Changes in DNA methylation patterns in mammals with senescence, ageing and energy restriction

Abstract

DNA methylation is a reversible and inheritable chemical modification which involves the addition of a methyl group to DNA catalyzed by DNA methyltransferases (DNMTs), resulting in the conversion of the cytosine to 5’-methylcytosine, where the cytosine residue is followed by a guanine residue (CpG). In mammals, there are unusually dense clusters of CpG dinucleotides in the promoters of genes which are called “CpG islands”. For many housekeeping genes, these CpG islands are unmethylated in normal healthy tissue. However, CpG islands methylation is often associated with gene silencing and changed patterns of DNA methylation, associated with altered patterns of gene transcription, contribute to the aetiology of several diseases and to ageing. The overall aim of this project was to characterise the changes in DNA methylation which are observed during cell senescence (in the human cell line MRC-5) and during ageing and in response to dietary energy restriction (in various mice tissues). For this purpose: i) global DNA methylation was quantified using various techniques (ELISA, LINE-1, B1 and LUMA assays and HPLC); ii) site-specific genome-wide screening for methylation changes was performed using the MeDIP technique followed by hybridization to DNA microarrays; iii) validation of the results for selected candidates was performed by pyrosequencing and iv) investigation of mitochondrial DNA methylation was conducted using bisulphite-modified-DNA PCR direct sequencing. Effects of senescence on gene expression were assessed by transcriptome microarrays and by RT-qPCR studies. During the study, previously established methods for the investigation of global DNA methylation (LUMA) and site-specific methylation (MeDIP) were improved. Whilst global DNA methylation changes were detectable in senescence and after short-term dietary energy restriction, DNMTs/Dnmts expression changes were observed in senescence, ageing and following dietary energy restriction and were tissue- and treatment-specific. In parallel, site-specific aberrant DNA methylation was found in the promoters of the genes CTTN, GLIPR2, NPTX1 and SLC39A14 in replicative senescent MRC-5 human fibroblasts. These changes were validated by pyrosequencing and were accompanied by changes in expression of the corresponding genes. Also, in a pilot study, promoter methylation of several cell cycle genes was altered in replicative senescence with associated changes in gene expression. Concordant methylation changes were found in the promoters of 47 gene in ageing mouse and heart tissues including the genes Wnt5a, Map4k5, Apcdd1, Chp2 and Rasgrp2. In addition, dietary energy restriction counteracted the age-related DNA methylation changes in the promoters of 40 genes, including Aifm1, Irf8, Rarg, Nmi, Maf1, Rab33a and Fxn in mouse liver. Finally, mitochondrial DNA methylation studies revealed that senescence affected the DNA methylation patterns of the MT-COI and MT-ND1 gene coding sequences in MRC-5 fibroblasts whilst ageing affected the DNA methylation pattern of the D-Loop region in mouse liver, but this was not reversed by dietary energy restriction. Pathway analysis revealed that senescence- and age-related aberrant DNA methylation affected genes involved in inter-cellular communication, stress response, malignant transformation, cellular development/proliferation control, cell growth/differentiation and survival, apoptosis and immune response. As these genes contribute to the maintenance of cellular and tissue homeostasis, these findings suggest a potential role for altered DNA methylation in the aetiology of senescence and ageing. On the other hand, short-term dietary energy restriction modulated some of the age-related aberrant DNA methylation patterns of the ageing mouse liver, in particular those in promoters of genes involved in apoptosis regulation, inflammatory and immune response to viral infections, transcription regulation, vesicle trafficking and mitochondrial iron transport and respiration. Finally, mitochondrial DNA aberrant methylation - found to occur at genes belonging to Complex IV and to Complex I - may contribute to the accumulation of hazardous superoxide species in senescent cells whilst DNA aberrant methylation at the D-Loop mitochondrial regulatory region may contribute to age-related mitochondrial dysfunction. In conclusion, these findings suggest that altered DNA methylation may have a role in the aetiology of senescence and ageing and that some of the effects of dietary energy restriction in slowing down the ageing process and also delaying the onset of age-related diseases may occur via epigenetic mechanisms, including amelioration of age-related aberrations in patterns of DNA methylation.