Our previous data have shown that in L929 mouse fibroblasts the control of methylation pattern depends in part on poly(ADP-ribosyl)ation and that ADP-ribose polymers (PARs), both present on poly(ADP-ribosyl)ated PARP-1 and/or protein-free, have an inhibitory effect on Dnmt1 activity. Here we show that transient ectopic overexpression of CCCTC-binding factor (CTCF) induces PAR accumulation, PARP-1, and CTCF poly(ADP-ribosyl)ation in the same mouse fibroblasts. The persistence in time of a high PAR level affects the DNA methylation machinery; the DNA methyltransferase activity is inhibited with consequences for the methylation state of genome, which becomes diffusely hypomethylated affecting centromeric minor satellite and B1 DNA repeats. In vitro data show that CTCF is able to activate PARP-1 automodification even in the absence of nicked DNA. Our new finding that CTCF is able per se to activate PARP-1 automodification in vitro is of great interest as so far a burst of poly(ADP-ribosyl)ated PARP-1 has generally been found following introduction of DNA strand breaks. CTCF is unable to inhibit DNMT1 activity, whereas poly(ADP-ribosyl)ated PARP-1 plays this inhibitory role. These data suggest that CTCF is involved in the cross-talk between poly(ADP-ribosyl)ation and DNA methylation and underscore the importance of a rapid reversal of PARP activity, as DNA methylation pattern is responsible for an important epigenetic code.

PARylation [poly(ADP-ribosyl)ation] is involved in the maintenance of genomic methylation patterns through its control of Dnmt1 [DNA (cytosine-5)-methyltransferase 1] activity. Our previous findings indicated that Ctcf (CCCTC-binding factor) may be an important player in key events whereby PARylation controls the unmethylated status of some CpG-rich regions. Ctcf is able to activate Parp1 [poly(ADP-ribose) polymerase 1], which ADP-ribosylates itself and, in turn, inhibits DNA methylation via non-covalent interaction between its ADP-ribose polymers and Dnmt1. By such a mechanism, Ctcf may preserve the epigenetic pattern at promoters of important housekeeping genes. The results of the present study showed Dnmt1 as a new protein partner of Ctcf. Moreover, we show that Ctcf forms a complex with Dnmt1 and PARylated Parp1 at specific Ctcf target sequences and that PARylation is responsible for the maintenance of the unmethylated status of some Ctcf-bound CpGs. We suggest a mechanism by which Parp1, tethered and activated at specific DNA target sites by Ctcf, preserves their methylation-free status.

Aberrant hypermethylation of CpG islands in housekeeping gene promoters and widespread genome hypomethylation are typical events occurring in cancer cells. The molecular mechanisms behind these cancer-related changes in DNA methylation patterns are not well understood. Two questions are particularly important: (i) how are CpG islands protected from methylation in normal cells, and how is this protection compromised in cancer cells, and (ii) how does the genome-wide demethylation in cancer cells occur. The latter question is especially intriguing since so far no DNA demethylase enzyme has been found. Our data show that the absence of ADP-ribose polymers (PARs), caused by ectopic over-expression of poly(ADP-ribose) glycohydrolase (PARG) in L929 mouse fibroblast cells leads to aberrant methylation of the CpG island in the promoter of the Dnmt1 gene, which in turn shuts down its transcription. The transcriptional silencing of Dnmt1 may be responsible for the widespread passive hypomethylation of genomic DNA which we detect on the example of pericentromeric repeat sequences. Chromatin immunoprecipitation results show that in normal cells the Dnmt1 promoter is occupied by poly(ADP-ribosyl)ated Parp1, suggesting that PARylated Parp1 plays a role in protecting the promoter from methylation. In conclusion, the genome methylation pattern following PARG over-expression mirrors the pattern characteristic of cancer cells, supporting our idea that the right balance between Parp/Parg activities maintains the DNA methylation patterns in normal cells. The finding that in normal cells Parp1 and ADP-ribose polymers localize on the Dnmt1 promoter raises the possibility that PARylated Parp1 marks those sequences in the genome that must remain unmethylated and protects them from methylation, thus playing a role in the epigenetic regulation of gene expression.

Aging is associated with alterations in the content and patterns of DNA methylation virtually throughout the entire human lifespan. Reasons for these variations are not well understood. However, several lines of evidence suggest that the epigenetic instability in aging may be traced back to the alteration of the expression of DNA methyltransferases. Here, the association of the expression of DNA methyltransferases DNMT1 and DNMT3B with age has been analysed in the context of the MARK‐AGE study, a large‐scale cross‐sectional study of the European general population. Using peripheral blood mononuclear cells, we assessed the variation of DNMT1 and DNMT3B gene expression in more than two thousand age‐stratified women and men (35–75 years) recruited across eight European countries. Significant age‐related changes were detected for both transcripts. The level of DNMT1 gradually dropped with aging but this was only observed up to the age of 64 years. By contrast, the expression of DNMT3B decreased linearly with increasing age and this association was particularly evident in females. We next attempted to trace the age‐related changes of both transcripts to the influence of different variables that have an impact on changes of their expression in the population, including demographics, dietary and health habits, and clinical parameters. Our results indicate that age affects the expression of DNMT1 and DNMT3B as an almost independent variable in respect of all other variables evaluated.

Quantitative data from experiments of gene expression are often normalized through levels of housekeeping genes transcription by assuming that expression of these genes is highly uniform. This practice is being questioned as it becomes increasingly clear that the level of housekeeping genes expression may vary considerably in certain biological samples. To date, the validation of reference genes in aging has received little attention and suitable reference genes have not yet been defined. Our aim was to evaluate the expression stability of frequently used reference genes in human peripheral blood mononuclear cells with respect to aging. Using quantitative RT-PCR, we carried out an extensive evaluation of five housekeeping genes, i.e. 18s rRNA, ACTB, GAPDH, HPRT1 and GUSB, for stability of expression in samples from donors in the age range 35-74 years. The consistency in the expression stability was quantified on the basis of the coefficient of variation and two algorithms termed geNorm and NormFinder. Our results indicated GUSB be the most suitable transcript and 18s the least for accurate normalization in PBMCs. We also demonstrated that aging is a confounding factor with respect to stability of 18s, HPRT1 and ACTB expression, which were particularly prone to variability in aged donors.