Transactivation of a Ribosomal Gene by Simian Virus 40 Large-T Antigen Requires at Least Three Activities of the Protein

Expression of the simian virus 40 (SV40) large-T antigen (or, for simplicity, T antigen) is sufficient to initiate and maintain transformation of cells in culture and tumorigenesis in experimental animals (see references 21 and 40 for recent reviews). Regions and activities of the multifunctional protein that are involved in specific growth property changes that accompany transformation have been defined (40). The aggressive growth that characterizes transformed and tumor cells places increased demands on protein synthesis. In support of this contention, current evidence indicates that increased protein synthesis, brought about by overexpression of translation initiation factors, can result in transformation (for a review, see reference 30). Increased expression of ribosomal genes might provide a second mode of increasing protein synthesis. One consequence of T-antigen expression is transactivation of ribosomal genes (36, 53, 55, 68). The relationship of this activity to transformation remains to be determined. Genetic analysis to identify the regions of T antigen required for transactivation of the ribosomal genes in vivo is a first step in correlating this capability with the oncogenic activities of the protein. The ribosomal genes are transcribed by RNA polymerase I (pol I) in a species-specific manner with the aid of at least two transcription factors, the upstream binding factor (UBF) and the species selectivity factor SL1. The rat ribosomal gene (rDNA) contains a core promoter element (CPE) located between nucleotides (nt) −31 and +6 (6, 20, 27, 36, 69, 70), an upstream promoter element (UPE), whose exact location varies slightly between species (nt −50 to −186) (27, 50), an enhancer (nt −2357 to −2183) (16), and terminator sequences (29). UBF binds to specific sequences in both the UPE and the CPE (2, 42) and stimulates pol I-mediated transcription. SL1 has low DNA-binding affinity unless it is accompanied by UBF (2). SL1 is needed for efficient transcription of the ribosomal genes and is responsible for conferring the species-specific nature of the transcription (3, 25, 35). Human SL1 is a complex composed of the TATA-binding protein (TBP; 38 kDa) and three TATA-associated factors, TAFI48 (48 kDa), TAFI63 (63 kDa), and TAFI110 (110 kDa) (12, 18). TAFI48 and TBP efficiently bind to UBF, whereas TAFI110 and TAFI63 contact the promoter directly (1). Thus, during transcription, UBF and pol I interact with cis elements in the promoter, whereas SL1 associates with these proteins to form the active initiation complex (1, 12). T antigen is a promiscuous transcriptional transactivator. It transactivates the ribosomal genes, which are transcribed by pol I (36, 53, 54), as well as genes that are dependent on either pol II or pol III. T antigen’s ability to transactivate pol II- and III-dependent promoters (38, 47, 63) and the regions of the protein involved have been investigated in detail (4, 26, 31, 56, 72). However, less is known concerning the T-antigen activities required for transactivation of pol I-dependent promoters. In HeLa cell extracts, purified T antigen increases in vitro transcription from a human ribosomal promoter (36). Recently, Zhai et al. (71) showed that T antigen binds to the SL1 complex through interactions with TAFI48, TAFI110, and TBP and that the T antigen-SL1 association is crucial to activation of the ribosomal gene promoter. They showed further that T-antigen amino acids 1 to 436 were sufficient to bind SL1 and that amino acids 1 to 538 were sufficient to stimulate pol I-mediated transcription of the human ribosomal gene in HeLa cell extracts. Early investigations into T antigen’s ability to override the species specific nature of ribosomal transcription in vivo assessed reactivation silent rDNA promoters within mouse-human hybrid cell lines (53, 55). These assays defined the T-antigen region necessary for the reactivation of a heterologous rRNA gene as amino acids 1 to 509 (54). The role of specific T-antigen activities in pol I-dependent transactivation remains to be determined. It was shown recently that the retinoblastoma gene product, Rb, plays a role in the regulation of ribosomal transcription (7, 67). In vitro Rb binds to UBF (7) and inhibits its ability to bind to the UPE. This interaction represses transcription from the ribosomal promoter (67). It is not known whether T-antigen binding to Rb is involved in activation of ribosomal genes. The study reported here focused on the ability of T antigen to transcriptionally activate the rat ribosomal gene promoter when transiently transfected into monkey cells and investigated the specific regions and activities of T antigen necessary for this heterologous ribosomal gene transactivation. The results indicate that at least three activities cooperate to transactivate the rat ribosomal gene and that Rb binding is not essential.