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The levels of genomic DNA methylation in vertebrate species display a wide range of developmental dynamics. Here, we show that in contrast to mice, the paternal genome of the amphibian, Xenopus laevis, is not subjected to active demethylation of 5-methyl cytosine immediately after fertilization. High levels of methylation in the DNA of both oocyte and sperm are maintained in the early embryo but progressively decline during the cleavage stages. As a result, the Xenopus genome has its lowest methylation content at the midblastula transition (MBT) and during subsequent gastrulation. Between blastula and gastrula stages, we detect a loss of methylation at individual Xenopus gene promoters (TFIIIA, Xbra, and c-Myc II) that are activated at MBT. No changes are observed in the methylation patterns of repeated sequences, genes that are inactive at MBT, or in the coding regions of individual genes. In embryos that are depleted of the maintenance methyltransferase enzyme (xDnmt1), these developmentally programmed changes in promoter methylation are disrupted, which may account for the altered patterns of gene expression that occur in these embryos. Our results suggest that DNA methylation has a role in regulating the timing of gene activation at MBT in Xenopus laevis embryos. Key Words: Xenopus; development; demethylation; xDnmt1.

DNA methylation patterns in preimplantation mouse development have been studied by analysis of monoclonal antibody-to-5-methylcytosine (5-MeC) binding on metaphase chromosomes. At each cell stage certain chromosome patterns appeared. It appears that chromosome demethylation is associated with each DNA replication. A passive mechanism appears to be predominant in early development.

Antibodies directed against N7-methylguanosine (m7Guo) were prepared and added to a wheat germ cell-free protein-synthesizing system programmed with RNA extracted from monkey cells persistently infected with measles virus. A dose-dependent inhibition of [35S]methionine incorporation was observed when RNA was preincubated with anti-m7Guo immunoglobulins. Antibodies preincubated with m7Guo did not show any inhibiting activity. The inhibitory effect of antibodies was abolished when RNA was preincubated with immunoglobulins in the presence of spermine and spermidine. When polyamines were added to the assay programmed with the IgG-RNA complex, no inhibition was observed. [ABSTRACT FROM AUTHOR]

Antibodies specifically directed to 5-methylcytidine were raised in rabbits and purified by affinity chromatography. The accessibility of 5-methyldeoxycytidine (m5dCyd) to such antibodies was studied with DNAs from various origins. The reaction was followed by measuring the retention of radiolabelled DNA by antibodies on nitrocellulose filters, by immunoprecipitation, by gel filtration and was visualized with the electron microscope. Antibodies did not bind to Escherichia coli B DNA, which is deficient in m5dCyd. Denatured and native DNA from calf thymus, which contains m5dCyd as a minor nucleoside, was weakly retained on the filters whereas DNA extracted from Xanthomonas oryzac XP12 bacteriophage, which is rich in m5dCyd, was well recognized even in the native form. [ABSTRACT FROM AUTHOR]

A computer-assisted assay based on the quantitative analysis of DNA methylation in individual interphase nuclei by indirect immunolabelling with anti-5-methylcytosine antibodies was recently developed in our laboratory. In situ analyses were performed on individual nuclei from normal and experimentally hypo- or hypermethylated cultured cells as well as on human peripheral blood B-lymphocytes from normal and chronic lymphoid leukemia (CLL) samples. We present the results obtained on cells from patients affected by different degrees of preneoplastic or neoplastic changes of the uterine cervix as compared to normal controls. The analysis of DNA methylation in individual cells from cytofuge samples was performed as follows: within each nucleus the eu- and heterochromatin methylation levels were quantified in the grey scale range by dedicated software in terms of numbers, areas and optical densities (ODs) of the immunolabeled dense heterochromatic regions ("spots"), and of the optical density of nuclear background, i.e., of nuclear euchromatin. Analogously, in randomly chosen microscope fields of tissue sections from paraffin-embedded samples, progressive tissue demethylation was observed in dysplastic and cancer cells as compared to normal ones. Both methods showed significant and progressive DNA hypomethylation in dysplastic and cancer cells as compared to control specimens.

The DNA methylation pattern is an important component of the epigenome that regulates and maintains gene expression programs. In this paper, we test the hypothesis that vertebrate cells possess mechanisms protecting them from epigenomic stress similar to DNA damage checkpoints. We show that knockdown of DNMT1 (DNA methyltransferase 1) by an antisense oligonucleotide triggers an intra-S-phase arrest of DNA replication that is not observed with control oligonucleotide. The cells are arrested at different positions throughout the S-phase of the cell cycle, suggesting that this response is not specific to distinct classes of origins of replication. The intra-S-phase arrest of DNA replication is proposed to protect the genome from extensive DNA demethylation that could come about by replication in the absence of DNMT1. This protective mechanism is not induced by 5-aza-2'-deoxycytidine, a nucleoside analog that inhibits DNA methylation by trapping DNMT1 in the progressing replication fork, but does not reduce de novo synthesis of DNMT1. Our data therefore suggest that the intra-S-phase arrest is triggered by a reduction in DNMT1 and not by demethylation of DNA. DNMT1 knockdown also leads to an induction of a set of genes that are implicated in genotoxic stress response such as NF-kappaB, JunB, ATF-3, and GADD45beta (growth arrest DNA damage 45beta gene). Based on these data, we suggest that this stress response mechanism evolved to guard against buildup of DNA methylation errors and to coordinate inheritance of genomic and epigenomic information.