The ITGAL promoter is methylated in fibroblasts but not T cells.

(A) DNA was isolated from 2 fibroblast cell lines, treated with bisulfite, and the ITGAL promoter amplified in 5 overlapping regions. For each amplified region, 5 fragments were cloned and sequenced. The filled circles on the x-axis represent each potentially methylatable dC residue, and the filled circles with error bars represent the average methylation (mean ± SEM) for each site of the 5 sequenced fragments from both fibroblast lines. (B) T-cell DNA was similarly isolated, treated with bisulfite, amplified, and sequenced. The region from −1261 to −68 represents the average methylation (mean ± SEM) of 5 fragments from each of 6 donors, while the remainder of the sequence represents the average methylation of 5 fragments from each of 4 healthy donors. The horizontal line indicates the region containing Alu elements. (C) DNA was isolated from the T cells of 3 healthy donors, and then bisulfite sequencing of 5 fragments from each donor was performed as above for the region from bp −1261 to −68 (identified by the arrows). Results are presented as in panel A. (D) T cells from the same donors shown in panel C were stimulated with PHA and DNA similarly isolated, treated with bisulfite, and the region from bp −1261 to −68 (identified by the arrows) sequenced. Results again represent the mean ± SEM of 5 determinations from each of the 3 donors. (E) DNA was isolated from CD8⁺ T cells and bisulfite sequencing was performed on the region from bp −1261 to −68 (identified by the arrows) as in panel A. Results represent the mean ± SD of 5 determinations for each dC residue. (F) DNA was isolated from CD4⁺ T cells and analyzed as in panel E. Results again represent the mean ± SD of 5 determinations for each dC residue.