DNA methylation of the extraembryonic tissues: an in situ study on human metaphase chromosomes Nadja Kokalj-Vokac, Andreja Zagorac, Marija Pristovnik, Claire A. Bourgeois & Bernard Dutrillaux Received 13 October 1997; received in revised form and accepted for publication by H. Macgregor 16 December 1997 DNA methylation level and pattern of human meta- phase chromosomes from extraembryonic tissues (chorionic villi and placental ®broblasts) were ana- lysed in situ. The DNA methylation global level of these tissues was studied by comparing them with the one observed in fetal ®broblasts and adult lymphocytes. In order to assess the tissue speci®- city and signi®cance of the observed differences, chromosomal preparations were then treated in parallel. They were ®rst stained with distamycin A/ DAPI and pictured, then treated with immuno¯uores- cent staining using monoclonal antibodies raised against 5-methylcytosine. Compared with meta- phases from lymphocytes or placental and fetal ®broblasts, distamycin-A/DAPI stained metaphases and constitutive heterochromatic regions with very similar intensities. In contrast, in chorionic villi, the immuno¯uorescent intensities revealing the pre- sence of 5-methylcytosine was much duller than in the other tissues. In addition, in both chorionic villi and placental ®broblasts, large differences were ob- served between various chromosome structures within individual metaphases. In particular, the sec- ondary constriction of chromosome 9, the distal segment of chromosome Y and the short arms of acrocentric chromosomes exhibited a much lower staining than the one observed for the secondary constrictions of chromosome 1 and 16 of the same metaphases. Because all these structures are known to be deeply methylated in other somatic tissues, this suggests that in extraembryonic tissues DNA methylation level remained hypomethylated and the pattern is under precise control. Key words: DNA methylation, embryogenesis, extra- embryonic tissues, heterochromatin Introduction In mammalian cells, about 4% of all cytosine residues are methylated as 5-methylcytosine (5mC), almost exclusively at CpG dinucleotides. The haploid mamma- lian genome contains approximately 5 3 10 7 CpG dinu- cleotides, of which about 60% are methylated. The 5mC are distributed throughout the genome in cell type-speci®c patterns, which are faithfully transmitted by clonal inheritance during DNA replication (Bird 1987, Bestor 1990). These methyl groups are known to affect protein DNA interactions by either preventing or facilitating protein binding to the DNA. The functional signi®cance of this modi®cation of the DNA is re¯ected in a number of important biological processes: gene expression, cell differentiation, tumorigenesis and par- ental imprinting (Razin & Kofri 1994). DNA methyla- tion is thus essential for normal embryonic development (Li et al. 1992). Several approaches were used to study DNA methylation, mostly at the mole- cular level (Saluz & Jost 1993). Using monoclonal antibodies to 5mC (anti-5mC), the sites of methylcyto- sine-rich DNA were detected on metaphase chromo- somes of the normal human lymphocytes and different types of cell lines (Barbin et al. 1994, Montpellier et al. 1994, Bernardino et al. 1996), demonstrating a recurrent pattern of immuno¯uorescent signals. This DNA methylation pattern was modi®ed in lymphoblastoid cell lines and cancer cells (Barbin et al. 1994, Montpellier et al. 1994, Veilleux et al. 1996). While cell-speci®c methylation patterns are quite stable in somatic cells, methylation is subject to dramatic changes in the developing embryo. Embryonic cells perform active demethylation and de novo methylation (Frank et al. 1991, Shemer et al. 1991). In mouse, methylation patterns of extraembryonic tissues occur independently and differently from those in the em- bryonic genome. Overall, genomic methylation in extra- embryonic lineages does not occur to the ®nal extent as observed in the fetal somatic tissue (Monk et al. 1987, Frank et al. 1991, Shemer et al. 1991). In this study, we looked for differences in DNA methylation levels and patterns between chorionic villus samples or placental ®broblasts as extraembryonic tissues and normal human lymphocytes or fetal skin ®broblasts as controls using an in situ analysis on metaphase chromosomes. Chromosome preparations were made from material obtained after termination of pregnancy at various times of gestation to search for possible changes occurring during fetal development. Chromosome Research 1998, 6, 161±166 # 1998 Rapid Science Publishers N. Kokalj-Vokac (corresponding author), A. Zagorac and M. Pristovnik are at Maribor Teaching Hospital, Cytogenetic Laboratory, Ljubljanska 5, 2000 Maribor, Slovenia. C. A. Bourgeois and B. Dutrillaux are at Institut Curie-CNRS UMR 147, Section de Recherche, 26 rue d'Ulm, F75248 Paris Cedex 05, France. Chromosome Research Vol 6 1998 161