COOPERATIVITY BETWEEN AN UPSTREAM TATA-LIKE SEQUENCE AND A CAA REPEATED ELEMENT MEDIATES E1A-DEPENDENT NEGATIVE REPRESSION OF THE H-2K(B) CLASS-I GENE

In primary rodent cells transformed by the E1A region of the highly on cogenic adenovirus type 12, repression of transcription mediated by th e far upstream TATA-like element was observed only in conjunction with either possible juxtaposition of a CAA repeated element in the presen ce of E1A and was dependent upon the relative arrangement of both the TATA-like and CAA repeated motifs in both homologous and heterologous promoter constructs, A gel shift competition study demonstrated that t he TATA-binding protein (TBP) or a TBP-like protein can bind to both t he upstream TATA-like sequence and the regular TATA box on the H-2K(b) basal promoter, Moreover, employing immunoselection and cyclic amplif ication and selection of targets (CASTing) methods with nuclear extrac ts derived from Ad12-E1A transformants, we have identified a high affi nity binding site in the H-2K(b) class I promoter for E1A-associated D NA-binding proteins. The sequences of the binding sites were identifie d and were found to contain both the upstream TATA like motif and the CAA repeated motifs. Our results suggest that the TATA like sequence i n the far upstream region of the H-2K(b) gene is one of the elements t hat is required for Ad12-E1A-mediated negative repression.