Cell-specific regulation of transcription of the malic enzyme gene: characterization of cis-acting elements that modulate nuclear T3 receptor activity.

Stimulation of malic enzyme transcription by triiodothyronine (T3) is robust (> 60-fold) in chick embryo hepatocytes, weak (5-fold) in chick embryo fibroblasts that stably overexpress the nuclear T3 receptor-alpha, and still weaker (1-fold) in chick embryo fibroblasts which contain nuclear T3 receptor levels that are similar to those of chick embryo hepatocytes. Using DNase I hypersensitivity, functional transfection, and in vitro DNA-binding analyses, four cis-acting elements were identified in the malic enzyme 5'-flanking DNA that conferred differences in nuclear T3 receptor activity between chick embryo hepatocytes and chick embryo fibroblasts. These cell-specific regulatory elements are located at -3895/-3890, -3761/-3744, -3703/-3686, and -3474/-2715 bp and overlap with DNase I hypersensitive sites that are observed in chromatin of chick embryo hepatocytes. Each element enhances T3 responsiveness of the malic enzyme promoter in chick embryo hepatocytes but has no effect on T3 responsiveness in chick embryo fibroblasts. Three of the cell-specific regulatory elements flank a previously identified DNA fragment (-3889 to -3769 bp; Hodnett et al., Arch. Biochem. Biophys. 334, 309-324, 1996) that contains one major and four minor T3 response elements. The cell-specific regulatory element at -3703/-3686 bp binds to the liver-enriched factor, CCAAT/enhancer-binding protein-alpha, whereas cell-specific regulatory elements at -3895/-3890 and -3761/-3744 bp bind proteins of unknown identity. While the cell-specific regulatory element at -3761/-3744 bp contains sequences that resemble binding sites for CCAAT/enhancer-binding protein, activator protein-1, cyclic AMP response element binding protein, and NF-1, none of these proteins appear to bind to this DNA fragment. These data suggest that cell-specific differences in T3 responsiveness of the malic enzyme gene are mediated in large part by nonreceptor proteins that augment the transcriptional activity of the nuclear T3 receptor in hepatocytes.