Cooperative transcription activation between ad1, a CRE-like element, and other elements in the CYP11B gene promoter

We previously reported the presence of six different cis.-acting elements (Ad1 to Ad6) in the promoter region of the bovine CYP11B gene. Although the Ad1 site (TGACGTGA) was similar to a palindromic CRE (TGACGTCA), two other upstream sequences, Ad3 and Ad4, were identified as the cAMP response sequences of the gene. We analyzed the functional relationship between the Ad1 site and the upstream elements. Mutation analyses of the Ad1 site indicated that the 5′ half of the site (TGACG) was important for the transcription of the gene in vitro. In Y-l cells, a plasmid with a mutated Ad1 showed no response to cAMP. The effect of the mutation at the Ad1 site on the cAMP response was almost the same as that of the deletion of Ad3 and Ad4, although the role of each element seemed to be different. These results indicated that both the Ad1 site and the upstream elements, Ad3 and Ad4, were necessary for the full response to cAMP of the CYP11B gene. When the Ad1 site in the promoter region was replaced with a palindromic CRE, elevated transcription activity was detected both in vitro and in vivo. Two kinds of CREBs (43 and 47 kDa) purified from a HeLa cell nuclear extract bound to the Ad1 site. The binding of the palindromic CRE to the nuclear factors) was stronger than that of Ad1.