Consensus AP-1 and CRE motifs upstream from the human cytotoxic serine protease B (CSP-B/CGL-1) gene synergize to activate transcription

Abstract

The human CGL-1/cytotoxic serine protease B gene (CSP-B; also known as granzyme B) is transcriptionally activated during cytotoxic T- lymphocyte maturation. Activation can be mimicked in the PEER T-cell leukemia cell line by treatment with 12-O-tetradecanoylphorbol-13- acetate (TPA) and N6–2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate (bt2cAMP). In this report, we show that a consensus AP-1 element and a consensus cAMP response element (CRE) located 5′ to the CSP-B transcriptional start site are both required for transcriptional activation of the CPS-B promoter in TPA + bt2cAMP-stimulated PEER cells. A 94-bp fragment containing both elements activates a heterologous promoter in an orientation-independent fashion. Several single nucleotide substitutions in the AP-1 site abolish activity of the 94-bp fragment. Several point mutations in the consensus CRE substantially reduce promoter activity, but one CRE mutation increases activity fourfold. Replacement of the CRE with a second copy of the AP- 1 site results in a level of transcriptional activity comparable with that of the wild-type sequence, but replacement of the AP-1 site with a CRE abolishes activity. Neither the AP-1 site nor the CRE can be effectively replaced with an SP-1 site. Deletions between the AP-1 site and the CRE retain full activity only if helical spacing is preserved, suggesting that synergism between these two elements is either the result of cooperative binding of factors to the DNA or of cooperative binding of DNA-bound factors to another protein.

Type 1 Interferons Potentiate Human CD8+ T-Cell Cytotoxicity Through a STAT4- and Granzyme B–Dependent Pathway

“…Previous studies investigating the transcriptional regulation of GZMB revealed that T-cell activation induces mRNA expression, which was mapped to a 243 bp promoter element upstream of the GZMB TSS ( 54 , 55 ). Mutational analysis and electrophoretic mobility shift assay studies confirmed the binding sites for several transcription factors involved in the activation and differentiation of T lymphocytes including AP-1, Runx1, Ikaros, and CREB1 ( 56 , 57 ). Apart from these studies, very little is known regarding the transcriptional regulation of GZMB.…”

Characterization of the MN/CA 9 promoter proximal region: a role for specificity protein (SP) and activator protein 1 (AP1) factors

“…Mutational analysis of the cytotoxic serine protease B gene promoter proved that this is not the case. In this promoter, an AP1 site and cAMP-response element were required for optimal activity, and the replacement of neither element with an SP1 site restored the activity [38]. Thus instead of entering into indiscriminate synergistic co-operation each time, the outcome of AP1 and SP1 combination depends on the promoter context.…”

“…The widespread AP1 sites require cooperation with other transcription factor(s) for optimal activity in promoters of many genes, e.g. the neighbouring upstream regulatory sequence in the stromelysin promoter [36], polyoma virus enhancer domain element in the collagenase promoter [37], cAMP-response element in the cytotoxic serine protease B promoter [38], ETS1 (v-ets avian erythroblastosis virus E26 oncogene homologue 1) and nuclear factor κB in the granulocytemacrophage colony-stimulating factor promoter [39] or SP1 in the involucrin [40] and VEGF [41] promoters. AP1, in an enhancer mode, was found to stimulate activity of nuclear factor 1, CP1, ATF\cAMP-response-element-binding protein and GCbox (SP1 site) element in promoter-element compatibility tests [30].…”

Regulation of Murine Granzyme B Gene Transcription in Activated Primary T Cells

“…These include T cell-specific transcription factor binding sites such as Ikaros and core binding factor (CBF 1 /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). These sequences have been shown to be sufficient to induce reporter gene expression in immortalized T cell lines in which many of these transcription factors are constitutively active (Frégeau and Bleackley, 1991;Hanson et al, 1993).…”