. Pop,R., Shearstone, J.R., Shen, Q., Liu, Y., Hallstrom, K., Koulnis, M., Gribnau, J., Socolovsky, M. A key commitment Step in Erythropoiesis Is synchronized with the cell cycle clock Through mutual inhibition between PU.1 and S-phase progression. PLoS Biology 2010; 8(9): e1000484
2. Shearstone, J.R., Pop, R., Bock, C., Boyle, P., Meissner, A., Socolovsky, M. Global DNA Demethylation During Erythropoiesis in vivo. Science, 2011: Vol. 334 no. 6057 pp. 799-802.
System-level investigation of erythroblast survival
The size of the erythroid progenitor pool determines erythropoietic rate. It may increase ten-fold in response to stress, a dynamic property of clinical importance whose regulation is incompletely understood.
Using a combined experimental and mathematical modeling approach we found that the death receptor Fas, and its ligand, FasL, are negative regulators of erythropoiesis in the fetus and adult. Further, signaling by Epo and its receptor, EpoR, suppresses expression of the pro-apoptotic Fas, FaL and Bim proteins, and induces the anti-apoptotic Bcl-xL. These Epo-activated survival pathways appear redundant in vitro. However, we found that they each impart unique system-level functions in vivo (Figure 6). Thus, Fas and FasL are unique in that they alone amongst these proteins exert negative autoregulation of erythroblasts within erythropoietic tissue. We showed that this autoregulatory loop stabilizes the erythroid progenitor pool, and in addition, accelerates its stress response.
By contrast, Epo-mediated induction of Bcl-xL is unique in that it undergoes classical adaptation, a dynamic response that is well known in sensory pathways or bacterial chemotaxis. Thus, the acute onset of erythropoietic stress induces a rapid but transient Bcl-xL response that quickly resets, ready to respond afresh to any further change in stress. Adaptation in the Bcl-xL pathway extends the dynamic range of the erythropoietic stress response.
Figure 6: In response to an increase in Epo during hypoxia stress, the EpoR in early erythroblasts transduces both a rapid, adapting survival signal via Bcl-xL induction, and a slower persistent survival signal through Bim, Fas and FasL suppression.
This work is described in the following publications:
1. Liu, Y., Pop, R., Sadegh, C., Brugnara, C., Haase, V.H., Socolovsky, M. Suppression of Fas-FasL co-expression by erythropoietin mediates erythroblast expansion during the erythropoietic stress-response in vivo. Blood, 2006; 108:123-133.
2. Socolovsky, M*., Murrell, M., Liu, Y., Pop, R., Porpiglia, E., and Levchenko, A*. Negative Autoregulation by Fas Mediates Robust Fetal Erytrhopoiesis. PLoS Biology, 2007; 5(10): e252. *Joint corresponding authors
This paper was highlighted in the ‘Research Roundup’ section of the Journal of Cell Biology (‘Red Blood Cells have a Killer Touch’, Robinson, R., JCB 179(2): 172 2007).
3. Socolovsky, M. Molecular Insights into Stress Erythropoiesis. Current Opinion in Hematology, 2007,14(3):215-224.
4. Koulnis, M., Liu, Y., Hallstrom K., Socolovsky. M. Negative autoregulation by Fas stabilizes adult erythropoiesis and Accelerates its Stress response. PLoS One. 2011;6(7):e21192.
5. Koulnis, M., Porpiglia, E., Porpiglia, P.A., Liu, Y., Hallstrom, K., Hidalgo, D., Socolovsky, M. Contrasting Dynamic Responses in vivo of the Bcl-xL and Bim Erythropoietic Survival Pathways. Blood, 2011 Nov 15. [Epub ahead of print]. This work will be highlighted in the Cover of Blood.