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)