FAST-MUSCLE-SPECIFIC DNA-PROTEIN INTERACTIONS OCCURRING IN-VIVO AT THE HUMAN ALDOLASE-A M-PROMOTER ARE NECESSARY FOR CORRECT PROMOTER ACTIVITY IN TRANSGENIC MICE

The human aldolase A tissue-specific M promoter (pM) has served as a m odel system for identifying pathways that lead to fast-muscle-speciali zed expression. The current study has delimited the sequences necessar y and sufficient for fast-muscle-specific expression in transgenic mic e to a short 209-bp fragment extending from bp -164 to +45 relative to the pM transcription start site. Genomic footprinting methods shelved that in this proximal region, the same elements that bind muscle nucl ear proteins in vitro are involved in DNA-protein interactions in inta ct muscle nuclei of transgenic mice. Furthermore, these experiments pr ovided the first evidence that different DNA-binding activities exist between slow, and fast muscles in vivo. Fast-muscle-specific interacti ons occur at an element named M1 and at a muscle-specific DNase I-hype rensitive site that was previously detected by in vitro methods. The f ormation of the muscle-specific DNase I-hypersensitive site reflects b inding of proteins to a close element, named M2, which contains a bind ing site for nuclear factors of the NF1 family. Mutational analysis pe rformed with transgenic mice confirmed the importance of the M1 elemen t for high-level fast-muscle-specific pM activity and suggested that t he M2/NF1 element is differently required for correct pM expression in distinct fast muscles. In addition, two other protein binding sites, the MEF3 motif and the USF site, seem to act as stage-specific activat ors and/or as participants in the establishment of an active chromatin configuration at pM.