Plant 45S rDNA Clusters Are Fragile Sites and Their Instability Is Associated with Epigenetic Alterations

PLOS ONE, Dec 2019

Our previous study demonstrated that 45S ribosomal DNA (45S rDNA) clusters were chromosome fragile sites expressed spontaneously in Lolium. In this study, fragile phenotypes of 45S rDNA were observed under aphidicolin (APH) incubation in several plant species. Further actinomycin D (ActD) treatment showed that transcriptional stress might interfere with chromatin packaging, resulting in 45S rDNA fragile expression. These data identified 45S rDNA sites as replication-dependent as well as transcription-dependent fragile sites in plants. In the presence of ActD, a dramatic switch to an open chromatin conformation and accumulated incomplete 5′ end of the external transcribed spacer (5′ETS) transcripts were observed, accompanied by decreased DNA methylation, decreased levels of histone H3, and increased histone acetylation and levels of H3K4me2, suggesting that these epigenetic alterations are associated with failure of 45S rDNA condensation. Furthermore, the finding that γ-H2AX was accumulated at 45S rDNA sites following ActD treatment suggested that the DNA damage signaling pathway was associated with the appearance of 45S rDNA fragile phenotypes. Our data provide a link between 45S rDNA transcription and chromatin-packaging defects and open the door for further identifying the molecular mechanism involved.

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Plant 45S rDNA Clusters Are Fragile Sites and Their Instability Is Associated with Epigenetic Alterations

et al. (2012) Plant 45S rDNA Clusters Are Fragile Sites and Their Instability Is Associated with Epigenetic Alterations. PLoS ONE 7(4): e35139. doi:10.1371/journal.pone.0035139 Plant 45S rDNA Clusters Are Fragile Sites and Their Instability Is Associated with Epigenetic Alterations Min Huang 0 Hui Li 0 Lu Zhang 0 Fei Gao 0 Pu Wang 0 Yong Hu 0 Shihan Yan 0 Lin Zhao 0 Qi Zhang 0 Junjun Tan 0 Xincheng Liu 0 Shibin He 0 Lijia Li 0 Mary Bryk, Texas A&M University, United States of America 0 State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University , Wuhan , China Our previous study demonstrated that 45S ribosomal DNA (45S rDNA) clusters were chromosome fragile sites expressed spontaneously in Lolium. In this study, fragile phenotypes of 45S rDNA were observed under aphidicolin (APH) incubation in several plant species. Further actinomycin D (ActD) treatment showed that transcriptional stress might interfere with chromatin packaging, resulting in 45S rDNA fragile expression. These data identified 45S rDNA sites as replicationdependent as well as transcription-dependent fragile sites in plants. In the presence of ActD, a dramatic switch to an open chromatin conformation and accumulated incomplete 59 end of the external transcribed spacer (59ETS) transcripts were observed, accompanied by decreased DNA methylation, decreased levels of histone H3, and increased histone acetylation and levels of H3K4me2, suggesting that these epigenetic alterations are associated with failure of 45S rDNA condensation. Furthermore, the finding that c-H2AX was accumulated at 45S rDNA sites following ActD treatment suggested that the DNA damage signaling pathway was associated with the appearance of 45S rDNA fragile phenotypes. Our data provide a link between 45S rDNA transcription and chromatin-packaging defects and open the door for further identifying the molecular mechanism involved. - Funding: This work was supported by the National Natural Science Foundation of China (nos. 30870261 and 31171186) and the Research Fund for the Doctoral Program of Higher Education (no. 20090141110031). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Eukaryotic genomes contain hundreds of tandemly arranged 45S ribosomal RNA (rRNA) genes (45S rDNAs) which are transcribed by RNA polymerase I to generate 18S, 5.8S and 28S rRNAs. The 45S rDNA region shows some typical characteristics of genetic instability, which often results in variable copy number and distribution among closely related species and even intraspecies. The studies revealed that the whole 45S rDNA repeats in Allium and subgenus Mus chromosomes could free shift from one locus to another, suggesting that 45S rDNA might serve as a transposable element [1,2]. In Neurospora, nucleolus organized region (NOR) breakage caused large terminal deletions and such 45S rDNA termini were healed by the addition of telomeric repeats to prevent terminus fusion [3]. In Lolium rigidum, in vivo 45S rDNA breakage led to chromosome rearrangement and varied position and number of 45S rDNA sites [4]. It has been reported that 45S rDNA clusters are preferred sites for gene rearrangements and chromosome breakage-fusion-bridge cycles in telomerase-deficient Arabidopsis [5]. The 45S rDNA instability might be related to its transcriptional ability [3]. This hypothesis was tested using a transcription inhibitor actinomycin D (ActD). ActD is an anti-tumor antibiotic and forms a stable complex with GpC-rich nucleotide clusters particularly in transcriptionally active regions, thus inhibiting the elongation of growing RNA chains [6]. For example, the NORs seemed to be the most likely regions of stretching and breaks in indian muntjac cells following treatment with ActD [7]. Recent work has demonstrated that 45S rDNA regions as the chromosome fragile sites are spontaneously expressed in vitro on metaphase chromosomes in Lolium [8] and this fragility is likely to account for the observed genetic instability. Fragile sites are large and highly sensitive regions that are inclined to form abnormal poorstaining lesions (gaps, constrictions or breaks) in metaphase chromosomes [9,10]. Currently, the well characterized fragile sites are thought to be caused by DNA replication-dependent defects, which therefore can be induced and enhanced when cells are partially inhibited by replication stress that retards DNA replication fork progression at fragile sites, such as exposure to the DNA polymerase inhibitor aphidicolin (APH) [11,12]. The alteration of DNA sequence motifs and epigenetic modifications interfere indirectly or directly with the higher-order chromatin packing, thus may be involved in the appearance of the fragile sites [13,14]. Two replication checkpoint proteins (ATR and BRCA1) were found to participate in maintaining the stability of fr (...truncated)


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Min Huang, Hui Li, Lu Zhang, Fei Gao, Pu Wang, Yong Hu, Shihan Yan, Lin Zhao, Qi Zhang, Junjun Tan, Xincheng Liu, Shibin He, Lijia Li. Plant 45S rDNA Clusters Are Fragile Sites and Their Instability Is Associated with Epigenetic Alterations, PLOS ONE, 2012, Volume 7, Issue 4, DOI: 10.1371/journal.pone.0035139