The DREAM complex represses growth in response to DNA damage in Arabidopsis

Cytoscape representation of a meta-analysis of 182 FP-IPs using different bait proteins (35 × E2FA-GFP [blue], 39 × E2FB-GFP [red], 20 × E2FC-GFP [green], 32 × RBR1-GFP [yellow], 3 × DPA-GFP [orange], and 3 × DPB-CFP [pale-orange], 50 × GFP [control]). To reduce complexity, only prey proteins which were enriched in at least one third of the IPs of one bait are shown. For the more comprehensive dataset, see Table S4. The thickness of the edges corresponds to the frequency of positive IPs by which a bait/prey interaction was found. Proteins that were also identified in the tandem affinity purification experiments are represented by an ellipse. Prey proteins are grouped according to their occurences in different E2F-IPs. DREAM complex homologs are shown in dark blue. Additional proteins that display an interaction pattern like the DREAM component homologs, that is, do not interact with E2FA, but interact with E2FB/C and the DPs, are shown in bold with a thick outline.

Results of Y2H assays testing the here-identified DREAM complex components and selected additional proteins for binary interactions. AD, activating domain; BD, DNA-binding domain. (A) Interaction matrix. Signal strength was classified according to yeast growth on different dropout media in two categories and is indicated by shades of blue. Dark blue, signal on QDO; light blue, signal on TDO but not on QDO; orange, signal not stronger than with mEGFP control; dark grey, strong auto-activation observed using the BD construct and an AD-mEGFP control. (B) Cytoscape representation of the observed interaction network. Interactions are indicated by an edge between two protein nodes and were classified according to yeast growth in two categories. If yeast growth was observed with a pair of proteins in both AD/BD combinations, the stronger signal is shown. Thick line, growth on QDO; thin line, growth on TDO but not on QDO. Dark blue, MuvB-core proteins; light blue, other DREAM components; orange, known DNA damage regulators; grey, other interactors.

Y2H interaction assays to test for binary interaction of wild type as well as mutant TCX5 and TCX6 with RBR1, TCX6, and mEGFP as auto-activation control. AxAxA replaces the LxCxE motif in the mutant proteins. AD, activating domain; BD, DNA-binding domain. Yeast cells were diluted as shown on top and spotted on different dropout media as indicated below. Growth on TDO and QDO indicates interaction.

Y2H interaction assays to test for binary interaction of wild type as well as mutant NAC044 with RBR1, LIN37B, and mEGFP as auto-activation control. AxAxA replaces the LxCxE motif in the mutant NAC044. AD, activating domain; BD, DNA-binding domain. Yeast cells were diluted as shown on top and spotted on different dropout media as indicated below. Growth on TDO and QDO indicates interaction.

(A, B, C, D) Using genomic reporter lines to include the native regulatory sequences, expression of mEGFP-NAC044 (A), TCX5-EGFP (B), LIN37A-EGFP (C), and LIN37B-EGFP (D) was followed for 24 h after transfer of 6-d-old seedlings to 50 μM cisplatin-containing plates. Representative images of root tips at different time points, as indicated in the upper left corner of each panel, are shown here using the royal LUT in ImageJ. PI staining of the corresponding part of the root tip is shown as inset in the upper right corner of each panel. Scale bar = 30 μm. Microscopic settings were kept constant for each line, but not necessarily between lines.

(A, B) Root growth of the wild type, lin37a, lin37b, and lin37a lin37b double mutants on control plates and in the presence of cisplatin (A: representative pictures; B: quantification). Plants grown for 5 d in the absence of cisplatin were transferred to medium containing 0 or 10 μM cisplatin, and grown for further 7 d. Data are presented as mean ± SD (n = 10). Significant differences as determined by two-way ANOVA and Tukey–Kramer post hoc test (P < 0.05) are indicated by differing letters over the bars.

(A) Confocal analysis of seedling roots expressing pgE2FA-3xvYFP or pgE2FB-3xvYFP indicates an overall increase in E2FB-3xvYFP but not E2FA-3xvYFP fluorescence after 24 h treatment with 50 μM cisplatin. Scale bar = 50 μm. (B, C, D) Quantification by FP-IP. (B, C, D) 1 d of cisplatin treatment (50 μM) of 6-d-old E2F-GFP-expressing seedlings increases the quantity of DREAM components specifically in the protein complexes containing E2FB (B) but not E2FA (C) or E2FC (D). Interacting protein partners were immunopurified from the corresponding E2F-GFP translational lines by using anti-GFP-containing magnetic beads, and components were identified with mass spectrometry. Graphs show fold change calculated as a ratio of immunoprecipitated components after cisplatin treatment relative to untreated conditions. Values represent the mean of three biological replicates ± SE.

(A) Whole-plant photographs of Col-0, e2fb-1, and e2fb-2 treated with 0.05% vol/vol DMF (mock) or 15 μM cisplatin. Scale bar = 10 mm. (B) Primary root length Col-0, e2fb-1, and e2fb-2 seedlings after 6 d of 15 μM cisplatin or mock treatment. An average of 20 roots were measured for each genotype and condition. A box plot of the replicate means is shown. Significant differences were determined by ANOVA and Tukey post hoc test (P < 0.05). (C) Representative confocal images of EdU-labelled root tips of Col-0, e2fb-1, and e2fb-2 treated with 50 μM cisplatin or 0.16% vol/vol DMF (mock) for 3 h. Scale bar = 50 μm. (D) Percentage of EdU-positive S phase cells relative to DAPI-stained nuclei in the root meristems of Col-0, e2fb-1, and e2fb-2 treated with 50 μM cisplatin or 0.16% DMF (mock) for 3 h. An average of 25 roots were imaged for each genotype and condition. A box plot of the replicate means is shown, outlier values are shown as circles. Significant differences were determined by ANOVA and Tukey post hoc test (P < 0.05).