Structure and function of the murine perforin promoter and upstream region. Reciprocal gene activation or silencing in perforin positive and negative cells.

Abstract

Gene expression of the cytolytic protein perforin is restricted to and tightly regulated in cytolytic lymphocytes. To begin to understand the molecular basis of perforin gene transcription, we cloned and analyzed 5.1 kb of the genuine murine perforin promoter and upstream region. The murine perforin promoter is located approximately 2.1 kb upstream of the translation start codon in the genomic DNA due to an intron in the 5' untranslated sequence. Although the sequenced murine promoter and upstream region was found to be quite homologous to that of the human gene, most of the interspecies conserved sequences lacked obvious consensus to known regulatory elements. Functional analysis of this region, however, indicated that it contains regulatory elements that may determine the cell-type-specific expression of this killer protein. After transient transfection into several cell lines, the perforin promoter and upstream region was used to drive the expression of the chloramphenicol acetyltransferase (CAT) reporter gene. High levels of CAT activities, exceeding 110 times the expression of a promoterless reporter gene construct, were expressed in CTL. In contrast, in perforin-negative cell types the perforin promoter and upstream region mediated barely detectable transcription of the CAT gene. Analysis of the immediate proximal perforin promoter, -120 to +2, revealed that it was ubiquitously active and that it expressed in all cells tested 20- to 50-fold higher CAT activity than the promoterless reporter gene construct. The cell-type restricted transcriptional activity of the perforin promoter and upstream region, however, was controlled by at least four negative and positive cis-acting upstream regions that spread over the entire 5 kb of the cloned DNA and acted reciprocally in different cells. Thus, in perforin-negative cells, the transcriptional activity of the immediate proximal perforin promoter was dominantly suppressed by several upstream negative regulatory elements, whereas in perforin-positive cells, the promoter activity was enhanced more than fivefold by several upstream regulatory elements.