Atypical E2F activity restrains APC/CCCS52A2 function obligatory for endocycle onset

Abstract

The endocycle represents an alternative cell cycle that is activated in various developmental processes, including placental formation, Drosophila oogenesis, and leaf development. In endocycling cells, mitotic cell cycle exit is followed by successive doublings of the DNA content, resulting in polyploidy. The timing of endocycle onset is crucial for correct development, because polyploidization is linked with cessation of cell division and initiation of terminal differentiation. The anaphase-promoting complex/cyclosome (APC/C) activator genes CDH1, FZR, and CCS52 are known to promote endocycle onset in human, Drosophila, and Medicago species cells, respectively; however, the genetic pathways governing development-dependent APC/C(CDH1/FZR/CCS52) activity remain unknown. We report that the atypical E2F transcription factor E2Fe/DEL1 controls the expression of the CDH1/FZR orthologous CCS52A2 gene from Arabidopsis thaliana. E2Fe/DEL1 misregulation resulted in untimely CCS52A2 transcription, affecting the timing of endocycle onset. Correspondingly, ectopic CCS52A2 expression drove cells into the endocycle prematurely. Dynamic simulation illustrated that E2Fe/DEL1 accounted for the onset of the endocycle by regulating the temporal expression of CCS52A2 during the cell cycle in a development-dependent manner. Analogously, the atypical mammalian E2F7 protein was associated with the promoter of the APC/C-activating CDH1 gene, indicating that the transcriptional control of APC/C activator genes by atypical E2Fs might be evolutionarily conserved.

Fig. 1.

Exclusive transcription of E2Fe/DEL1 in nonendoreplicating dividing cells. (A) GUS activity in the shoot apical meristem, root apex, young vascular tissue, and leaf of an 8-day-old seedling. (B) Detail of shoot apical meristem and young leaf. (C) Detail of expression in the root tip.

Fig. 2.

Regulation of endocycle onset by E2Fe/DEL1 through control of CCS52A2. (A) Advanced onset of the endocycle in del1–1 plants, as demonstrated by a more rapid increase in the EI (mean number of duplication cycles; for calculation, see Materials and Methods). Values are mean ± standard error (n > 5). (B) Ploidy maps of 12-day-old abaxial epidermal cells of wild-type (Col-0) and del1–1 plants. 4′,6-Diamidino-2-phenylindole (DAPI) stains (left) were translated into color maps (right). (C) Transcript cluster positively correlated with the endoreplication phenotypes of E2Fe/DEL1^OE and del1–1 plants. Data are the average of three independent experiments. (D) Quantification of CCS52A2 expression in del1–1, del2–1, and del3–1 mutants. Transcript levels were measured by real-time PCR. All values were normalized to the ACT2 housekeeping gene. The ΔCt method was used for relative quantification of transcripts. Data are mean ± standard deviation (n = 3).

Fig. 3.

E2Fe/DEL1-dependent CCS52A2 transcription. (A) Effects of CCS52A1 and CCS52A2 knockout on the EI of mature first leaves. Values are mean ± standard deviation (SD) (n = 3). (B) CCS52A1 and CCS52A2 transcript levels in 8-day-old del1–1 seedlings. Transcript levels were measured by real-time PCR. All values were normalized to the ACT2 housekeeping gene. The ΔCt method was used for relative quantification of transcripts. Measurements were made relative to wild-type and are mean ± SD (n = 3). (C) ChIP analysis showing binding of E2F/DEL1 to the CCS52A2 promoter in vivo, but not to the CCS52A1 promoter. Data represent two independent assays. (D) ChIP scanning of the CCS52A2 promoter showing the strongest E2Fe/DEL1 association around the putative E2F cis-acting element. (E) ChIP analysis illustrating that E2Fe/DEL1 binding requires a functional E2F-binding site within the CCS52A2 promoter.

Fig. 4.

Control of development-dependent expression of CCS52A2 by E2Fe/DEL1. (A) Kinetics of E2Fe/DEL1 and CCS52A2 transcription during leaf development. Transcript levels were measured by real-time PCR. All values were normalized to the ACT2 housekeeping gene. The ΔCt method was used for relative quantification of transcripts. Values are means ± SD (n = 3). Note that transcription of CCS52A2 peaked at day 10, marking the endocycle onset. (B and C) CCS52A2 and CCS52A1 mRNA levels during leaf development in wild-type (Col-0) and del1–1 mutants, respectively. Data are mean ± SD (n = 3). (D) Ploidy maps of 12-day-old abaxial epidermal cells of wild-type (Col-0) and CCS52A2^OE plants. DAPI stains (left) were translated into color maps (right). (E) Simulation of CCS52A2 accumulation during leaf development showing a progressive increase in CCS52A2 transcript levels during the S and G₂ phases.

Fig. 5.

Evolutionarily conserved transcriptional control of APC activator genes by atypical E2F proteins. ChIP analysis on extracts prepared from U2OS cells showed that E2F7 binds the CDH1 promoter in vivo. A 10% fraction of the chromatin served as input (IN). Immunoprecipitations were carried out with an E2F7 or control antibody (NS). The E2F1 and albumin genes were used as positive and negative controls, respectively.