A minimal serpin promoter with high activity in haematopoietic progenitors and activated T cells.

INTRODUCTION: The serine protease inhibitor Serpin 2A is highly expressed in ex vivo bipotent granulocyte/macrophage progenitor cells and in cultured myeloid stem cells. The gene undergoes rapid down-regulation as these cells are induced to differentiate, and constitutive expression in cultured myeloid stem cells retards maturation. Serpin 2A is also expressed in T cells as a consequence of activation. We now report analysis of the upstream regulatory elements that control Serpin 2A transcription. MATERIALS AND METHODS: Using primer extension and rapid amplification of cDNA ends the transcription start site of the Serpin 2A gene was mapped, and a 1.2 Kb genomic upstream fragment cloned and sequenced. Promoter activity and protein binding of deletion and site-directed mutant constructs were analysed by transient transfection and by electrophoretic mobility shift assays. RESULTS: A minimal promoter fragment was identified with high activity dependent on NF-kappa and Moloney murine leukaemia enhancer factor LVa binding sites in both myeloid stem cells and activated T cells. NF-kappa was shown to be the main DNA binding protein in T cells, whereas that in haematopoietic stem cells appears to be novel. CONCLUSION: Serpin 2A promoter activity in T cells is due predominantly to NF-kappa binding to its consensus site. Activity in haematopoietic stem cells appears to be mediated by a novel protein, which recognises the NF-kappa consensus only in the context of flanking sequences. This concise regulatory element may be of potential value in gene therapeutic applications.

Upstream elements bestow T-cell and haemopoietic progenitor-specific activity on the granzyme B promoter.

Cytotoxic T cells and early haemopoietic progenitors share the expression of a number of specific genes. Of these, granzyme B has attracted particular interest because of its role in inducing apoptosis during cytotoxic T cell-mediated target cell killing, and its potential role in the mobilisation and homeostasis of haemopoietic stem cells. Studies of granzyme B regulation should therefore yield valuable information concerning the molecular control of these processes, and also identify elements capable of directing gene expression to two cell types of relevance to gene therapy. Here we show that proximal regulatory elements already known to direct promoter activity in T cells are similarly active in haemopoietic progenitors. However, this activity is not strictly specific, since the promoter regions also direct low levels of reporter gene expression in fibroblasts. More importantly, we also report the presence of two previously unidentified clusters of DNaseI hypersensitive sites upstream from the murine granzyme B gene, and show that these regions impart both increased transcriptional activity and the appropriate cell type specificity on the granzyme B promoter. These upstream regulatory regions are therefore likely to play a key role in the coordination of granzyme B expression in vivo.

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

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.

In vivo regulation of murine granzyme B gene transcription in activated primary T cells.

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.