Mutational analysis of the murine granzyme B gene promoter in primary T cells and a T cell clone

Abstract

The granzyme B gene is induced in cytotoxic T lymphocytes in response to antigenic stimulation. Previous studies have identified several distinct regions in the granzyme B promoter which may be important in either the induction or the T cell specificity of the gene. These regions contain the canonical transcription factor binding sites AP1, cyclic AMP-responsive element (CRE), Ikaros, and core-binding factor (CBF/PEBP2). Each protein binding site was disrupted by site-directed mutagenesis to investigate its role in granzyme B promoter function. Mutations were introduced alone, or in various combinations, within the context of a 243-base pair promoter fragment known to confer high levels of reporter gene expression. Transfection assays revealed that all of the single binding site mutant promoters were capable of sustaining moderate to high levels of transcriptional activity in primary activated T lymphocytes, whereas certain mutants were more impeded in a T cell clone. A quadruple mutant promoter, with only the CRE binding site intact, showed background expression levels. This drop in expression was found to be mostly due to mutations in AP1 and the 3' CBF binding sites. Their close proximity and requirement in promoter function suggest an important role for protein-protein interaction between these two factors.