In Cell, Reis and Edgar shed light on the regulation of cell-cycle timing by showing that the E2F1 transcription factor can modulate cell-cycle-phase length in Drosophila melanogaster wing-disc cells.

The regulation of cell-cycle-control proteins has been studied extensively, but how the rate of progression through cell-cycle phases is coordinated to control overall cell-cycle length is poorly understood. Overexpression of the cell-cycle regulators Cyclin E (CycE) and Cdc25 (known as String (Stg) in D. melanogaster) shortens G1 and G2 phases, respectively. However, the overall rate of cell division is maintained by making other cell-cycle phases longer. If an active mechanism that monitors, and maintains, the overall duration of the cell cycle is responsible for this regulation, then experimentally lengthening cell-cycle phases would similarly result in the maintenance, rather than the elongation, of the rate of cell division.

The authors lengthened G1 phase in larval tissues by overexpressing the CycE–Cdk2 inhibitor dacapo ( dap ; the D. melanogaster homologue of p21CIP1 and p27KIP1). Analysis of wing-disc cells showed no overall lengthening of the cell cycle due to a compensatory shortening of the subsequent S and G2 phases. Transcription of stg, which is the rate-limiting factor for the G2–M transition, was upregulated in the dap-overexpressing cells, which explained the increase in the rate of G2–M progression. stg transcription is affected by E2F1 activity and other E2F1 target genes, ribonucleotide reductase-2 ( rnr2 ) and cycE, were also upregulated in these cells. Immunostaining of these cells showed increased amounts of E2F1 protein, which correlated with the upregulation of E2F1 target genes.

Next, the authors overexpressed wee1, which lengthens G2 phase by inhibiting Cdk1. mRNA and protein levels of the E2F1 targets stg, rnr2 and cycE were increased in these cells, which, again, correlated with increased amounts of E2F1 protein. The overall cell-cycle length was maintained by shortening G1 phase, an effect that is attributable to the increased amounts of CycE. Similar results were observed in cells in which G2 phase was lengthened by overexpressing another cell-cycle regulator, Tribbles. So, a reduction in Cdk activity extends a specific gap phase, increases E2F1 levels and results in the compensatory truncation of the complementary gap phase.

Manipulating Cdk activity by overexpressing activators or inhibitors of Cdks confirmed that Cdk activity regulates the levels of the E2F1 protein. Double labelling of wing-disc cells for E2F1 protein and for markers that are specific for different cell-cycle phases also showed that the level of E2F1 oscillates with the cell cycle — it is present in G1, G2 and M phases but absent in S phase. However, the cell-cycle-specific distribution of E2F1 was not affected by Cdk activity. Furthermore, when e2f1 was artificially expressed throughout the cell cycle, the E2F1 protein was still absent during S phase, which indicates that E2F1 protein is removed from S-phase cells.

Next, the authors manipulated the cell cycle in cells that lacked E2F1 activity due to a mutation in DP, which is an essential coactivator of E2F1. They found that E2F1 activity is required for stg, rnr2 and cycE gene upregulation in Cdk2-inhibited cells and cycle compensation in Cdk1- and Cdk2-inhibited cells. Moreover, the overexpression of proteins that promote cell growth (such as Rheb or Myc) normally accelerates the G1–S transition and elongates G2 phase, but in DP-mutant cells the effect was much less pronounced. These cells also had high rates of apoptosis, which indicates that, in the absence of E2F1 activity, cells cannot tolerate changes in cell-cycle phasing that are induced by physiological growth promoters.

The authors conclude that “...in Drosophila, E2F1 is a central component of a regulatory circuit that allows cells to sustain alterations in the lengths of individual cell cycle phases without compromising overall cell cycle timing.” And, they propose that the phosphorylation of E2F1 by Cdks might be responsible for targeting E2F1 for protein degradation.