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.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) M22526
Final activities are given as luciferase/b-galactosidase values. It was previously established by in vitro footprinting that within the 243-bp granzyme B promoter, there existed five distinct regions that were protected from DNase I digestion [9]. These regions correspond to transcription factor binding sites for ATF/CREB, activator protein-1 (AP1), CBF, and Ikaros, and specific DNA-protein interactions were observed as in vivo footprints in activated CD81 T cells and MTLs
To determine whether any of these binding sites were important for the high levels of reporter gene expression observed from the 243-bp promoter, we constructed a series of promoters with three nucleotide substitutions in each binding site
Summary
Mutagenesis—Site-directed mutagenesis was performed using the SculptorTM in vitro mutagenesis system (Amersham Corp.). Plasmids—Granzyme B promoter fragments were obtained by restriction enzyme digestion or polymerase chain reaction amplification They were inserted upstream of the promoterless luciferase reporter gene of the basic pGL2 vector (Promega), and orientation was confirmed by sequencing or directional primer amplification. Primary splenocytes were cultured at 3.0 3 106 cells/ml in RHFM plus 60 units/ml IL-2, 1:750 dilution aCD3, and 5 mg/ml ConA for 20 h prior to transfection. 1.0 3 107 exponentially growing cells (MTL) or 2.0 3 107 (whole splenocytes) were washed twice in serum-free medium and resuspended in 1.0 ml of TBS (25 mM Tris-HCl (pH 7.5), 137 mM NaCl, 5 mM KCl, 0.6 mM Na2HPO4, 0.7 mM CaCl2, and 0.5 mM MgCl2 (pH 7.0)) with 500 mg/ml DEAE-dextran (Sigma), 15 mg of covalently closed circular luciferase reporter plasmid, and 5 mg of b-galactosidase control plasmid.
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