Repetitive sequences, including transposable elements, represent approximately half of the mammalian genome. Epigenetic mechanisms evolved to repress these potentially deleterious mobile elements. However, such elements can be variably silenced between individuals – so called ‘metastable epialleles’. The best known example is the Avy locus where an endogenous retrovirus (ERV) of the intracisternal A-particle (IAP) class was spontaneously inserted upstream of the agouti coat colour gene, resulting in variable IAP promoter DNA methylation, variable expressivity of coat phenotype, and environmentally modulated transgenerational epigenetic inheritance within genetically identical individuals. It is not known whether the behaviour exhibited by the ERV at Avy represents a common occurrence throughout the genome or is unusual. Taking a genetic approach in purified cell populations, I have conducted a systematic genome-wide screen of murine metastable epialleles. I have identified over 100 murine IAPs with properties of metastable epialleles. Like Avy, each exhibits a stable epigenetic state within an individual but epigenetic variability between individuals. Methylation levels are locus-specific within an individual, suggesting cis-acting control. The same screening strategy was applied for identification of metastable epialleles associated with other types of LTR-retroelements. However, many of identified candidates showed no inter-individual methylation variation upon experimental validation. These results suggest that IAPs are the dominant class of ERVs capable of acquiring epigenetic states that are variable between genetically identical individuals. I have conducted an analysis of IAP induced initiation and termination of transcription events using de novo assembled transcriptomes generated for B and T cells. 143 IAPs have been identified to overlap de novo assembled transcripts. 33 IAPs are metastable epialleles. Several of them show an inverse correlation between LTR promoter methylation and adjacent gene expression. In addition, I have shown that metastable epialleles have a characteristic pattern of histone modification and are flanked by the methylation sensitive binding factor CTCF, providing testable hypotheses concerning the establishment and/or maintenance of the variable methylation state. My findings indicate that metastability is, in general, specific to the IAP class of ERVs, that only around 1% of these elements have this unusual epigenetic property and that the ability to impact transcription, such as at agouti in Avy, is not a ubiquitous feature of these loci.

<p>Low-dose ionizing radiation (LDIR) exposure (under 10.0 centigray (cGy)) from man-made sources, such as diagnostic imaging, predominates in the US population and comprises nearly 50% of an average individual's yearly radiation exposure (Ullrich, Brooks et al. 2009). The increase in such exposures has led to public and government alarm about the impact of LDIR on human health (Ullrich, Brooks et al. 2009). Besides the mutational effects of radiation exposure, there is concern it might also result in modifications of the epigenome. Such aberrations can disrupt normal development and are involved in the progression of numerous diseases, including cancer (Gasser and Li 2011). High doses of radiation (>100.0 cGy) have been shown to cause epigenetic disruption (Kaup, Grandjean et al. 2006; Tamminga, Koturbash et al. 2008; Ilnytskyy, Koturbash et al. 2009), which is necessary for the persistence of radiation-induced genomic instability (Rugo, Mutamba et al. 2011); however, it is presently unclear to what extent LDIR in vivo alters the epigenome. </p><p>The viable yellow agouti (Avy) mouse was used here to characterize the dose-dependent epigenetic response to LDIR. The Avy mouse is a unique biological model that functions as a biosensor for environmentally induced epigenetic changes and disease susceptibility due to the presence of a metastable epiallele that modulates coat color (Waterland and Jirtle 2003). Pregnant dams were whole-body exposed to one of five doses of X-ray radiation ranging from 0-10.0 cGy on gestational day 4.5. Using a phantom mouse model, the intrauterine doses were estimated to be 0.0 cGy, 0.4 cGy, 0.7 cGy, 1.4 cGy, 3.0 cGy, and 7.6 cGy, respectively. At weaning, offspring coat colors were assessed and tissues were collected for methylation analysis. First, methylation changes at CpG sites in the Avy and Cdk activator binding protein (CabpIAP) metastable epialleles and at intracisternal a particle (IAP) elements across the genome were quantified using Sequenom technology. Second, three imprinted genes, Peg3, Nnat, and H19, were assessed for methylation changes in differentially methylated regions (DMRs) that regulate their parent-of-origin monoallelic expression using Sequenom technology. Lastly, it was postulated that the epigenetic changes at the Avy locus could be counteracted with dietary alterations. To test this hypothesis, female mice were placed on an antioxidant-supplemented diet prior to pregnancy and throughout gestation and lactation. Pregnant dams were irradiated with 3.0 cGy of whole-body X-rays. Offspring coat colors were assessed and methylation changes at the Avy allele were measured with the Sequenom platform. </p><p>Herein, I demonstrate that in utero LDIR exposure induced epigenetic changes in the Avy mouse in a dose-dependent and sex-specific manner. Acute, whole-body exposure to 0.7 cGy, 1.4 cGy, 3.0 cGy or 7.6 cGy X-rays significantly shifted offspring coat color distribution toward pseudoagouti. Acute exposure to 1.4 cGy, 3.0 cGy, and 7.6 cGy significantly increased methylation at multiple CpG sites in the Avy metastable epiallele in male offspring, but not female offspring. Methylation changes at DMRs in Nnat, Peg3, and H19 also occurred in a dose-dependent manner. Furthermore, inhibition of the phenotypic and Avy methylation changes with an antioxidant-supplemented diet suggests that the mechanisms to induce epigenetic changes are mediated by oxidative stress. These results demonstrate that relevant, low doses of radiation can elicit epigenetic changes that lead to a persistent phenotype, but can be mitigated with dietary supplementation. The successful completion of this project has resulted in the first in vivo epigenetic characterization of LDIR exposure and will contribute to the development of more relevant risk assessment strategies for protecting human populations.</p> / Dissertation

