Functional Dissection of a cAMP-Response Element Using Recombinant Mutations

Abstract

The human CG-a gene expressed in the placenta is transcriptionally activated by cAMP. Two copies of a palindrome, 5′-TGACGTCA-3′, similar to sequences in other cAMP-responsive genes, reside within two identical 18-bp sequences arranged as adjacent direct repeats in the CG-a 5′-flanking region. DNase footprint and gel mobility shift analyses identify interactions of binding proteins with the 18-bp repeats at -146 to -111, with a more upstream expression sequence (UES) at -178 to -156, and with a more downstream sequence, including a CCAAT-box-like element (DES) at -100 to -72. One or two synthetic copies of the 18-bp sequence fused to the CG-a promoter enhance transcription of a reporter chloramphenicol acetyl transferase (CAT) gene in placental choriocarcinoma (JEG-3) cells when inserted in either orientation, both 5′ to the cap site or 3′ of the coding sequence. When paired with the CGa promoter the 18-bp enhancer exhibits strong preference for JEG-3 cells. Transcription of fusion genes containing the 18-bp enhancer is stimulated 30- to 40-fold by 8-br-cAMP, indicating that the 18-bp enhancer is a cAMP-responsive enhancer (CRE). A single CRE stimulates transcriptional activity 10-fold less than the double CRE and 100-fold less than the double CRE fused to the UES; the transcriptional enhancement of the two CREs and the UES are synergistic and not additive. Synthetic oligonucleotide expression cassettes encompassing the single CRE, double CRE and UES sequences were used to assess the effects of mutations on the individual functional elements. Mutations of sequences adjacent to, as well as point substitutions and deletional mutations within the CRE indicate that the octamer motif is essential for expression and cAMP responsivity but that synergistic transcriptional responses are highly dependent upon the surrounding contextual sequences. Co-transfection and expression of a-gene CAT reporters with increasing amounts of specific competitor DNA sequences localize the interaction of rate-limiting stimulatory transacting factors predominantly to the region containing the CREs and UES (-236 to -100). Saturation expression and competitive inhibition of expression were indistinguishable in the presence and absence of 8-br-cAMP, suggesting that the cAMP-mediated enhancement of transcription does not alter affinities of the binding of factors to the DNA but rather increases the efficiency of transcription. These studies indicate that the cooperative cis-trans interaction of cAMP-responsive enhancer-like elements with the homologous promoter and an upstream element in a constrained cis-context mediated by transacting DNA binding proteins, represents part of the mechanism of cell-specific expression of the CG-a gene.