The mechanism of down-regulation of major histocompatibility complex (MHC) class I antigens in highly oncogenic adenovirus 12-transformed cells.

Abstract

Down-regulation of major histocompatibility complex (MHC) class I antigens by expression of the ElA gene of highly oncogenic adenovirus 12 (Ad12) may permit Adl2-transformed cells to evade the T-cell mediated immune system and elicit tumours. Much, evidence points to a major role of E1A-mediated transcriptional repression of class I genes in this process. In contrast, neither Ad2 nor Ad5 EIA participates in transcriptional repression, providing a possible explanation for the normal or elevated levels of class I antigens found on these cells and the non-oncogenic phenotype of these viruses [l, 21. This system also provides an interesting example of a selective effect of E1A from different viral serotypes on transcription of a cellular gene. Ad12 El A mediates down-regulation by interacting with several regulatory elements in the MHC class I promoter. The best characterised of these elements is the class I regulatory element (CRE), located between -159 to -201, that is highly conserved between murine and human class I promoters and contains two domains which have been reported to be targets for Ad12 ElA. The region I1 domain (CRE 11) contains a nuclear hormone consensus sequence 5'-AGGTCA-3' and binds cellular transcription factors belonging to the family of nuclear hormone receptors, such as RXR-p and COUP-TF and the region I domain binds members of the NF-KBI c-re1 family. Nuclear extracts fiom Ad 12-transformed cells contain a protein factor that binds the CRE I1 and is absent or present at reduced levels in Ads-transformed or non-transformed cells. It has also been shown that Ad12transformed cells have increased binding of the factor COUP-TF to the CRE I1 and reduced binding of NF-KB to the CRE I (3,4,5). Site directed mutagenesis using PCR has been used to introduce specific mutations into both the CRE I1 and CRE I regions which ablate the sites for factors that bind to these regions. The effect of these mutations on promoter activity was then assayed by transfecting the mutant constructs into Ad12 or Ad5transformed mammalian cell lines and measuring the activity of a chloramphenicol acetyltransferase gene product. Point mutations in the nuclear hormone consensus motif of CRE I1 which abrogated binding reduced promoter activity to a basal level whereas deletion of a 17 base pair region spanning the entire CRE I1 actually increased activity of the promoter above wild type level. Point mutations in the CRE I which ablated the NF-KB binding site also reduced promoter activity to a basal level. However when these mutations were combined with either the point mutations or deletion in the CRE 11, promoter activity was increased to levels approaching those shown by the wild-type promoter, suggesting a synergistic interaction between factors bound at the CRE I and CRE I1 sites. Our recent work and that of others has also identified Ad1 2 El A-mediated negative regulatory elements located further upstream at approximately 1.1 to 1.4kb and -1.5 to -1.8kb, respectively in the mouse H-2Kb promoter [6]. 1.