Arabidopsis R1R2R3-Myb proteins are essential for inhibiting cell division in response to DNA damage

Inhibition of cell division is an active response to DNA damage that enables cells to maintain genome integrity. However, how DNA damage arrests the plant cell cycle is largely unknown. Here, we show that the repressor-type R1R2R3-Myb transcription factors (Rep-MYBs), which suppress G2/M-specific genes, are required to inhibit cell division in response to DNA damage. Knockout mutants are resistant to agents that cause DNA double-strand breaks and replication stress. Cyclin-dependent kinases (CDKs) can phosphorylate Rep-MYBs in vitro and are involved in their proteasomal degradation. DNA damage reduces CDK activities and causes accumulation of Rep-MYBs and cytological changes consistent with cell cycle arrest. Our results suggest that CDK suppressors such as CDK inhibitors are not sufficient to arrest the cell cycle in response to DNA damage but that Rep-MYB-dependent repression of G2/M-specific genes is crucial, indicating an essential function for Rep-MYBs in the DNA damage response.


Supplementary
(a) T-DNA insertion sites in myb3r4-3. Exons and introns are indicated by boxes and solid lines, respectively. Protein-coding regions are shown by black boxes. Black triangles indicate the sites of primers used in (b). Note that T-DNA is inserted into exon 4 and intron 4. (b) Quantitative RT-PCR analysis of MYB3R4. Total RNA was isolated from two-week-old WT and myb3r4-3 seedlings, and subjected to quantitative RT-PCR using primers shown in (a). The expression levels were normalized to that of ACTIN2, and are indicated as relative values, with that of WT set to 1. Data are presented as mean ± SD of three biological replicates. Significant difference from WT was determined by Student's t-test: ***, P < 0.001. Five-day-old seedlings were treated with or without 1, 2 or 4 µM zeocin for 24 h, and root tips were observed after staining with propidium iodide. Blue and white arrowheads indicate the quiescent centre and the first elongated cell in the cortex cell file, respectively. Scale bars, 100 µm. Five-day-old seedlings of WT, myb3r3 and myb3r5 were transferred to medium containing 0.25 µg ml -1 bleomycin, 0.06 µg ml -1 bleocin, 5 mM boron, 1 mM hydroxyurea (HU) or 5 µg ml -1 aphidicolin, and root length was measured for 6 days. Root growth was also observed after irradiation with gamma rays (100 Gy). Data are presented as mean ± SD (n > 30). Significant differences from WT were determined by Student's t-test: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Seeds were irradiated with gamma rays (200 or 400 Gy), and the number of true leaves was counted using 10-day-old seedlings. The numbers of true leaves are expressed as relative values, with that of the nontreated seedlings set to 1. Data are presented as mean ± SD (n > 80). sog1-1 and lig4 were used as controls. Five-day-old WT seedlings were transferred to medium with or without 2 µM zeocin, and grown for the indicated times. Total RNA was isolated from a 1-cm region at the root tip. Expression levels were normalized to that of ACTIN2, and are indicated as relative values, with that for the time of transfer to new medium set to 1. Data are presented as mean ± SD of three biological replicates. Significant differences from the control without zeocin were determined by Student's t-test: ***, P < 0.001. Five-day-old WT seedlings were transferred to medium with 25 µM roscovitine, and grown for the indicated times. Total RNA was isolated from a 1-cm region at the root tip. Expression levels were normalized to that of ACTIN2, and are indicated as relative values, with that for the time of transfer to roscovitine-containing medium set to 1. Data are presented as mean ± SD of three biological replicates.

Supplementary
Six-day-old seedlings of WT, myb3r3 and myb3r5 were transferred to medium containing 2 µM zeocin, and grown for the indicated times. Total RNA was isolated from the region 1 cm from the root tip and subjected to quantitative RT-PCR analysis. The expression levels were normalized to that of ACTIN2, and are indicated as relative values, with that for the time of transfer to zeocin-containing medium set to 1. Data are presented as mean ± SD of three biological replicates. Figure 16. Expression of MYB3R4 in the sog1-1 mutant.

Supplementary
Five-day-old seedlings of WT and sog1-1 were transferred to medium with or without 6 µM zeocin, and grown for 24 h. Total RNA was isolated from roots and subjected to quantitative RT-PCR analysis. The expression levels were normalized to that of ACTIN2, and are indicated as relative values, with that for WT grown in the absence of zeocin set to 1. Data are presented as mean ± SD of three biological replicates.
Significant differences from WT grown in the absence of zeocin were determined by Student's t-test: ***, P < 0.001. (a) ChIP-PCR analysis of G2/M-specific genes. Chromatin bound to MYB3R3 was collected by immunoprecipitation with (+Ab) or without (-Ab) anti-GFP antibodies using roots of 10-day-old myb3r3 plants carrying ProMYB3R3::MYB3R3-GFP. Fold enrichment of each promoter region was determined by normalizing the recovery rate against that of samples immunoprecipitated without the antibody. CDKA;1 was used as a control that is expressed throughout the cell cycle. Data are presented as mean ± SD of three biological replicates. Significant differences from the control immunoprecipitated without the antibody were determined by Student's t-test: ***, P < 0.001. (b) ChIP-PCR analysis using zeocin-treated roots.

Supplementary
Ten-day-old myb3r3 seedlings carrying ProMYB3R3::MYB3R3-GFP were treated with or without 2 µM zeocin for 24 h, and used for ChIP assay. Fold enrichment of each promoter region was determined by normalizing the recovery rate against that of samples without zeocin treatment. Data are presented as mean