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 bipotentgranulocyte/macrophage progenitor cells and in cultured myeloid stem cells. The geneundergoes rapid down-regulation as these cells are induced to di•erentiate, and constitutiveexpression in cultured myeloid stem cells retards maturation. Serpin 2A is also expressed in Tcells as a consequence of activation. We now report analysis of the upstream regulatoryelements that control Serpin 2A transcription.Materials and methods: Using primer extension and rapid amplification of cDNA ends thetranscription start site of the Serpin 2A gene was mapped, and a 1.2 Kb genomic upstreamfragment cloned and sequenced. Promoter activity and protein binding of deletion and site-directed mutant constructs were analysed by transient transfection and by electrophoreticmobility shift assays.Results: A minimal promoter fragment was identified with high activity dependent on NF-kand Moloney murine leukaemia enhancer factor LVa binding sites in both myeloid stem cellsand activated T cells. NF-k 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-k binding toits consensus site. Activity in haematopoietic stem cells appears to be mediated by a novelprotein, which recognises the NF-k consensus only in the context of flanking sequences. Thisconcise regulatory element may be of potential value in gene therapeutic applications.The Hematology Journal (2001) 2, 150–160Keywords: Serpin; T cells; promoter; NF-k; LVa; stem cells