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24653号 / 農博第2536号 / 新制||農||1097(附属図書館) / 学位論文||R5||N5434(農学部図書室) / 京都大学大学院農学研究科農学専攻 / (主査)教授 土井 元章, 教授 田尾 龍太郎, 教授 那須田 周平 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Orientador: Michel Georges Albert Vincentz / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T03:47:47Z (GMT). No. of bitstreams: 1 Silveira_AmandaBortolini_D.pdf: 6025388 bytes, checksum: 97930bc131f2a0eac6926dae1eeb9319 (MD5) Previous issue date: 2012 / Resumo: Modificações epigenéticas do DNA ou da cromatina atuam principalmente no controle da atividade de elementos de transposição, podendo também silenciar genes, geralmente quando estes estão associados a elementos de transposição ou sequências repetidas. Em plantas, alguns alelos epigenéticos afetando caracteres como morfologia floral, florescimento, estatura ou amadurecimento do fruto foram descritos, revelando o potencial deste tipo de regulação para gerar variabilidade fenotípica herdável não necessariamente vinculada a alterações da sequência de DNA. No entanto, o impacto de mecanismos epigenéticos em processos de evolução adaptativa é ainda bastante desconhecido, em parte, pela falta de informação sobre variação epigenética em populações naturais. Identificamos Qua-Quine Starch (QQS) de Arabidopsis thaliana como um gene sob um controle epigenético flexível e, portanto, particularmente propenso a variações epialélicas frequentes na natureza. QQS é um gene recente, que provavelmente originou-se de novo em Arabidopsis thaliana em uma região rica em elementos de transposição. Mostramos que QQS apresenta-se diferencialmente expresso entre acessos naturais assim como entre indivíduos diretamente coletados na natureza e que estas diferenças de expressão estão negativamente correlacionadas com o nível de metilação de sequências repetidas localizadas em sua região promotora e 5' UTR, não estando relacionadas a variação genética em cis ou trans. Mostramos ainda que variação epialélica em QQS é independente do nível de metilação de transposons vizinhos e que pode ser estavelmente herdada entre gerações. Considerando o impacto potencial de padrões de expressão contrastantes de QQS no metabolismo de amido, um importante componente para produção de biomassa e crescimento, sugerimos que variação epialélica em QQS possa ter implicações adaptativas. Nossos dados também apontam pela primeira vez uma ligação potencial entre mecanismos epigenéticos e o processo de evolução de genes novos. Propomos que genes novos, especialmente os de origem de novo, poderiam ser mais propensos a variar epigeneticamente, o que permite um ajuste fino de seu padrão de expressão até que o estado mais vantajoso seja fixado geneticamente / Abstract: Epigenetic modifications of DNA or chromatin control of the activity of transposable elements and can also silence genes which are associated to transposons or repetitive sequences. In plants, epigenetic alleles affecting characters such as floral morphology, flowering, stature or fruit ripening have been described, highlighting the potential of this type of regulation in generating heritable phenotypic diversity, not necessarily linked to DNA sequence alterations. However, the impact of epigenetic mechanisms in adaptative evolution is still largely unknown, in part, due to the lack of information about epiallelic variation in natural populations. We have identified Qua-Quine Starch (QQS) of Arabidopsis thaliana as a gene under flexible epigenetic control and thus particularly prone to epiallelic variation in nature. QQS is a recent gene that likely originated de novo in Arabidopsis thaliana in a transposon-rich region. We show that QQS is differentially expressed among natural accessions as well as among individuals directly sampled from the wild and that these expression differences are negatively correlated with the DNA methylation level of repeat sequences located on QQS promoter and 5'UTR region and are not correlated with cis or trans genetic variation. We also show that epiallelic variation at QQS is independent of the methylation status of nearby transposable elements and can be stably inherited across generations. Considering the potential impact of contrasting QQS expression patterns on starch accumulation, an important component of biomass production and growth, we suggest that epiallelic variation at QQS may have adaptative implications. Our data also points for the first time to a potential link between epigenetic mechanisms and the evolution of novel genes. We suggest that novel genes, more specifically those created de novo, could be endowed with an increased potential for epigenetic variation and thus for adjusting their expression pattern until the most adaptive state becomes genetically fixed / Doutorado / Genetica Vegetal e Melhoramento / Doutor em Genetica e Biologia Molecular