116 TRANSCRIPTIONAL REGULATION OF THE HUMAN IL-7 RECEPTOR ALPHA-CHAIN GENE IN CD8 T-CELLS J Kakal, E Faller, C Prematunga, P MacPherson Objective: We have demonstrated that the HIV Tat protein specifically down regulates expression of the IL-7 receptor alpha-chain (CD127) on CD8 T-cells and that this down regulation occurs at the level of transcription initiation. To better understand how Tat influences the expression of this gene, the putative CD127 promoter/enhancer was cloned and analyzed for Tat-responsive elements.
Ottawa Health Research Institute, Ottawa, Ontario
Methods: Using human genomic DNA, we first cloned two fragments, one extending 1 kb and the other extending 3 kb upstream of the start site of transcription and transferred these into the luciferase reporter plasmid pGL3b. A series of deletion mutations as well as site-specific mutants within each construct were then created by PCR. These constructs were introduced into Jurkat cells using the Transit transfection kit and luciferase expression was measured in the presence and absence of exogenous Tat protein. Transfection efficiencies were normalized by co-transfecting with CMV-lacZ.
Results: Sequence analysis of the putative CD127 gene promoter reveals the presence of a number of binding sites for transcription factors known to influence CD127 gene expression and whose activity is known to be affected by Tat. Transient transfection of the 1 kb CD127 promoter fragment drove luciferase expression 20-fold above background, indicating all the necessary transcription control elements required for CD127 expression in T-cells are contained within the first kb. Deletion mutants are being used to map out the minimal promoter sequences and site-specific mutations are being constructed to identify Tat-responsive elements.
Conclusions: We are the first to clone the human CD127 gene promoter and define minimal regulatory sequences. Given that IL-7 plays a crucial role throughout T-cell development, this analysis may suggest important regulatory pathways governing T-cell survival and activity. These results may also suggest molecular targets to circumvent Tat-induced down regulation of CD127 and T-cell inactivation.