REPETITIVE ALU ELEMENTS FORM A CRUCIFORM STRUCTURE THAT REGULATES THEFUNCTION OF THE HUMAN CD8-ALPHA T-CELL-SPECIFIC ENHANCER

We previously identified a T cell-specific enhancer in the last intron of the human CD8 alpha gene that is adjacent to a sequence element th at significantly represses enhancer function. This negative regulatory region consists of a half-Alu sequence that has potential to base-pai r with a downstream Alu element, which is part of the fully active enh ancer, to form a cruciform structure. The activity of this half-Alu si lencer sequence is position and orientation-dependent, suggesting that DNA structure plays an important role in its function. Using site-dir ected mutational analysis and P-1 nuclease mapping, we directly demons trate that formation of a cruciform structure is required for repressi on of enhancer function in transient transfection assays. Finally, a P -1 nuclease-sensitive site is present in the endogenous CD8 alpha gene in T cell lines providing indirect evidence that the stem-loop may fo rm in vivo. Taken together, these results suggest that Alu elements ma y contribute to the regulation of the CD8 alpha gene enhancer through the formation of secondary structure that disrupts enhancer function.