c-Myc binds to the distal KIR promoter element in NK cells. (A) The intergenic region preceding each of the KIR genes on chromosome 19 was analyzed for transcription factor binding sites. The sequence of the distal promoter element, −1150 bp upstream of the translational start site, was aligned for each KIR gene, and a predicted Myc-binding site was identified. The sequence of the putative Myc-binding region of the KIR3DL1 gene is shown, and polymorphisms found in the other genes are highlighted, as well as the consensus Myc probe used for EMSA analysis (Cons). The sequence of the distal promoter element −1190 bp upstream of the translational start site for the KIR2DL2/2DS2/2DL3/3DL2 is also shown. (B) The location of the EMSA probes spanning the Myc-binding sequence 1150 bp upstream of selected KIR translational start sites. The location of the EMSA probe spanning the consensus Myc site 1190 bp upstream of the KIR2DL2 translational start site is also shown. (C) EMSA analysis of the predicted Myc-binding sites. Probes corresponding to the nucleotide sequences shown in panel A were used for an EMSA with YT cell extracts. A Myc consensus probe alone control is shown in lane 1. Complexes formed by individual ³²P-labeled KIR probes are shown in lanes 2 to 7. A cold competitor control for probe specificity is shown in lane 8. (D) Inhibition of the complex formed by the KIR2DL2⁻¹¹⁹⁰ probe with anti-c-Myc and anti-Max antibodies is shown in lanes 10 and 11. The vertical line has been inserted between lanes 11 and 12 to indicate a repositioned gel lane. Inhibition of the complex formed by the KIR2DL2⁻¹¹⁹⁰ probe with anti-CREB and anti-YY1 control antibodies is shown in lanes 12 and 13. (E) Because of a 406-bp deletion and a 317-bp Alu insertion in the intergenic region of KIR2DL2/2DS2/2DL3/3DL2, a Myc-binding consensus is located at position −1190 relative to the transcriptional start site instead of −1150, where the site is located for the rest of the KIR genes.