Adenovirus 12-mediated down-regulation of the major histocompatibility complex (MHC) class I promoter: identification of a negative regulatory element responsive to Ad12 E1A

Abstract

In highly oncogenic adenovirus (Ad) 12-transformed cells, major histocompatibility complex (MHC) class I gene expression is down-regulated by the products of the viral E1A oncogene at the level of initiation of transcription. However, class I gene expression is unaltered or elevated in non-oncogenic Ad2- or Ad5-transformed cells. These changes in class I expression may permit Ad12-transformed cells to escape host immune surveillance and elicit tumour formation. Here we show that the 2kb of 5' flanking region of the mouse H-2Kb class I gene is sufficient to mediate down-regulation of transcription driven from homologous or heterologous (HSV thymidine kinase) basal promoter elements in cells expressing Ad12 E1A, but not in Ad2 E1A-expressing cells. Deletion analysis of the 2kb region showed that sequences from -1.18 to -1.44kb (relative to the cap site) were a target for Ad12 E1A-mediated transcriptional down-regulation. Deletion of this entire region from the 2kb flanking sequence of the H-2Kb gene abolished Ad12 E1A-mediated down-regulation of transcription. Computer analysis of the -1.18 to -1.44kb sequence identified two 6/7bp matches with the AP-1 transcription factor consensus sequence and two matches with the pig MHC class I PD1 repressor element. Gel retardation analysis using overlapping DNA fragments derived from the -1.18 to -1.44kb sequence revealed several DNA:protein complexes formed using nuclear extract derived from Ad12-, but not from Ad2- or Ad5-transformed cells. Some of these DNA:protein complexes were also present, but at lower levels, in nuclear extracts from untransformed rat cells suggesting the possible involvement of cellular factors in the mechanism of down-regulation mediated by Ad12 E1A. A binding site for the AP-1 factor failed to compete for protein binding to fragments within the -1.18 to -1.44 sequence, while the PD1 site competed for binding only in the -1.15 to -1.23 region. These results indicate that novel factors (as well as a previously identified class I repressor, PD1) may be involved in Ad12 E1A-mediated down-regulation of MHC class I transcription.

Transcriptional regulation of the major histocompatibility complex (MHC) class I heavy chain, TAP1 and LMP2 genes by the human papillomavirus (HPV) type 6b, 16 and 18 E7 oncoproteins

“…Adenoviruses, another major group of DNA tumour viruses, have been shown to interfere with the expression of the major components of the antigen presentation pathway. The highly oncogenic adenovirus (Ad) 12 E1A represses transcription driven from both the class I heavy chain promoter (Pro tt et al, 1994) and the TAP1/LMP2 bidirectional promoter (Pro tt and Blair, 1997). The E7 protein of the high-risk HPVs exhibits a signi®cant degree of structural and functional similarities to the Ad E1A protein.…”

“…A total of 11 mg of DNA was used, comprising 1 mg pRSVluc reference plasmid along with 5 mg of reporter gene plasmid and 5 mg of test vector (or control plasmid). The reporter constructs pH2KCAT (a) and pE2ACAT (b) have been previously described (Pro tt et al, 1994;Dery et al, 1987, respectively) and contain the 2 kb 5' region of the mouse H-2K b gene and the Ad 2 E2 early promoter, respectively, cloned upstream of the chloramphenicol acetyltransferase (CAT) reporter gene. These were co-transfected with the HPV E7 expression plasmids pMo6bE7, pMo16E7 and pMo18E7 that contain the HPV types 6b, 16 and 18 E7 ORFs, respectively (Barbosa et al, 1991), as well as pAsc 4.7, which is the AccI-H fragment of Ad 12 (comprising the E1A coding region plus the 5' end of the E1B gene) cloned between the AccI and EcoRI sites of pBR322 (Byrd et al, 1982).…”

“…These involved the co-transfection of the pH2KCAT construct, containing the 2 kb 5'¯anking region of the mouse H-2K b gene fused to the CAT gene in the pBLCAT3 reporter vector (Pro tt et al, 1994), along with HPV 6b, 16 or 18 E7 (Barbosa et al, 1991) or Ad 12 E1A (Byrd et al, 1982) expression vectors in established rat 3Y1 ®broblasts (Figure 1a). Expression of either HPV 16 or 18 E7 or Ad 12 E1A resulted in a 2.5 ± 3-fold down-regulation of reporter gene expression, whereas HPV 6b E7 caused a 1.5 ± 2-fold increase in CAT activity.…”

“…We have previously demonstrated that the target site for Ad 12 E1A-mediated repression of the H-2K b promoter lies within the 71.07 to 71.44 kb region (Pro tt et al, 1994). To establish whether the downregulation of class I heavy chain promoter by HPV 18 E7 could be ascribed to the Ad 12 E1A targeted region of the class I promoter, co-transfection assays were carried out using the p(71.…”

“…To establish whether the downregulation of class I heavy chain promoter by HPV 18 E7 could be ascribed to the Ad 12 E1A targeted region of the class I promoter, co-transfection assays were carried out using the p(71. 07/71.44)tkCAT,p(71.07/ 71.18)tkCAT and p(71.18/71.44)tkCAT constructs that contained the PCR-generated 71.07/71.44, 71.07/71.18 and 71.18/71.44 fragments of the upstream class I regulatory region (Figure 3), linked to a basal thymidine kinase (tk) promoter driving expression of the CAT reporter gene (Pro tt et al, 1994), together with the HPV 18 E7 and Ad 12 E1A expression plasmids. As shown in Figure 3, expression of E7 resulted in an approximately 3.5-fold decrease in CAT activity of p(71.07/71.44)tkCAT, comparable with the fourfold repression mediated by E1A ( Figure 3a), whereas CAT activity directed by the p(71.07/ 71.18)tkCAT was slightly elevated above basal levels (Figure 3b), suggesting that this sequence was not involved in repression of the H-2K b promoter by HPV 18 E7, in agreement with previous ®ndings for Ad 12 E1A (Pro tt et al, 1994).…”