The Chicken Malic Enzyme Gene: Structural Organization and Identification of Triiodothyronine Response Elements in the 5′-Flanking DNA

In vivo,feeding stimulates and starvation inhibits transcription of the malic enzyme gene. In chick-embryo hepatocytes in culture, triiodothyronine (T3) stimulates and glucagon inhibits transcription of this gene. As a first step in the characterization of the involved regulatory mechanisms, fragments of genomic DNA spanning the structural and 5′-flanking regions of the chicken malic enzyme gene were cloned. The coding region of the gene is organized into 14 exons and 13 introns and is greater than 106 kb in length. The size of the gene, the number and lengths of the exons, and positions at which introns are inserted into the coding regions are virtually identical in the chicken and rat genes. When transiently transfected into chick-embryo hepatocytes, 5800 bp of 5′-flanking DNA conferred T3 responsiveness to a linked chloramphenicol acetyltransferase (CAT) reporter gene. Using deletion and site-specific mutations of 5′-flanking DNA, we identified a complex T3 response unit that contains one major T3 response element (T3RE) and several minor ones. The major element contains two degenerate copies of the hexamer, RGGWMA, separated by 4 bp and was a strong repressor in the absence of ligand. Endogenous levels of T3 receptor are sufficient to allow the T3 response elements in the upstream region of the malic enzyme gene to confer responsiveness to T3, suggesting that they are physiologically relevant.