In situ alterations of DNA methylation were studied between 14 d postcoitum and 4 d postpartum in Sertoli cells and germ cells from mouse testis, using anti-5-methylcytosine antibodies. Compared to cultured fibroblasts, Sertoli cells display strongly methylated juxtacentromeric heterochromatin, but hypomethylated chromatids. Germ cells always possess hypomethylated heterochromatin, whereas their euchromatin passes from a demethylated to a strongly methylated status between days 16 and 17 postcoitum. This hypermethylation occurs in the absence of DNA replication, germ cells being blocked in the G₀-G₁ phase from day 15 postcoitum to birth. The DNA hypermethylation of germ cells is maintained until birth and could be visualized on both chromatids of metaphase chromosomes at the first postpartum cell division. Subsequently, the DNA hypermethylation is lost semiconservatively, being replaced by a methylation pattern recalling the typical fibroblast pattern. These alterations of DNA methylation follow a strict chronology, are chromosome structure and cell-type dependent, and may underlie profound changes of genome function.

In order to gain more insight into the relationships between DNA methylation and genome stability, chromosomal and molecular evolutions of four Epstein-Barr virus–transformed human lymphoblastoid cell lines were followed in culture for more than 2 yr. The four cell lines underwent early, strong overall demethylation of the genome. The classical satellite-rich, heterochromatic ,juxtacentromeric regions of chromosomes 1, 9, and 16 and the distal part of the long arm of the Y chromosome displayed specific behavior with time in culture. In two cell lines, they underwent a strong demethylation, involving successively chromosomes Y, 9, 16, and 1, whereas in the two other cell lines, they remained heavily methylated. For classical satellite 2–rich heterochromatic regions of chromosomes 1 and 16, a direct relationship could be established between their demethylation, their undercondensation at metaphase, and their involvement in non-clonal rearrangements. Unstable sites distributed along the whole chromosomes were found only when the heterochromatic regions of chromosomes 1 and 16 were unstable. The classical satellite 3–rich heterochromatic region of chromosomes 9 and Y, despite their strong demethylation, remained condensed and stable. Genome demethylation and chromosome instability could not be related to variations in mRNA amounts of the DNA methyltransferases DNMT1, DNMT3A, and DNMT3B and DNA demethylase. These data suggest that the influence of DNA demethylation on chromosome stability is modulated by a sequence-specific chromatin structure.

We show that in a series of eight breast cancer cell lines, a direct relationship exists between the overall DNA demethylation and the percentage of rearranged chromosomes, except for cell lines with a highly rearranged genome which can be weakly demethylated. A real time fluorescent detection method was used to quantify by reverse transcription-PCR the expression of the DNA methyltransferase 1 and of the newly discovered DNA demethylase. The overall DNA methylation status seems to result from a complex interplay between the expression of these two genes. Our results suggest that in these tumor cells, the overall DNA demethylation is implicated in one of the mechanisms at the origin of the genome instability and that besides the role of the DNA methyltransferase 1, that of the DNA demethylase may be essential in the control of DNA methylation.z 1999 Federation of European Biochemical Societies.