Early genomic response to X-ray induced DNA damage and transposon regulation in Arabidopsis thaliana

Journal Article / 2014

Wang, Z.

In this study two major topics were investigated. The first part deals with the early genomic response to DNA damage with particular emphasis on transposable elements (TEs). The second part aims to improve Ac transposition by suppressing the formation of mis-processed Ac transcripts by introducing point mutations in the Ac transposase coding sequence. 1) DNA damage and TEs Double-strand breaks (DSBs) can lead to genome instability and transcriptional and transpositional reactivation of TEs. However the mechanism underlying this process is not well understood. Two different strategies were applied to generate DSBs and to follow transposon reactivation activity. Initially four coding sequences of meganuclease (ISI-opA, I-Sce I, ZFN3 and QQR), under the control of an estradiol-inducible promoter, and their respective target sites were introduced into the genome of Arabidopsis. Only the I-Sce I endonuclease, encoded by the Arabidopsis codon-optimized sequence ISI-opA, successfully generated DSBs after estradiol induction. However, the expected downstream response to DSBs was not observed probably due to a low frequency of introduced breaks into the genome. As an alternative strategy, X-rays were used to generate DSBs in the Arabidopsis genome. To compare the transcriptome-wide early response to DSBs in WT and the DNA-repair-defective mutant atm, the RNA-seq technology was used as a tool. 1315 and 644 genes were found to be e2-fold regulated in WT and atm respectively, largely consistent with previous microarray-based transcriptome studies. However, the RNA-seq technology allowed obtaining additional information concerning previously undetected events in the genome response. RNA-Seq data revealed that, in contrast to the large number of regulated genes, TEs/TE-related elements were less responsive on transcriptional level to DSBs, at least in the early response (3h after the treatment). The DSBs induced transcriptional activation of TEs/TE-related elements observed in this study is consistent with previous published data. However, in this work DNA damage induced transcriptional down-regulation of TEs/TE-related elements was detected at transcriptome level for the first time. Among the regulated TEs, retrotransposons were more responsive to X-ray than DNA transposons. In WT plants, 68 % of the regulated TEs/TE-related elements were associated with long non-coding RNAs (lncRNAs). In addition, several differentially expressed novel transcripts were detected and were also identified as lncRNAs. In total, 91 regulated lncRNAs were identified in WT, but only 18 in the atm mutant. The observation indicated that lncRNAs were regulated by ATM in response to X-ray induced DSBs in Arabidopsis. The epigenetic machinery is associated with DNA damage repair and the regulation of TEs. In this study, the de novo DNA methylation gene DRM1 was the only up-regulated DNA methyltransferase gene induced by X-ray. The demethylase gene JMJ30 and two RNA-directed DNA methylation (RdDM) genes AGO2 and AGO7 were also upregulated. In order to further investigate the roles of these epigenetic-related genes in regulating TEs/TE-related elements and lncRNAs in response to DSBs, the Arabidopsis T-DNA insertion knock-out lines drm1, jmj30, ago2 and ago7 mutant were X-ray treated. BRCA1 repairs DSBs via homologous recombination. DRM2, the homolog of DRM1, is the mainly active methyltransferase for de novo DNA methylation and AGO4 is the key player in the RdDM pathway. The corresponding T-DNA insertion lines were also irradiated. The phenotypical analysis of mutants and the quantitative expression analysis of a subset of TEs/TE-related elements and lncRNAs via qRT-PCR indicated that DNA damage repair genes and genes involved in epigenetic control might selectively regulate TEs/TE-related elements and lncRNAs in response to DSBs. 2) Improvement of Ac transposition in Arabidopsis Previous studies have reported that the expression of Ac in Arabidopsis results in a low germinal excision rate which might be due to the occurrence of cryptic introns and early terminated Ac transcripts. Site-directed mutagenesis of Ac was used in the present study in order to prevent mis-processing and incorrect splicing of Ac transcripts and to improve Ac transposition frequency. However, the modified AcTPase coding sequence did not increase transposition frequency. Additional studies are needed to determine if the different transcription-controlling mechanisms in Arabidopsis and maize might be responsible for the occurrence of mis-processed Ac transcripts in Arabidopsis.