5 × 10^5 YTS cells were seeded per well in a 24-well plate the day before transfection; 4.5 µg of reporter construct DNA, 400 ng of Renilla DNA, and 6 µl of jetOPTIMUS were diluted in 150 µl buffer and incubated at room temperature for 10 min before addition to each well. The DNA mixture containing plasmid DNA was then added to each well and incubated at 37 °C in 5% CO2 for 48 h before luciferase activity analysis. Briefly, the culture medium was removed 48 h post-transfection, and cells were washed with 0.5 ml of phosphate-buffered saline (PBS, pH 7.4). Cells were then lysed with passive lysis buffer. The suspensions were centrifuged at 14,000 rpm for 1 min. A total of 20 μl of cell lysate supernatant was mixed with 100 μl of luciferase substrate, and the light units were measured on a luminometer.

The human KIR genes encode a family of class I MHC receptors that are expressed on subsets of NK cells. The expression of KIR proteins is controlled by a stochastic process, and competition between sense and antisense promoter elements has been suggested to program the variegated expression of these genes. Previous studies have demonstrated distinct roles of distal, intermediate, and proximal sense promoter/enhancer elements in gene activation and expression. Conversely, proximal and intronic antisense promoter transcripts have been associated with gene silencing at different stages of NK cell development. In the current study, we examine the effect of intermediate promoter deletion on KIR2DL1 expression in the YTS cell line. Homozygous deletion of the KIR2DL1 intermediate element did not affect proximal promoter activity but resulted in increased detection of upstream transcripts. No significant changes in alternative mRNA splicing or expression levels of KIR2DL1 protein were observed. However, intermediate element deletion was associated with a reduced frequency of gene activation by 5-azacytidine. Taken together, these results indicate that the intermediate element is not an enhancer required for KIR expression; however, it is required for the efficient activation of the gene.