Técnicas de procesamiento y análisis de datos

Méthylation of cytosine residues in vertebrate DNA has a regulatory role in that méthylation of specific sites in the vicinity of certain genes suppresses transcription of those genes (reviewed by Doerfler, 1983). However, little is known about how specific patterns of méthylation are established in early development due to the lack of sufficiently sensitive techniques to analyse the DNA in the amount of tissue available. Variation in overall DNA méthylation clearly occurs during normal development. Monk et al (1987) described an initial loss of overall genomic méthylation during preimplantation development, followed by de novo méthylation occurring to different degrees in the embryonic, extraembryonic and germ cell lineages. Other studies show that repetitive and low copy number DNA sequences are substantially undermethylated in all derivatives of two extraembryonic cell lineages in early mouse embryos, whereas DNA of the postimplantation embryonic lineage is highly methylated (Chapman et al, 1984; Sanford et al, 1985; Monk et al, 1987; Sanford et al, 1987). This tissue-specific méthylation pattern correlates with the specificity of X-inactivation (preferential paternal X-inactivation) in these lineages in female conceptuses. However, it is not known whether the overall méthylation difference is associated with the imprinting of the X chromosomes in extraembryonic lineages.

Marsupial development differs from early development of placental mammals in that the blastocyst is unilaminar, so that both the embryonic and extraembryonic cells are derived from a single layer of cells (protoderm) which faces the blastocyst cavity. Also, all cells in female marsupial conceptuses so far examined show preferential paternal X-inactivation. Stevens et al (1988) demonstrated that the pattern of DNA méthylation of the marsupial genome during development is similar to that of the mouse, with embryonic DNA being more highly methylated than extraembryonic DNA, despite the occurrence of paternal X-inactivation and the outside position of marsupial embryonic cells.

The basis of these large scale changes in méthylation is uncertain. One possibility that should be considered is the availability and activity of MTases in development. During preimplantation development, the activity of MTase will be determined by the level of enzyme inherited in the egg, the stability of maternally inherited enzyme and the timing of activation of transcription of the embryonic gene for the enzyme.

Monk et al (1991) show a large decrease in methylase activity in early mouse development. If one assumes that the level of activity measured in vitro is correlated with the activity of the enzyme in vivo, the marked decrease in enzyme activity might account for the overall decrease in méthylation at the blastocyst stage. A decrease in methylase activity during de-differentiation of Chlamydomonas gametes appears to be the reason for a loss of chloroplast méthylation (Sano et al, 1984). The decrease in methylase in this case was consistent with dilution of the enzyme during cell division.

However, the results in the mouse embryo are not so easily interpreted owing to the fact that the level of methylase in the egg is very high at the start. A four hundredfold increase in the concentration of DNA methylase is found in fully grown oocytes and MIX stage oocytes as compared to somatic cells (Howlett & Reik, 1991). High DNA methylase activity is also associated with unfertilized eggs (Monk et al, 1991). With this high level of methylase, de novo méthylation may be occurring from the onset of development of the fertilised egg. The efficiency of the de novo méthylation may be low in early development (see Adams & Burdon, 1985; Adams, 1990) so that an

overall increase in méthylation only becomes detectable by the ICM stage (Monk, 1988). Alternatively, the high methylase in the egg may ensure that sufficient enzyme survives dilution (due to cell division) so as to be available for the onset of de novo

méthylation at a later stage (Monk et al, 1991).

There is also some evidence that de novo méthylation may be occuiing during oocyte growth. The Intracistemal A Particle (lAP) gene and the Murine Urinary Protein (MUP) gene are highly methylated in unfertilized eggs (Howlett & Reik, 1991) and the RSV-Ig-myc transgene becomes methylated during oocyte growth (Chaillet et al,

1991).

A targeted mutation of the DNA MTase gene results in a threefold reduction in 5mC in mutant mice (Li et al, 1992); homozygous mutant embryos are stunted and die at midgestation suggesting that DNA méthylation is an essential requirement for normal mammalian development. Significant morphogenesis and tissue differentiation are nonetheless observed in these homozygous mutants and several explanations could account for their apparently normal development up to organogenesis. Firstly, normal méthylation patterns may be maintained in early development by additional MTases; however, the evidence for more than one species of MTase is unconvincing (see section 1.7.4). An alternative and more likely explanantion is that early development proceeds normally in the presence of high levels of maternal MTase inherited in the egg (see above). Only when the embryo begins to acquire adult levels of méthylation, starting at the time of gastrulation (Monk et al, 1987), do the reduced levels of MTase become limiting for development. Genes that are normally repressed by méthylation may be activated in the presence of limiting levels of MTase; expression of many genes at high levels or in inappropriate tissues might result in cell death or a reduced ability of affected cells to participate in tissue formation. The targeted mutation of MTase may elucidate the proposed role of méthylation in other developmental processes, including X-inactivation and imprinting (see below).