Exemption of Satellite DNA from Demethylation in Immortalized Differentiated Derivatives of F9 Mouse Embryonal Carcinoma Cells

Abstract

DNA methylation in F9 embryonal carcinoma cells at various stages of retinoic acid-induced differentiation was compared to that in immortalized differentiated derivatives of this cell line. Different repetitive sequences, such as L1 LINE, GAPDH pseudogenes, and major and minor satellite DNA, lost their methylation to similar extents during F9 differentiation into parietal endoderm cells. In the immortalized derivatives 8a and P1, which phenotypically resemble F9 cells at intermediate stages of differentiation, methylation patterns diverged: Methylation of L1 and GAPDH sequences was strongly and moderately diminished, respectively, whereas methylation of satellite DNA was almost as high as that in stem cells. P19 embryonal carcinoma cells and D3 embryonic stem cells possessed methylation patterns similar to those of F9 stem cells. In immortalized cell lines with differentiated phenotypes derived from P19, methylation was diminished uniformly throughout the genome, but the overall level of methylation remained higher than that in terminally differentiated F9 cells. Treatment with the methylation inhibitor deoxyazacytidine halted the proliferation of 8a, P1, and F9 cells and elicited marked changes in morphology. However, markers of differentiation were not induced in F9 cells. The finding that all immortalized differentiated derivatives of embryonal carcinoma cells retained a higher level of DNA methylation—at least in parts of their genomes—than did terminally differentiated cells may indicate a function of demethylation of DNA and of satellite sequences in particular in terminal differentiation of extraembryonic tissues.