Modification of CIITA-PIII affects CIITA and MHC class II expression. (A) Jurkat T-ALL cells were treated with 0.1, 0.5, or 1 μM of the demethylation agent 5-AZA-2′-deoxycytidine for 3 days and mRNA was used for the detection of CIITA-PIII and HLA-DRA expression by RT-PCR analysis. Raji cDNA is used as a control for CIITA-PIII and HLA-DRA amplification. (B) Isolated CIITA-PIII and SV40 promoter fragments were either unmethylated (▪) or in vitro methylated (▦) , ligated back into the pGL3-vector, and transfected in Jurkat T cells. Unmethylated and methylated CIITA-PIII was also cotransfected with a CREB-1 expression vector.28 Promoter activity was measured 2 days after transfection and was normalized with a β-actin–Renilla construct. Promoter activity is indicated as relative luciferase units (RLUs). Error bars represent SD.
