Light-dependent regulation of DEL1 is determined by the antagonistic action of E2Fb and E2Fc

Abstract

Endoreduplication represents a variation on the cell cycle in which multiple rounds of DNA replication occur without subsequent chromosome separation and cytokinesis, thereby increasing the cellular DNA content. It is known that the DNA ploidy level of cells is controlled by external stimuli such as light; however, limited knowledge is available on how environmental signals regulate the endoreduplication cycle at the molecular level. Previously, we had demonstrated that the conversion from a mitotic cell cycle into an endoreduplication cycle is controlled by the atypical E2F transcription factor, DP-E2F-LIKE1 (DEL1), that represses the endocycle onset. Here, the Arabidopsis (Arabidopsis thaliana) DEL1 gene was identified as a transcriptional target of the classical E2Fb and E2Fc transcription factors that antagonistically control its transcript levels through competition for a single E2F cis-acting binding site. In accordance with the reported opposite effects of light on the protein levels of E2Fb and E2Fc, DEL1 transcription depended on the light regime. Strikingly, modified DEL1 expression levels uncoupled the link between light and endoreduplication in hypocotyls, implying that DEL1 acts as a regulatory connection between endocycle control and the photomorphogenic response.

Figure 1.

Interaction of E2Fb and E2Fc with the DEL1 promoter. A, Sequence of the DEL1 promoter with the two putative E2F cis-acting sites (red) and the primers used for ChIP (black arrows) indicated. B and C, E2Fb and E2Fc interaction with the DEL1 promoter in yeast (B) and in planta (C) as shown by Y1H and ChIP, respectively. Interactions observed by Y1H are positive when both HIS3 (grown on +3-aminotriazole [3-AT] medium) and LacZ (X-gal positive) expression were induced. IP, Immunoprecipitation; NO AB, no antibody. D and E, Protoplast transactivation activity assays with a ProDEL1:fLUC reporter construct, a Pro-35S:rLUC normalization construct, and a 35S:E2Fa, 35S:E2Fb, or 35S:E2Fc effector construct, showing stimulation of DEL1 promoter activity by E2Fb (D) being counteracted by E2Fc (E). Luciferase activity of control cells was arbitrarily set to 1. Data are means ± se (n = 8; *** P ≤ 0.001, two-sided t test).

Figure 2.

Competition of E2Fb and E2Fc for binding to the E2F-2 site in the DEL1 promoter. A, Interaction of E2Fb and E2Fc with E2F-2 in yeast shown by Y1H. 3-AT, 3-Aminotriazole. B and C, Protoplast transactivation activity assay with a ProDEL1:fLUC reporter construct, a Pro-35S:rLUC normalization construct, and a 35S:E2Fb or 35S:E2Fc effector construct. An intact E2F-2 binding site was required for activation of the DEL1 promoter by E2Fb (B). Both E2Fb and E2Fc bound E2F-2 in a competitive manner (C). Luciferase activity of control cells was arbitrarily set to 1. Data are means ± se (n = 8; *** P ≤ 0.001, two-sided t test).

Figure 3.

Requirement of E2F-2 for DEL1 expression in dividing tissues and binding of E2Fb and E2Fc in vivo. A to J, ProDEL1:GUS (A–E) versus ProDEL1-Mut2:GUS (F–J) expression patterns. K and L, In vivo analysis by ChIP of E2Fb (E2Fb-IP) and E2Fc (E2Fc-IP) binding to the endogenous DEL1 and inserted ProDEL1:GUS (K) or ProDEL1-Mut2:GUS (L) promoter, with a reverse primer, specific for the endogenous DEL1 or GUS gene. WT, Wild type.

Figure 4.

Changes in DEL1 expression levels in E2Fb and E2Fc transgenic lines. A, DEL1 expression levels in control (Columbia [Col-0]), E2Fc^OE, e2fb-1, and E2Fb^OE lines. Data are means ± sd (n = 3). B to G, ProDEL1:GUS in Col-0 (B–D) or e2fb-1 (E–G) background. H to K, ProDEL1:GUS in Col-0 (H) or E2Fb^OE (I–K) background.

Figure 5.

Dependence of DEL1 expression levels on light and COP1. A to D, ProDEL1:GUS plants grown for 3 d in the dark (A) and exposed for 4 h (B), 24 h (C), or 48 h (D) to light. E, ProDEL1-Mut2:GUS plant switched to continuous light for 48 h after 3 d of germination in the dark. F, DEL1 expression levels in control (Columbia [Col-0]) and cop1-4 lines. Data are means ± sd (n = 3).

Figure 6.

Influence of DEL1 transcript levels on the response to light of hypocotyl ploidy levels. A and B, Ploidy distribution in hypocotyls of plants grown under long-day (A) and short-day (B) conditions. C, Endoreduplication index in Columbia (Col-0), del1-1, and DEL1^OE lines under short-day (SD) and long-day (LD) conditions after 12 d of growth. D to F, Ploidy distribution in Col-0 (D), del1-1 (E), and DEL1^OE (F) lines under SD and LD conditions after 12 d of growth. Data are means ± sd (n = 3).

Figure 7.

Model for light-controlled hypocotyl endoreduplication. In the presence of light, E2Fb activates DEL1 expression, preventing cells from entering the endoreduplication program. Under dark conditions, E2Fb is marked by COP1 for degradation, by which E2Fc becomes the most abundant E2F binding the DEL1 promoter. The decrease in DEL1 transcript level allows cells to enter the endoreduplication cycle.