Abstract
A murine granzyme B promoter fragment that extends 243 base pairs upstream of the transcription start site confers high levels of luciferase reporter gene activity in transient transfection assays into T cells and mouse L cell fibroblasts. This promoter fragment contains canonical binding sites for the transcription factors AP-1, core binding factor (CBF), Ikaros, and the cyclic AMP responsive element binding protein (CREB). Oligonucleotides containing the granzyme B AP-1 or CBF elements form specific complexes with proteins present in nuclear extracts from activated CD8(+) splenocytes, MTL cells, EL4 T cells, and L cells. A strong DNase1 hypersensitive site that coincides with the closely associated AP-1, CBF, Ikaros, and CRE elements is present in activated CD8(+) T cells but not in resting T cells or L cells. Both in vitro and in vivo footprints are observed at these sequence elements in activated cytotoxic T cells (CTL) but not in resting T cells. The endogenous granzyme B gene is CTL-specific as no mRNA is detectable in EL4 or L cells. We propose that a condensed chromatin structure at the granzyme B promoter is responsible for transcription factor inaccessibility and repression of transcription in non-T cells.
Highlights
The body’s major defense against viral infections is mediated by cytotoxic T lymphocytes
Studies of the human and murine granzyme B proximal promoters reveal that they share several conserved sequences. These include T cell-specific transcription factor binding sites such as Ikaros and core binding factor (CBF1/PEBP2) (Haddad et al, 1993; Kamachi et al, 1990; Wang and Speck, 1992) as well as recognition sequences for the ubiquitous transcription factors AP-1 and the cyclic AMP response element binding factor (CREB)
We have developed a method for the transfection of reporter gene plasmids into primary mouse splenocytes and show that the minimal granzyme B promoter is able to induce significant levels of luciferase activity in activated CD8ϩ T cells
Summary
Cells—Primary splenocytes were obtained from 6- to 12-week-old Balb/c mice. Spleen tissue was ground through a fine wire screen in RHFM/IL-2 media, and the cells were pelleted. Red blood cells were lysed with buffered ammonium chloride lysis buffer. The IL-2-dependent cytotoxic T cell line MTL 2.8.2 was generated from CBA/J mice as described (Bleackley et al, 1982). The antigen- and IL-2-dependent CTL21.9 (Type 1) line was generated as described (Havele et al, 1986). EL4 is an IL-2-producing T lymphoma cell line (Paetkau et al, 1986),
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