Rapid methylation of genomic DNA in proliferating T-cells from bovine thymocytes

Two subpopulations of bovine calf thymus cells were separated by buoyant density centrifugation. The low-density cells (L-cells) showed high response to T-cell mitogens, while the high density cells (H-cells) did not. The DNA-metabolizing enzyme activities were elevated 10-fold in L-cells in comparison with those in H-cells. L-cells contained a DNA-replicating complex, DNA replitase, while H-cells did not. These observations suggested that L-cells were proliferating, mature T-cells and H-cells were dying intrathymic cells. A DNA methyltransferase was associated with DNA replitase in L-cells. In order to determine whether replitase-associated DNA methyltransferase functions at replicating regions, the methylation pattern of genomic DNA of L-cells was compared with that of H-cells. No significant difference was found in the extent of CpG dinucleotide methylation, and in the location of mC in the satellite I DNA sequence as identified by Southern hybridization and direct sequencing. Thus the majority of methylation patterns of genomic DNA did not change during T-cell development in the thymus. The results indicated that the methylation patterns were rapidly maintained in proliferating T-cells. Although methods employed in the present study might not be sensitive enough to detect transient hemimethylation, it is suggested that the rapid methylation might be catalyzed, albeit not completely, by a DNA methyltransferase associated with the DNA replitase complex.