Mechanisms of transcriptional regulation for the rat carbamyl phosphate synthetase I gene

The proximal promoter of the rat carbamyl phosphate synthetase I gene contains 4 cis-acting regulatory regions, designated sites GAC, I, II, and III. Transient transfection assays revealed that the GAG element functions as the main activator element, whereas sites I and III bind repressor proteins and site II binds an anti-repressor. We propose that sites I and III repress the CPS I promoter either by quenching the sole activator(s) bound to the GAG element, or by directly repressing the basal transcription apparatus. Footprint analyses of the promoter with and without a mutation within site II suggested that the site II-anti-repressor may interfere with the site I and site III repressors by a similar quenching mechanism. Although the elements within the proximal promoter exhibit the potential for complex regulation, they are unable to confer tissue-specific promoter activity when analyzed either by in vitro transcription employing nuclear extracts from tissues that do not express the endogenous gene (i.e. brain, kidney, and spleen), or by transient transfection assays in non-CPS I-expressing cells (i.e. Rat1 and C6). However, we identified a cell-type specific enhancer that lies 10 kb upstream of the promoter. Unexpectedly, the enhancer functions as well in a gene-specific manner because enhancer-stimulated promoter activity was exclusively dependent on the GAG element within the CPS I proximal promoter. As a result, the GAG element is required to couple enhancer activity to the proximal promoter and exemplifies an efficient method whereby enhancers can be constrained to activate only their cognate promoters