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 methyla tion inhibits transcription process. An interesting question that has been discussed but not solved is how the CpG islands maintain their characterist ic unmethylated status even though they are rich in CpG dinucleotides. Our previous in vitro and in vivo research has shown that poly(ADP-ribosyl)atio n is involved in protecting CpG dinucleotides from full methylation in geno mic DNA and that a block of poly(ADP-ribosyl)ation is also involved in modi fying the methylation pattern in the promoter region of Htf9 housekeeping g ene. In this study we locked for cytological evidence that in the absence o f an active poly(ADP-ribosyl)ation the DNA methylation pattern in L929 and NIH/3T3 mouse fibroblast cell lines is altered. For this purpose, differenc es 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 a fter indirect immunolabeling with anti-5MeC antibodies. The quantitative an alysis allowed us to demonstrate that blocking of the poly(ADP-ribosyl)atio n results in a higher number, size, and density of antibody binding regions in treated cells when compared to the controls. Analogously, sequential Gi emsa staining and indirect immunolabeling of the same slides showed the het erochromatic regions colocalized with the extended methyl-rich domains.