Transcription of the E1A gene of the highly oncogenic adenovirus 12 (Ad12) initiates at two start sites (TS1 and TS2). We have previously shown that the E2F and ATF motifs distal of TS1 co-operatively participate in E1A autostimulation from the TS1 promoter region. Here we report the identification of a second E2F-like target region (E2DFII) immediately upstream of the E1A-stimulating factor 1 binding site (ESF-1), important for 13S-mediated autoactivation from TS2. Reporter constructs lacking distinct TS2 cis-acting elements were analysed for their levels of CAT expression in the absence and presence of the E1A 13S protein in transient expression assays. In the absence of 13S, full promoter activity was observed only for a construct containing all elements (the E2F-like motif, and E-Box and the TATA element). Promoter activation increased significantly in Ad12 E1A-co-transfected cells. Induction by the 13S protein was also detected for the construct containing a non-functional ESF-1 sequence. Our results indicate that the E2F-like motif is responsible for activation medicated by the 13S protein from TS2, while ESF-1-or TATA-binding protein activity were not involved. Additionally, the TATA sequence appeared to be dispensable for transactivation. Gel-shift experiments using the E2F-like promoter element as a probe indicated the binding of an E2F-5 or E2F-5-like transcription factor to this region. We conclude that transcription through the TS1 as well as the TS2 promoter region is stimulated by the Ad 12 13S protein. Moreover, transfection of the construct including both TS1 and TS2 indicates an E2F-site-mediated synergism between both regions with respect ot E1A-induced transactivation.


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