Endoreplication

Abstract

Developmentally programmed polyploidy occurs by at least four different mechanisms, two of which (endoreduplication and endomitosis) involve switching from mitotic cell cycles to endocycles by the selective loss of mitotic cyclin-dependent kinase (CDK) activity and bypassing many of the processes of mitosis. Here we review the mechanisms of endoreplication, focusing on recent results from Drosophila and mice.

Figure 1.

Sustaining endocycles in Drosophila salivary glands. (A) Schematic of a larval salivary gland with attached fat body. Both salivary glands and fat bodies undergo endocycles resulting in cells with DNA contents of ∼1350C and ∼256C, respectively, whereas the diploid cells of the imaginal ring proliferate mitotically. (B) Diagram of the regulatory network proposed to control salivary gland endocycles. The counterplay of E2F1 and its antagonist CRL4·Cdt2 ensures that CycE activity peaks in late G phase. This initiates DNA replication and inhibits the APC, so that Geminin (Gem) can accumulate and prevent relicensing of replication origins during S phase. (C) Endocycle oscillations in wild-type salivary glands predicted by computational modeling. Peaks of E2F1 protein were always followed by increases in CycE concentration that subsequently led to Geminin accumulation.

Figure 2.

Sustaining endocycles in mouse TG cells. Oscillation of APC activity and the levels of CDK-specific inhibitors and Geminin (Gmnn) are inversely related to oscillation of cyclin E (broken red line). APC activity and CDK inhibitor levels are high in G phases but low in S phases, whereas cyclin E is low in G phases but high in S phases. CcnE·Cdk2 activity is required to begin S phase. Endocycles result from a sequence of feedback loops, resulting from phosphorylation events by CcnE·Cdk2 and ubiquitination events by CRL1 and the APC. Ubiquitination by CRL1 requires CDK-dependent phosphorylation of its substrate. These events inhibit the activity of their protein targets and cause them to be degraded by the 26S proteasome (→). Protein symbols are those for mammals.

Figure 3.

Initiation of endoreplication in Drosophila follicle cells. (A) The follicle cells that encapsulate the ovary originate from stem cells and undergo three different cell cycle variants during their differentiation. Up to stage 6, follicle cells undergo normal mitotic divisions resulting in a final number of approximately 650 follicle cells. During stages 7–10A, follicle cells execute three endocycles. At stage 10B, follicle cells switch from endoreplication to gene amplification, in which specific loci become amplified by repeated origin activation. (B) At stages 6–7, up-regulation of the Notch ligand Delta (Dl) in germline cells activates the Notch pathway in follicle cells, which results in inhibition of Stg/Cdc25 and activation of the transcription factor Hnt. Activation of Hnt prevents the transcriptional repressor Cut from inhibiting APC^Fzr, thereby promoting proteasomal degradation of mitotic cyclins and Stg/Cdc25. Down-regulation of Stg/Cdc25 arrests the cell in G₂ phase, whereas the subsequent degradation of mitotic cyclins suppresses the initiation of mitosis.

Figure 4.

Initiation of endoreplication in the mouse trophectoderm lineage. Chk1 is the effector kinase for two checkpoints. The “DNA replication checkpoint” prevents premature mitosis in response to DNA damage and stalled replication forks. These events activate (+) the ATR kinase that activates the Chk1 kinase that inhibits (⊥) the CDC25 phosphatase through site-specific phosphorylation. Because CDC25 is required to activate Cdk1, inhibition of CDC25 prevents entrance into mitosis. The “G₂ restriction point” prevents expression of p57 and p21 in response to mitogen stimulation. In the case of TS cells, the mitogen is FGF4. In the presence of FGF4, TS cells express the p21 and p57 genes, but Chk1 phosphorylates the p21 and p57 proteins, thereby targeting them for degradation (Ø) by the 26S proteasome. This prevents TS cells from exiting their mitotic cell cycle. In the absence of FGF4, Chk1 protein is degraded, thereby up-regulating p57 and p21. P21 may constitute a feedback loop that sustains suppression of Chk1 expression in TG cells (Gottifredi et al. 2001). Inhibition of Cdk1 by p57 triggers endoreduplication and differentiation into TG cells. P21 could facilitate this event by down-regulating Emi1, a specific inhibitor of the APC (Lee et al. 2009b). The APC targets mitotic cyclins and Geminin for degradation, thereby promoting origin licensing. The absence of Chk1 in TG cells allows p57 protein levels to oscillate. As CcnE levels increase, Cdk2·CcnE will eventually phosphorylate p57 at a CDK-specific site, thereby targeting it for degradation.