dc.description.abstract | 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. | en_US |