Comment on Kinross et al, page 886

Lack of E2f4 causes macrocytic fetal anemia, which is due to the requirement of E2f4 for the proliferation of erythroid progenitors during differentiation.

Differentiation, in general, is associated with cell-cycle arrest. In the erythroid lineage, however, terminal differentiation is coupled to proliferation, and 2 distinct cell-cycle mechanisms can be discerned in erythroid cells. When expansion of the erythroid compartment is required during fetal erythropoiesis or following hypoxic stress, erythroid progenitors can undergo “renewal divisions” (ie, divisions that double the number of erythroid progenitors while maintaining the differentiation potential). In addition, differentiation of progenitors to mature, enucleated, and hemoglobinized erythrocytes requires 3 to 4 “differentiation divisions.” During the differentiation divisions, the cell size is reduced from a 12-μm proerythroblast to a 4-μm erythrocyte (mouse).

Expansion and differentiation of the erythroid compartment can be recapitulated in vitro when erythroid progenitors undergo renewal divisions in the presence of erythropoietin (Epo), stem cell factor (SCF), and glucocorticoids, whereas they can be induced to synchronous differentiation in the presence of Epo and insulin.1  Renewal and differentiation divisions can be sharply discriminated in this system. Renewal divisions conform to standard cell cycle characteristics including size control. The most striking change in the cell cycle that immediately follows induction of differentiation is a reduction of the G1 phase from 11 hours in renewal to 5 hours during differentiation accompanied by loss of size control.1,2 

In this issue of Blood, Kinross and colleagues used this erythroid cell culture system to examine why E2f4 deficiency causes macrocytic fetal anemia. Of interest, E2f4-deficient erythroid progenitors undergo normal renewal divisions. It is only when differentiation is induced that E2f4–/– erythroblasts fail to undergo differentiation divisions. The differentiation program per se is not affected, because E2f4–/– erythroblasts down-regulated early markers, up-regulated late markers of erythroid cells, and synthesized hemoglobin as efficiently as control cells. A specific role of E2f4 in the differentiation-type cell cycle resulting in size reduction may explain why Kinross and colleagues do not observe E2f4 expression in yolk sac–derived erythroid cells that do not undergo size reduction. Because renewal divisions of E2f4–/– erythroid progenitors are not affected, the loss of expansion during differentiation can be compensated by an increase in the progenitor population, which explains why anemia occurs only when maximal expansion of the erythroid compartment is required (ie, in the fetal liver).

Although E2f4 is generally known to be a transcriptional repressor, target gene identification in differentiating erythroblasts showed that E2f4 directly activates expression of several cell cycle genes such as Ccna2, Mcm2, and Emi1. This finding creates an opportunity to start to understand how differentiation divisions are regulated differently from a renewal-type cell cycle.

Adult E2f4–/– animals do not suffer from anemia, but peripheral erythrocytes are macrocytic, suggesting that differentiation divisions with their short G1 phase and loss of size control are functionally required to produce properly sized erythrocytes. Although size control is intimately linked to cell-cycle regulation, its regulation is hardly understood.3  Induction of differentiation (ie, the switch from a renewal to a differentiation type of cell cycle) results in a rapid change in cell-cycle regulators, most notably down-regulation of cyclin D1,4  suggesting that renewal divisions use cyclin D1, whereas differentiation divisions may use cyclin D2 and/or D3. This is supported by the observation that mice lacking cyclin D2 and D3, or mice lacking cdk4/cdk6, similarly suffer from macrocytic fetal anemia,5,6  whereas only the macrocytic phenotype remains after birth.

Kinross and colleagues report slow S phase progression and accumulation of 4n cells in G2/M in E2f4–/– erythroid cells, which seems in contrast to a role of E2f4 in a switch of cell cycle that involves shortening of the G1 phase. In yeast, important cell size decisions are taken in G2; in mammalian cells, cell size control seems to be regulated throughout the cell cycle.3  The differentiation-type cell cycle with its reduced G1 phase may have lost size control at START, the point in G1 where cell size is supposed to determine whether S phase can be initiated, or START is deliberately placed much earlier in G1, which may be determined before G1 (ie, during G2). Future work along the line set out by Kinross and colleagues may clarify some of these issues. ▪

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