The Arabidopsis SWR1 chromatin-remodeling complex is important for DNA repair, somatic recombination, and meiosis

Abstract

All processes requiring interaction with DNA are attuned to occur within the context of the complex chromatin structure. As it does for programmed transcription and replication, this also holds true for unscheduled events, such as repair of DNA damage. Lesions such as double-strand breaks occur randomly; their repair requires that enzyme complexes access DNA at potentially any genomic site. This is achieved by chromatin remodeling factors that can locally slide, evict, or change nucleosomes. Here, we show that the Swi2/Snf2-related (SWR1 complex), known to deposit histone H2A.Z, is also important for DNA repair in Arabidopsis thaliana. Mutations in genes for Arabidopsis SWR1 complex subunits photoperiod-independent Early Flowering1, actin-related protein6, and SWR1 complex6 cause hypersensitivity to various DNA damaging agents. Even without additional genotoxic stress, these mutants show symptoms of DNA damage accumulation. The reduced DNA repair capacity is connected with impaired somatic homologous recombination, in contrast with the hyper-recombinogenic phenotype of yeast SWR1 mutants. This suggests functional diversification between lower and higher eukaryotes. Finally, reduced fertility and irregular gametogenesis in the Arabidopsis SWR1 mutants indicate an additional role for the chromatin-remodeling complex during meiosis. These results provide evidence for the importance of Arabidopsis SWR1 in somatic DNA repair and during meiosis.

Figure 1.

At-SWR1 Mutants Are Hypersensitive to DNA-Damaging Agents. (A) Phenotypes of 10-d-old seedlings with (i) and without (ii) true leaves. (B) to (F) True leaf assay with seedlings treated with γ-irradiation (γ-IR) (B), BLEO (C), MMC (D), HU (E), or UV light (UV-C) (F) at the dosage indicated. The percentage of 10-d-old treated plants with true leaves was calculated in relation to mock populations. The DNA damage-sensitive mutant ku70-2 was used as a control in (B) and (D). Error bars indicate the se between three or more biological replicates with 50 to 80 seedlings each. Asterisks indicate significant differences according to P values from unpaired t tests: ***P < 0.001, **0.001 < P < 0.01, and *0.01 < P < 0.05. n.d., not determined; WT, the wild type.

Figure 2.

At-SWR1 Mutants Show Symptoms of DNA Damage Accumulation. (A) Transcript levels of DNA damage–induced genes RAD51, BRCA1, and PARP2 in At-SWR1 mutants, without DNA damaging treatment. Values are presented relative to untreated wild-type (WT) Columbia (set as 1). Transcript levels were measured by quantitative RT-PCR and normalized to UBC28. The values represent the average of two or more biological replicates. Error bars correspond to the standard deviation of the mean (sd). Asterisks indicate significant differences according to P values from unpaired t tests: ***P < 0.001, **0.001 < P < 0.01, and *0.01 < P < 0.05. (B) Distribution of DNA content in nuclei from the second pair of true leaves of 30-d-old wild-type and At-SWR1 mutant plants determined by flow cytometry. Error bars correspond to the sd in two replicates.

Figure 3.

Double Mutant Analysis Indicates a Role for At-SWR1 in HR. True leaf assay with seedlings of the wild type (WT), brca1-1 (impaired in HR), ku70-2 (impaired in NHEJ), arp6-3 and swc6-1 (At-SWR1 mutants), and double mutants arp6 brca, arp6 ku70, swc6 brca, and swc6 ku70 treated with 0.5 μg/mL BLEO. The percentage of 10-d-old treated plants with true leaves was calculated in relation to mock populations. Error bars indicate the se between two or more biological replicates with 50 to 80 seedlings each. Asterisks in single mutants indicate significant differences from the wild type. Asterisks in double mutants indicate significant differences from either the arp6-3 or the swc6-1 single mutant. P values were determined in unpaired t tests: ***P < 0.001 and **0.001 < P < 0.01.

Figure 4.

The Frequency of SHR Is Strongly Reduced in the At-SWR1 Mutant Background. (A) Recombination substrates in SHR reporter lines DGU.US-1 and IU.GUS-8 (Orel et al., 2003). P, 35S promoter from the Cauliflower mosaic virus; BAR and HPT, selection markers phosphinothricin acetyltransferase and hygromycin phosphotransferase; T, 35S terminator. (B) to (E) Distribution of seedlings with different numbers of blue spots ([B] and [C]) and total frequency ([D] and [E]) in line DGU.US-1 and line IU.GUS-8. Error bars correspond to the se. Each mutant population was significantly different to its wild-type (WT) counterpart, with a P value < 0.001. Asterisks indicate the significance between treated and mock populations according to P values from unpaired t tests: ***P < 0.001 and **0.001 < P < 0.01.

Figure 5.

Reduced Fertility of At-SWR1 Mutants Is Due to Defects in Both Male and Female Gametogenesis. (A) Reduced elongation of siliques in At-SWR1 mutants (arrows). WT, the wild type. Bar = 1 cm. (B) Average number of seeds per silique in selfed pie1-3, arp6-3, and swc6-1 or upon reciprocal crosses with wild-type plants. (C) Assay for pollen viability by Alexander staining of wild-type and mutant anthers. Bar = 100 μm. (D) Cytological analysis of megagametogenesis in wild-type and mutant ovules. The central cell (CC) and the egg cell (EC) are present only in wild-type ovules; mutant ovules have aborted structures (arrows) or are empty. Bar = 10 μm.

Figure 6.

Meiosis Is Perturbed in At-SWR1 Mutants. (A) and (B) Analysis of meiotic progression in DAPI-stained pollen mother cells of the wild type (WT) and pie1-3. (A) Wild-type cells in leptotene (I), zygotene (II), pachytene (III), diplotene (VI), diakinesis (V), metaphase I (VI), anaphase I (VII), interkinesis (VIII), metaphase II (IX), and tetrad stage (X). (B) pie1-3 cells in leptotene (I), zygotene (II), pachytene (III), diplotene (VI), diakinesis (V), metaphase I (VI), anaphase I (VII), interkinesis (VIII), metaphase II (IX), and tetrad stage. A regular tetrad is shown (X). (C) Representative pictures of aberrant meiotic products observed in addition to regular tetrads, in pie1-3, arp6-3, and swc6-1 mutant plants. Please note DAPI-stained bodies outside newly forming microspore nuclei. (D) Quantification of tetrads and aberrant meiotic products in the wild type, pie1-3, arp6-3, and swc6-1 in more than 80 samples each.