Author: Michelle Renee Newman
Newman, Michelle Renee, 2012 Investigation of the modulation of murine repeat element DNA methylation by ionising radiation in vivo, Flinders University, School of Medicine
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Mouse models that are used to investigate the biological effects of ionising radiation exposure have shown that different inbred strains respond differently to radiation exposure. Based on end-points such as time to lethality, repair of DNA damage and the development of cancers, these strains are defined as radiation-sensitive or resistant. Ionising radiation has been reported to induce a loss of DNA methylation, a modification of cytosine residues (predominantly when in sequence with a guanine; termed a CpG) that plays an important role in maintaining genome stability by influencing the expression of genes through chromatin structure. The most heavily methylated regions of the genome are found at transposable repeat elements, where a loss of methylation may result in transposition and increased genomic instability. It is not known whether the radiation sensitivity that these animals exhibit is influenced by the modulation of DNA methylation by ionising radiation. This thesis describes the investigation of the modulation of DNA methylation of a class of repeat elements known as LINE1 (L1), in three strains of laboratory mice that differ in radiosensitivity: the C57Bl/6 (radiation-resistant), BALB/c and CBA (radiation-sensitive) mouse strains. A sensitive PCR-based assay was developed in order to investigate the changes in L1 methylation following radiation exposure. The L1 assay utilised high resolution melt technology (HRM), which is able to distinguish between single nucleotide differences in sequences of DNA following PCR amplification. The L1-HRM assay was demonstrated to be able to detect differences in heterogeneous CpG methylation as small as 3%; and was also able to detect changes in methylation between samples that could not be detected by the gold standard method for total genomic 5mdC quantitation (liquid chromatography mass-spectrometry). Compared with other PCR-based methods for DNA methylation analysis, the L1-HRM assay was shown to be a sensitive, high through-put screening tool that did not require post-PCR manipulation in order to detect differences in methylation between samples. Following high dose irradiation (1 Gy), the radiosensitive mouse strains (BALB/c and CBA) exhibited early increases in spleen L1 methylation, which had returned to sham methylation levels by 14 days following irradiation. Differences in responses between male and female mice were also observed, with the male CBA mice demonstrating an earlier response in comparison with the female CBA mice. The radiation-resistant C57Bl/6 mice demonstrated a late change in methylation, where a loss of methylation was observed by 14 days following irradiation. The modulation of L1 DNA methylation was shown to only affect some CpGs within the L1-HRM assay target region, which was consistent across the three strains. This is the first analysis of the modulation of murine L1 element CpGs following radiation exposure. Furthermore, the loss of methylation in the C57Bl/6 mice did not result in an increase in L1 element transcripts. Other murine repeat DNA elements (B1 and Intracisternal-A particle long terminal repeat elements) were found to display similar modulation to that of the L1 elements following irradiation. These results show that strains that differ in radiosensitivity exhibit temporal differences in repeat element methylation responses following exposure to ionising radiation, highlighting the importance of timing of analysis, particularly when analysing the effects of a modulator of DNA methylation that does not appear to affect every CpG. This is the first direct comparison of the temporal DNA methylation response of three strains of mice that differ in radiosensitivity. Low doses of ionising radiation have been shown to demonstrate a protective role for endpoints such as DNA damage and tumour progression, termed the radioadaptive response. The exact mechanism(s) involved in the radioadaptive response are still being identified, and it has been suggested that stabilisation of the genome via the modulation of DNA methylation may be involved. Both radiation exposure and ageing are associated with increased genomic instability, shorter telomeres and reduced DNA methylation. Studies described in this thesis investigated whether a low dose radiation (10 mGy) exposure would modulate repeat element DNA methylation to induce an adaptive response. Following irradiation, the modulation of L1 and B1 DNA methylation of ageing mice was monitored over time using peripheral blood (PB) sampling. A decline in PB L1 and B1 element methylation levels was not observed by 420 days (~18 months of age) post-irradiation; however spleen L1 methylation levels increased with age. No effect of irradiation was detected on PB and spleen L1 and B1 methylation levels or telomere length in the ageing mice. These results indicate that there may be an age-threshold at which repeat element methylation levels decline in ageing animals. Furthermore, these results suggest that a low dose ionising radiation exposure does not elicit a long term effect on DNA methylation levels, nor is an adaptive response induced. This is the first study of the long term effect of a low dose ionising radiation exposure on DNA methylation levels. Very little is known about the effect of radiation exposure on repeat element DNA methylation at the doses used in this thesis. This is the first in vivo methylation study to use low doses of radiation that are in the adaptive response range. The results obtained using the L1-HRM assay exemplify the dynamic nature of DNA methylation over time, both in ageing animals and in response to ionising radiation exposure, highlighting the importance of timing of analysis, tissue type and age of an animal when interpreting DNA methylation responses to exogenous agents.
Keywords: ionising radiation,DNA methylation,DNA repeat elements
Subject: Medicine thesis, Haematology thesis
Thesis type: Doctor of Philosophy
School: School of Medicine
Supervisor: Professor Pamela Sykes