Identification of a Silencer, Enhancer, and Basal Promoter Region in the Human CD95 (Fas/APO-1) Gene

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Abstract
Genomic clones for the human CD95 (Fas/APO-1) and CD40 genes have been isolated and 2.3 kb of the CD95 and 0.8 kb of the CD40 gene 5′-flanking regions sequenced. Comparisons of the human CD95 gene with the human CD40 and the murine CD40 and TNFR-II genes showed a low degree of sequence similarity. However, dot matrix analyses revealed conservation of two stretches between human CD95 (−387 to −362 and −288 to 261 in CD95) and murine TNFR-II genes. Additionally, TCCTCC motifs are present within 400 bp upstream of the ATG of all genes examined. Repeated interferon-β (IFN-β) silencer B motifs and a lysozyme silencer 1 motif have been found in the CD95 gene at approximately −1,600 and −1,100, respectively. Sequence comparison of the 5′-flanking regions of the murine and human CD40 genes revealed the presence of a conserved AP-4 site and two SP-1 sites. CD95, CD40, and TNFR-II genes all lack classical TATA and CAAT boxes. However, a strongly increased frequency of CpG dinucleotides was found. Primer extension analysis revealed multiple transcriptional start sites in the CD95 gene, where the usage of individual start sites appeared to be cell type-specific. Functional analysis, using reporter constructs and transient transfections, identified a silencer activity residing between nucleotide positions −1,781 and −1,007 and a strong enhancer region between −1,007 and −425 in the human CD95 gene. The region between −425 and −1 retained a basal promoter activity. These data may be relevant to the cell type-specific and activation state-dependent transcriptional up-regulation of the CD95 gene during activation-induced cell death.