Paola Caiafa

Linker histone binding and displacement: versatile mechanism for transcriptional regulation

In recent years, the connection between chromatin structure and its transcriptional activity has attracted considerable experimental effort. The post‐translational modifications to both the core histones and the linker histones are finely tuned through interactions with transcriptional regulators and change chromatin structure in a way to allow transcription to occur. Here we review evidence for the involvement of linker histones in transcriptional regulation and suggest a scenario in which the reversible and controllable binding/displacement of proteins of this class to the nucleosome entry/exit point determine the accessibility of the nucleosomal DNA to the transcriptional machinery.—Zlatanova, J., Caiafa, P., van Holde, K. Linker histone binding and displacement: versatile mechanism for transcriptional regulation. FASEB J. 14, 1697–1704 (2000)

Epigenetics: poly(ADP-ribosyl)ation of PARP-1 regulates genomic methylation patterns

In the postgenome era, attention is being focused on those epigenetic modifications that modulate chromatin structure to guarantee that information present on DNA is read correctly and at the most appropriate time to meet cellular requirements. Data reviewed show that along the chain of events that induce DNA methylation‐dependent chromatin condensation/decondensation, a postsynthetic modification other than histone acetylation, phosphorylation, and methylation—namely poly(ADP‐ribosyl)ation (PARylation)—participates in the establishment and maintenance of a genome methylation pattern. We hypothesize that the right nuclear balance between unmodified and PARylated poly(ADP‐ribose) polymerase 1 (PARP‐1), which depends on the dynamics of PARPs/PARG activity, is key to maintaining genomic methylation pattern. According to our data, decreased or increased levels of PARylated PARP‐1 are responsible for diffuse hypermethylation or hypomethylation of DNA, respectively. In our model, polymers present on PARP‐1 interact noncovalently with DNA methyltransferase 1 (Dnmt1), preventing its enzymatic activity. In the absence of PARylated PARP‐1, Dnmt1 is free to methylate DNA; if, in contrast, high levels of PARylated PARP‐1 persist, Dnmt1 will be stably inhibited, preventing DNA methylation.—Caiafa, P., Guastafierro, T., Zampieri, M. Epigenetics: poly(ADP‐ribosyl)ation of PARP‐1 regulates genomic methylation patterns. FASEB J. 23, 672–678 (2009)

DNA methylation and chromatin structure: the puzzling CpG islands.

DNA methylation is the epigenetic modification, which introduces 5mC as fifth base onto DNA. As for the distribution of 5mCs, it is well known that they distribute themselves in a non‐random fashion in genomic DNA so that methylation pattern is characterized by the presence of methylated cytosines on the bulk of DNA while the unmethylated ones are mainly located within particular regions termed CpG islands. These regions represent about 1% of genomic DNA and are generally found in the promoter region of housekeeping genes. Their unmethylated state, which is an essential condition for the correct expression of correlated genes, is paradoxical if one considers that these regions are termed CpG islands because they are particularly rich in this dinucleotide, which is the best substrate for enzymes involved in DNA methylation. Anomalous insertion of methyl groups in these regions generally leads to the lack of transcription of correlated genes. An interesting scientific problem is to clarify the mechanism(s) whereby CpG islands, which remain protected from methylation in normal cells, are susceptible to methylation in tumor cells. How the CpG moieties in CpG islands become vulnerable or resistant to the action of DNA methyltransferases and can thus lose or maintain their characteristic pattern of methylation is still an open question. Our aim is to gather some mechanisms regarding this intriguing enigma, which, despite all energy spent, still remains an unresolved puzzle.

Modulation of DNMT1 activity by ADP-ribose polymers

We provided evidence that competitive inhibition of poly(ADP-ribose) polymerases in mammalian cells treated with 3-aminobenzamide causes DNA hypermethylation in the genome and anomalous hypermethylation of CpG islands. The molecular mechanism(s) connecting poly(ADP-ribosyl)ation with DNA methylation is still unknown. Here we show that DNMT1 is able to bind long and branched ADP-ribose polymers in a noncovalent way. Binding of poly ADP-ribose on DNMT1 inhibits DNA methyltransferase activity. Co-immunoprecipitation reactions indicate that PARP1 and DNMT1 are associated in vivo and that in this complex PARP1 is present in its ADP-ribosylated isoform. We suggest that this complex is catalytically inefficient in DNA methylation.

