CArG boxes in the human cardiac alpha-actin gene are core binding sites for positive trans-acting regulatory factors

Positively acting, rate-limiting regulatory factors that influence tissue-specific expression of the human cardiac alpha-actin gene in a mouse muscle cell line are shown by in vivo competition and gel mobility-shift assays to bind to upstream regions of its promoter but to neither vector DNA nor a beta-globin promoter. Although the two binding regions are distinctly separated, each corresponds to a cis region required for muscle-specific transcriptional stimulation, and each contains a core CC(A + T-rich)6GG sequence (designated CArG box), which is found in the promoter regions of several muscle-associated genes. Each site has an apparently different binding affinity for trans-acting factors, which may explain the different transcriptional stimulation activities of the two cis regions. Therefore, we conclude that the two CArG box regions are responsible for muscle-specific transcriptional activity of the cardiac alpha-actin gene through a mechanism that involves their binding of a positive trans-acting factor in muscle cells.