The use of alternative promoters in T cell development

Abstract Many mammalian genes, including a number involved in immune development and function, use multiple promoters encoding expression of the same protein product. To understand the function of alternative promotors during T cell development, we carried out genome-wide RNA-Seq analysis of DN, DP, CD4 and CD8 thymocytes. 26% (2469/9595) of genes expressed by thymocytes used two or more alternative promotors at some point in T cell development. However, only 3.2% (80/2469) of the genes which utilized two or more promoters showed the difference of promotor activity among different T cell developmental stages which were validated by selected representative genes with qPCR assay. To analyze mechanisms of alternative promoter regulation, DNase hypersensitivity sites were mapped, and ChIP-Seq with anti-H3K4me3 antibodies was performed in the same thymic subpopulations. To determine the in vivo functional role of alternative promotors for T cell development, several genes were selected for study by deletion of individual promoters by CRISPR technology. We have initially generated mice with deletion of distal or proximal promotor of the lck gene and demonstrated distinct functional roles of the two promoters: the proximal lck promoter is critical for early stages of thymic T cell development while the distal promoter was necessary for normal T cell activation. Ongoing studies will characterize more broadly the functional role of alternative promoter expression in thymocyte development, and the mechanisms mediating selective promoter activity.