CCCTC-binding factor activates PARP-1 affecting DNA methylation machinery

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.

Parp1 localizes within the Dnmt1 promoter and protects its unmethylated state by its enzymatic activity

Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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.

ADP-ribose polymers localized on Ctcf-Parp1-Dnmt1 complex prevent methylation of Ctcf target sites.

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.

DNA methylation-dependent chromatin fiber compaction in vivo and in vitro: requirement for linker histone

Dynamic alterations in chromatin structure mediated by postsynthetic histone modifications and DNA methylation constitute a major regulatory mechanism in DNA functioning. DNA methylation has been implicated in transcriptional silencing, in part by inducing chromatin condensation. To understand the methylation‐dependent chromatin structure, we performed atomic force microscope (AFM) studies of fibers isolated from cultured cells containing normal or elevated levels of m5C. Chromatin fibers were reconstituted on control or methylated DNA templates in the presence or absence of linker histone. Visual inspection of AFM images, combined with quantitative analysis of fiber structural parameters, suggested that DNA meth‐ylation induced fiber compaction only in the presence of linker histones. This conclusion was further substantiated by biochemical results.—Karymov, M. A., Tomschik, M., Leuba, S. H., Caiafa, P., Zlatanova, J. DNA methylation‐dependent chromatin fiber compaction in vivo and in vitro: requirement for linker histone. FASEB J. 15, 2631–2641 (2001)

Reduced levels of poly(ADP-ribosyl)ation result in chromatin compaction and hypermethylation as shown by cell-by-cell computer-assisted quantitative analysis

The unmethylated status of the CpG islands is important for gene expression of correlated housekeeping genes since it is well known that their methylation inhibits transcription process. An interesting question that has been discussed but not solved is how the CpG islands maintain their characteristic unmethylated status even though they are rich in CpG dinucleotides. Our previous in vitro and in vivo research has shown that poly(ADP‐ribosyl)ation is involved in protecting CpG dinucleotides from full methylation in genomic DNA and that a block of poly(ADP‐ribosyl)ation is also involved in modifying the methylation pattern in the promoter region of Htf9 housekeeping gene. In this study we locked for cytological evidence that in the absence of an active poly(ADP‐ribosyl)ation the DNA methylation pattern in L929 and NIH/3T3 mouse fibroblast cell lines is altered. For this purpose, differences in the methylation levels of interphase nuclei from control and treated cultures of two murine cell lines preincubated with 2 mM 3‐aminobenzamide, an inhibitor of poly(ADP‐ribosyl)ation, were measured in individual cells after indirect immunolabeling with anti‐5MeC antibodies. The quantitative analysis allowed us to demonstrate that blocking of the poly(ADP‐ribosyl)ation results in a higher number, size, and density of antibody binding regions in treated cells when compared to the controls. Analogously, sequential Giemsa staining and indirect immunolabeling of the same slides showed the hetero‐chromatic regions colocalized with the extended methyl‐rich domains.—de Capoa, A., Febbo, F. R., Giovannelli, F., Niveleau, A., Zardo, G., Marenzi, S., Caiafa, P. Reduced levels of poly(ADP‐ribosyl)ation result in chromatin compaction and hypermethylation as shown by cell‐by‐cell computer‐assisted quantitative analysis. FASEB J. 13, 89–93 (1999)

MARK-AGE biomarkers of ageing

Many candidate biomarkers of human ageing have been proposed in the scientific literature but in all cases their variability in cross-sectional studies is considerable, and therefore no single measurement has proven to serve a useful marker to determine, on its own, biological age. A plausible reason for this is the intrinsic multi-causal and multi-system nature of the ageing process. The recently completed MARK-AGE study was a large-scale integrated project supported by the European Commission. The major aim of this project was to conduct a population study comprising about 3200 subjects in order to identify a set of biomarkers of ageing which, as a combination of parameters with appropriate weighting, would measure biological age better than any marker in isolation.