Activation of the human thymidine kinase (TK) promoter by simian virus 40 large T antigen requires both the T antigen pRb family-binding domain and TK promoter sequences resembling E2F-binding sites.
Infection of quiescent cells with the DNA tumor virus simian virus 40 induces expression of the cellular thymidine kinase (TK) gene a minimum of 10- to 20-fold, and this induction depends upon the viral protein large T antigen (T-Ag). To define both human TK promoter elements and T-Ag functional domains required for transcriptional induction, we have established a system in which stable Rat-1 transfectants harboring TK promoter-luciferase hybrid genes are infected with recombinant adenoviruses expressing either wild-type or mutant forms of T-Ag and luciferase expression is measured as an indicator of promoter activity. The results show that (i) a 135-bp TK promoter fragment is activated 10- to 15-fold by viral infection; (ii) this activation is the result of both T-Ag-dependent and -independent mechanisms; (iii) the T-Ag pRb family-binding domain, but not the p53-binding, helicase, or ATPase domain, is required for activation; and (iv) activation is severely diminished with a TK promoter fragment in which E2F-like-binding sites have been removed. These data demonstrate a requirement for both an E2F-related factor and a pRb family member in activation of the TK promoter by T-Ag. This contrasts with the promiscuous activation of many cellular and viral genes by T-Ag, which is independent of its ability to bind pRb.
Anderson
M M
MM
Department of Microbiology, Michigan State University, East Lansing 48824, USA.
Chen
J
J
Cole
C N
CN
Conrad
S E
SE
D000818:Animals; D000957:Antigens, Viral, Tumor / Q000096:biosynthesis / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002460:Cell Line; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D015971:Gene Expression Regulation, Enzymologic; D015967:Gene Expression Regulation, Viral; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016160:Retinoblastoma Protein; D056465:Retinoblastoma-Binding Protein 1; D013539:Simian virus 40 / Q000235:genetics / Q000502:physiology*; D013937:Thymidine Kinase / Q000096:biosynthesis* / Q000235:genetics*; D050680:Transcription Factor DP1; D014157:Transcription Factors; D014162:Transfection
D000957:Antigens, Viral, Tumor; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; D008156:Luciferases; D013937:Thymidine Kinase