SOG1 activator and MYB3R repressors regulate a complex DNA damage network in Arabidopsis

Abstract

To combat DNA damage, organisms mount a DNA damage response (DDR) that results in cell cycle regulation, DNA repair and, in severe cases, cell death. Underscoring the importance of gene regulation in this response, studies in Arabidopsis have demonstrated that all of the aforementioned processes rely on SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1), a NAC family transcription factor (TF) that has been functionally equated to the mammalian tumor suppressor, p53. However, the expression networks connecting SOG1 to these processes remain largely unknown and, although the DDR spans from minutes to hours, most transcriptomic data correspond to single time-point snapshots. Here, we generated transcriptional models of the DDR from GAMMA (γ)-irradiated wild-type and sog1 seedlings during a 24-hour time course using DREM, the Dynamic Regulatory Events Miner, revealing 11 coexpressed gene groups with distinct biological functions and cis-regulatory features. Within these networks, additional chromatin immunoprecipitation and transcriptomic experiments revealed that SOG1 is the major activator, directly targeting the most strongly up-regulated genes, including TFs, repair factors, and early cell cycle regulators, while three MYB3R TFs are the major repressors, specifically targeting the most strongly down-regulated genes, which mainly correspond to G2/M cell cycle-regulated genes. Together these models reveal the temporal dynamics of the transcriptional events triggered by γ-irradiation and connects these events to TFs and biological processes over a time scale commensurate with key processes coordinated in response to DNA damage, greatly expanding our understanding of the DDR.

Keywords: DNA damage response; DREM; SOG1; transcriptional networks.

Fig. 1.

DNA damage response DREM analysis reveals coexpressed genes with distinct biological functions and regulatory features. (A) DREM model [see Source Data 1 (44)] showing 11 groups of coexpressed genes, termed wild-type paths W1–W11. Here, and in all other DREM models, the y axis indicates the log₂ FC in expression in response to γ-IR, the x axis indicates the time in minutes (’) and/or hours (h), and the number (N) of genes per path is indicated. All genes are listed in Dataset S3A. Comparisons with previously published DE gene sets are presented in SI Appendix, Fig. S1, expression patterns of the individual genes in each DREM path are shown in SI Appendix, Fig. S2A, and the TF families (i.e., NAC, TCP, HB, WRKY, and MYB) assigned to the DREM paths are indicated, with the lists of all of the TFs assigned to each path shown in SI Appendix, Fig. S4. (B) Screenshots showing the expression levels of representative genes from each DREM path. The gene indicated above is shown in blue and the neighboring genes are shown in gray. The difference between the mock and γ-IR–treated samples [(+γ-IR_av) − (−γ-IR_av)] is shown for each time point on a scale of ±125. (C) Heatmap showing the significance of all of the GO terms with a log₁₀ P < −1.7 in at least one path, across all of the DREM paths. Gray indicates a P > 0.5. See SI Appendix, Fig. S3 and Source Data 2 (44) for all enriched GO terms remaining after the REVIGO similarity filter (100). (D) Table of select genes associated with the processes indicated above each column based on GO category enrichments and/or manual curation. The gene names and/or the number of genes per category (in parentheses) are colored based on the path in which they reside. (E) Select motifs enriched in the indicated DREM paths. The full set of enriched motifs identified are presented in SI Appendix, Fig. S5 [Source Data 3 (44)]. Below each motif, E-values calculated against all Arabidopsis promoter sequences, and similar TF families, identified via comparisons with the motifs in the DAP-seq database (48) using Tomtom (98), are indicated. Three similar, but independently identified motifs that correspond to the previously described mitosis-specific activator (MSA) element (AACGG) (55) are marked with an asterisk.

Fig. 2.

SOG1 controls nearly all aspects of the transcriptional response to γ-IR. (A) sog1 DREM model [see Source Data 1 (44)] showing five sets of coexpressed genes, termed sog1 paths S1–S5. The expression profiles, enriched GO terms, and motifs are presented in SI Appendix, Figs. S6 and S9, respectively. All genes are listed in Dataset S3B. For comparison, the wild-type (wt) DREM model is shown as an inlay. (B) Scaled Venn diagrams showing the overlap of genes in DREM paths with similar trends in the wild-type and sog1 models. (C) Heatmaps showing the log₂ FC in expression (γ-IR vs. mock) of the genes present in paths W1–W11 (Fig. 1A) using either the wild-type or the sog1 expression data. For each path, the heatmaps were ranked based on the wild-type expression level. See also SI Appendix, Fig. S7.

Fig. 3.

SOG1 is a transcriptional activator that directly regulates nearly half of the genes strongly induced by γ-IR. (A) Heatmap showing SOG1 enrichment [log₂(SOG1/wt ChIP)] at the 307 peaks (±3 Kb) identified from both ChIP assays (20 min and 1 h) (SI Appendix, Fig. S10 and Dataset S4B). (B) Heatmaps showing the expression of SOG1 target genes (Datasets S4 C and D) during the wild-type (wt) and sog1 γ-IR time courses ranked based on the wild-type expression values at 1 h 30 min. (C) Expression profiles of the SOG1 target genes colored by gene paths. The fraction of genes in each path is indicated, with the percentage in parentheses. (D) Motif identified under the SOG1 ChIP peaks. The E-value, as reported by MEME (97, 102), and the fraction of peaks with the motif are indicated below (see also Dataset S4E).

Fig. 4.

Functional categorization of SOG1 target genes. Of the 206 SOG1 target genes in the DREM model, 141 were assigned to functional categories and colored based on the log₂ FC +/− γ-IR at the 3-h time point from the wild-type DREM model. Genes underlined in blue have a human and/or mouse ortholog shown to be targeted and up-regulated by p53. See also Materials and Methods, SI Appendix, Figs. S11 and S12, and Source Data 4 (44).

Fig. 5.

The Rep-MYB3R TFs are the master repressors of cell cycle genes in response to γ-IR. (A) Heatmaps showing the log₂ FC in expression (γ-IR vs. mock) of the genes present in paths W1–W11 of the wild-type (wt) DREM model, ordered as in Fig. 2, using either the wild-type or the myb3r1,3,5 expression data. For reference, the expression levels from the wild-type DREM model (wt_DREM) at the 3-h time point was included. (B) Heatmaps showing the log₂ FC in expression (γ-IR vs. mock) of the path W9, W10, and W11 genes from the wild-type DREM model that are significantly less repressed in the myb3r1,3,5 mutant than in the wild-type controls (“myb3r1,3,5 > wt”) (Dataset S5 B and C). (C and D) Scaled Venn diagrams showing the overlap between the genes shown in B and either genes expressed in the G2/M phase of the cell cycle (54, 57) (C) or genes associated with MSA motifs and/or MYB3R3 peaks (53, 90) (D).