Mechanism of tissue-specific transcription: interplay between positive and negative regulatory factors.

At least four regulatory cis-acting DNA sequences, CCAAAAGTGG (element A), TTATTTTTA (element B), TATTTATT (element C), and TATTACCTTTAT (element S), were identified in cardiac myosin light chain-2 (MLC2) proximal promoter as target sites for sequence-specific binding of nuclear proteins. For muscle-specific transcription, the proximal promoter (-53 to +1) consisting only of elements B and C is required. Addition of element A to this promoter results in a muscle-specific up-regulation, whereas the addition of element S exerts a negative effect on transcription. The negative and positive regulatory effects of elements S and A respectively were demonstrated by site-specific mutations of the promoter following transient transfection of cardiac muscle cells in culture. Elements S and A interact separately with distinct nuclear protein factor present in both muscle and non-muscle cells, even though their regulatory activities are restricted to muscle cells. Among the multiple complexes resulting from the interaction of nuclear proteins and elements S and A DNAs, one requires both S and A sequences together for binding. Element B, which exerts a muscle-specific positive effect on transcription, binds to a nuclear protein present in cardiac muscle, but not in non-muscle cells. DNA-protein binding assays and mutational analysis of the MLC2 promoter suggest that the contribution of the functionally opposed cis-elements depends upon an interplay between the positively and negatively acting DNA-binding proteins via protein-protein interactions to mediate opposite regulatory effects on gene transcription.