Dipartimento di Biologia, Ecologia e Scienze della Terra

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DNA methylation patterns in aging and aging phenotypes
(2019-04-11) Guarasci, Francesco; Bellizzi, Dina; Cerra, Maria Carmela
During my PhD program, my work has been addressed to the study of the role of epigenetic modifications in aging and in age-related phenotypes. Epigenetics is the study of changes in gene expression that do not involve changes to the underlying DNA sequence. These changes affect cellular phenotypic expression by regulating relative gene expression levels. They are a common and natural process in living cells and are tightly controlled by pre-programmed mechanisms. Epigenetics modifications can be influenced by multiple factors including environmental conditions, lifestyle, nutrition, use of drugs, disease state and age. Patterns of DNA methylation, the best known and characterized epigenetic modification, change during aging; indeed, with increasing aging, genome-wide methylation levels decrease, meanwhile genomic regions, including CpG islands, become more methylated. Analyses of the above patterns provided new perspectives for establishing powerful biomarkers of human aging which have the potential to generate accurate prediction not only of the chronological but also of the biological age. The first section of the PhD thesis consists in a comprehensive overview of the general features of DNA methylation and its implication in age and age-related diseases. The topic is addressed referring to the methylation patterns established not only at nuclear but also at mitochondrial genome level. In addition, the influence of a number of environmental factors on the above patterns is also discussed. In the second section, an original research work, carried out in order to identify novel biomarkers of aging, is reported. In this work, methylation status of nuclear genes involved in mitochondrial fusion, fission, biogenesis and mitophagy, fundamental components of the mitochondrial quality control process, was investigated in subjects of different ages of the Calabrian population. The methylation levels of RAB32 and RHOT2 genes were significantly associated with age and, in particular, those of RAB32 even with the risk of developing disability. The study, therefore, led to the identification of two new biomarkers for both chronological and biological aging. In the Appendix, research works already published are reported. The first one concerns the correlation between DNA methylation and nutrition during lifetime. Global DNA methylation profiles were examined in different tissues of rats of different ages, fed with a standard and hypocaloric diet, and their association with aging and nutrition was evaluated. The results obtained have shown that tissue-specific variations in methylation levels occur during aging and that nutrition influences the state of global DNA methylation during the course of life. The hypocaloric diet seems to influence more strongly the epigenetic status of the offspring when administered during the maternal pre-gestational period compared to the gestation and lactation period. Therefore, changes in the global DNA methylation status represent an epigenetic mechanism by which age and nutrition intersect each oth and, in turn, influence the plasticity of aging. The second one is a review on the current advances in mitochondrial epigenetics studies and the increasing indication of mtDNA methylation status as an attractive biomarker for peculiar physiological and pathological phenotypes. It comes from the increasing evidence on the fact that, similarly to nuclear DNA, also mtDNA is subject to methylation and hydroxymethylation and these modifications are influenced by multiple environmental factors.
Mitochondrial DNA a:nd epigenetics. unexpected complex interactions
(2012-11-30) Giordano, Marco; Bellizzi, Dina; Grandinetti, Lucio
In the research of fundamental processes underlying aging and longevity, an emerging field is represented by "aging epigenetics". In fact, different experimental evidences demonstrate as the rate and quality of human aging depend on a complex interplay among genetic, epigenetic and environmental factors. Epigenetics refers to the programmed changes, not involving alteration of DNA sequence, leading different genotypes to phenotypes. DNA methylation is the most studied epigenetic modification occurring in all prokaryotic and eukaryotic organisms. Although the occurrence of this modification and its effects in intracellular processes has been extensively documented for the nuclear genome, conflicting reports regarding the possible presence of methylated cytosines within the mitochondrial DNA (mtDNA) have emerged. In addition, in spite of the hypothesized role of ATP availability on the methylation process , little is known about the role of mitochondrial DNA variability on the methylation status of nuclear genome. The work presented here, has addressed the complex interactions between mitochondrial DNA and epigenetic changes, both investigating the methylation of mitochondrial genome and analyzing the effects of mtDNA variation on the Global methylation of nuclear genome. In fact, a series of in vivo and in vitro investigations are here reported in three sections. In the first two sections, experimental evidences about the presence of methylated cytosines within the mitochondrial control region (D-loop), containing regulator elements for replication and transcription of mtDNA, and within the gene encoding for ribosomal RNA 12S (12S rRNA), are reported. The methylation status of the D-loop was analyzed in both blood DNA collected from human subjects and in DNA from cultured cells by bisulfite sequencing and, consecutively, by methylated/hydroxymethylated DNA immunoprecipitation assays. We found the presence of methylated (5mC) and hydroxymethylated (5hmC) cytosines in all the samples analyzed. MtDNA methylation especially occurs within non-CpG sites. It also emerged that the methylation pattern of the D-loop is strictly cell type-dependent and that it might be an example of RNA-directed DNA methylation (RdDM). The methylation of D-loop occurred mainly in the promoter region of the heavy strand and in conserved sequence Summary blocks (CSBI-III), indicating the involvement of epigenetic modifications in regulating mtDNA replication and/or transcription. Bisulfite sequencing of 12S rRNA region showed the methylation of one CpG site located at 931nt. This site was then analyzed by real-time MGB Probe-based PCR reactions in bisulfite-treated DNA extracted from peripheral venous blood collected from subjects of different age and classified for frailty phenotype. We detected the co-presence of both unmethylated and methylated cytosines in most sample analyzed. Statistical analyses revealed that 12S rRNA methylation displays sex- and age-specific differences, and it is correlated with the health status. In the third section, it is reported that global DNA methylation levels are influenced by mitochondrial DNA inherited variants, probably via the different regulation of OXPHOS machinery. So that, we prove, through cybrid technology, which is an useful approach to reveal possible pathways of communication between mitochondrial and nuclear genomes, as epigenetics processes are modulated in response to the above pathways. In the appendix an additional published paper (Montesanto et al., The genetic component of human longevity: analysis of the survival advantage of parents and siblings of Italian nonagenarians, 2011, Eur J Hum Genet 19: 882-886.), which I worked to during my PhD appointment is reported.

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