IDENTIFICATION OF A SILENCER, ENHANCER, AND BASAL PROMOTER REGION IN THE HUMAN CD95 (FAS APO-1) GENE/

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'-flanki ng regions sequenced. Comparisons of the human CD95 gene with the huma n CD40 and the murine CD40 and TNFR-II genes showed a low degree of se quence similarity. However, dot matrix analyses revealed conservation of two stretches between human CD95 (-387 to -362 and -288 to 261 in C D95) and murine TNFR-II genes. Additionally, TCCTCC moths are present within 400 bp upstream of the ATG of all genes examined. Repeated inte rferon-beta (IFN-beta) silencer B moths and a lysozyme silencer 1 moth have been found in the CD95 gene at approximately -1,600 and -1,100, respectively. Sequence comparison of the 5'-flanking regions of the mu rine and human CD40 genes revealed the presence of a conserved AP-4 si te and two SP-1 sites. CD95, CD40, and TNFR-II genes all lack classica l TATA and CAAT boxes. However, a strongly increased frequency of CpG dinucleotides was found. Primer extension analysis revealed multiple t ranscriptional start sites in the CD95 gene, where the usage of indivi dual start sites appeared to be cell type-specific. Functional analysi s, 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 a ctivity. These data may be relevant to the cell type-specific and acti vation state-dependent transcriptional up-regulation of the CD95 gene during activation-induced cell death.