de Capoa A, Febbo F R, Giovannelli F, Niveleau A, Zardo G, Marenzi S, Caiafa P
Department of Genetics and Molecular Biology, University of Rome La Sapienza Rome, Italy.
FASEB J. 1999 Jan;13(1):89-93. doi: 10.1096/fasebj.13.1.89.
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 heterochromatic regions colocalized with the extended methyl-rich domains.
CpG岛的未甲基化状态对于相关管家基因的基因表达很重要，因为众所周知，它们的甲基化会抑制转录过程。一个已经被讨论但尚未解决的有趣问题是，尽管CpG岛富含CpG二核苷酸，但它们如何维持其特有的未甲基化状态。我们之前的体外和体内研究表明，聚（ADP-核糖基）化参与保护基因组DNA中的CpG二核苷酸免于完全甲基化，并且聚（ADP-核糖基）化的阻断也参与修饰管家基因Htf9启动子区域的甲基化模式。在本研究中，我们寻找细胞学证据，证明在缺乏活性聚（ADP-核糖基）化的情况下，L929和NIH/3T3小鼠成纤维细胞系中的DNA甲基化模式会发生改变。为此，在用聚（ADP-核糖基）化抑制剂2 mM 3-氨基苯甲酰胺预孵育的两种鼠细胞系的对照和处理培养物的间期核中，用抗5MeC抗体进行间接免疫标记后，在单个细胞中测量甲基化水平的差异。定量分析使我们能够证明，与对照相比，聚（ADP-核糖基）化的阻断导致处理细胞中抗体结合区域的数量、大小和密度更高。类似地，对同一张玻片进行连续吉姆萨染色和间接免疫标记显示，异染色质区域与扩展的富含甲基的结构域共定位。
