TRANSACTIVATION BY THYROID-HORMONE RECEPTORS OF THE 5' FLANKING REGION OF THE HUMAN CHAT GENE

Fusion gene constructs containing the human choline acetyltransferase 5' flanking region are stimulated by thyroid hormone (T3) in neuronal NG108-15 and NE1-115 cells but not in non neuronal COS-1 and JEG-3 cells. To identify potential T3 receptor binding elements (T3RE), chimeric plasmids containing various lengths of the 5' end of the hChAT gene linked to the CAT reporter gene were assayed by transient transfections into NG108-15, NE1-115 and COS-1 cells. We show that regulation is T3 specific as estrogen, dexamethasone, dihydrotestosterone, all-trans-retinoic acid and 9-cis-retinoic acid have no effect. We localized several potential T3REs and characterized the most proximal T3RE (position 3280-3291) which contains two hexameric half-sites arranged as a direct repeat without a base pair spacer. An oligonucleotide containing this sequence confers T3 responsiveness to a heterologous promoter. The transcriptional response of this T3RE is markedly reduced after mutation of the first or second half-site indicating that both half-sites are required for a maximal T3 response. We have found that RAR alpha, RXR alpha and COUP-TF do not enhance T3 responsiveness and therefore they may not interact with T3R alpha in NG108-15 cells on this regulatory sequence. T3R monomer and dimer specific binding to the proximal T3RE is demonstrated by gel-retardation DNA binding assays and by methylation interference experiments. In COS-1 cells, T3R inhibits transcriptional activation by the transcription factor AP-1 whereas in NE1-115 cells T3R enhances AP-1 mediated activation in a T3 dependant fashion. It is likely that these effects involve protein-protein interactions. These results suggest that the T3 receptor can act as a positive transcriptional regulatory factor on the hChAT gene.