Cyclic AMP-mediated Inhibition of Transcription of the Malic Enzyme Gene in Chick Embryo Hepatocytes in Culture CHARACTERIZATION OF A CIS-ACTING ELEMENT FAR UPSTREAM OF THE PROMOTER

Abstract Glucagon, acting via cAMP, inhibits transcription of the malic enzyme gene in chick embryo hepatocytes. In transiently transfected hepatocytes, fragments from the 5′-flanking DNA of the malic enzyme gene confer cAMP responsiveness to linked reporter genes. The major inhibitory cAMP response element at −3180/−3174 base pairs (bp) is similar to the consensus binding site for AP1. DNA fragments from −3134/−3115, −1713/−944, and −413/−147 bp also contain inhibitory cAMP response elements. The negative action of cAMP is mimicked by overexpression of the catalytic subunit of protein kinase A, inhibited by overexpression of a specific inhibitor of protein kinase A, and inhibited by overexpression of the T3 receptor; these results indicate involvement of the classical eukaryotic pathway for cAMP action and suggest interaction between the T3 and cAMP pathways. Sequence-specific complexes form between nuclear proteins and a DNA fragment containing −3192/−3158 bp of 5′-flanking DNA. In nuclear extracts prepared from cells treated with chlorophenylthio-cyclic AMP and T3, the complexes have different masses than those formed with extracts from cells treated with T3 alone. Antibodies to c-Fos or ATF-2 inhibit formation of the complex formed by proteins from cells treated with chlorophenylthio-cyclic AMP and T3 but not by those from cells treated with T3 alone. These results suggest an important role for c-Fos and ATF-2 in glucagon-mediated inhibition of transcription of the malic enzyme gene.