Analysis of human peripheral blood T cells and single-cell-derived T cell clones uncovers extensive clonal CpG island methylation heterogeneity throughout the genome

Methylation of cytosine residues in CpG dinucleotides is generally associat ed with silencing of gene expression. DNA methylation, as a somatic event, has the potential of diversifying gene expression in individual cells of th e same lineage. There is little quantitative data available concerning the extent of methylation heterogeneity in individual cells across the genome. T cells from the peripheral blood can be grown as single-cell-derived clone s and can be analyzed with respect to their DNA methylation patterns by res triction landmark genomic scanning. The use of the methylation sensitive en zyme NotI to cut and end-label DNA fragments before their separation in two dimensions provides a quantitative assessment of methylation at NotI sites that characteristically occur in CpG islands. We have undertaken quantitat ive analysis of two-dimensional DNA patterns to determine the extent of met hylation heterogeneity at NotI sites between peripheral blood single-cell-d erived T cell clones from the same individual. A total of 1,068 NotI-tagged fragments were analyzed. A subset of 156 fragments exhibited marked methyl ation heterogeneity at NotI sites between clones. Their average intensity a mong clones correlated with their intensity in uncultured, whole-blood-deri ved T cells, indicating that the methylation heterogeneity observed in clon es was largely attributable to methylation heterogeneity between the indivi dual cells from which the clones were derived. We have cloned one fragment that exhibited variable NotI-site methylation between clones. This fragment contained a novel CpG island for a gene that we mapped to chromosome 4. Th e methylation status of the NotI site of this fragment correlated with expr ession of the corresponding gene. Our data suggest extensive diversity in v ivo in the methylation and expression profiles of individual T cells at mul tiple unrelated loci across the genome.