STRUCTURAL CHARACTERIZATION AND REGULATORY ELEMENT ANALYSIS OF THE HEART ISOFORM OF CYTOCHROME-C-OXIDASE VIA

In order to investigate the mechanism(s) governing the striated muscle -specific expression of cytochrome c oxidase VIaH we have characterize d the murine gene and analyzed its transcriptional regulatory elements in skeletal myogenic cell lines. The gene is single copy, spans 689 b ase pairs (bp), and is comprised of three exons, The 5'-ends of transc ripts from the gene are heterogeneous, but the most abundant transcrip t includes a 5'-untranslated region of 30 nucleotides. When fused to t he luciferase reporter gene, the 3.5-kilobase 5'-flanking region of th e gene directed the expression of the heterologous protein selectively in differentiated So18 cells and transgenic mice, recapitulating the pattern of expression of the endogenous gene. Deletion analysis identi fied a 300-bp fragment sufficient to direct the myotube-specific expre ssion of luciferase in So18 cells. The region lacks an apparent TATA e lement, and sequence motifs predicted to bind NRF-1, NRF-2, ox-box, or PPAR factors known to regulate other nuclear genes encoding mito chon drial proteins are not evident. Mutational analysis, however, identifi ed two cis-elements necessary for the high level expression of the rep orter protein: a MEF2 consensus element at -90 to -81 bp and an E-box element at -147 to -142 bp. Additional E-box motifs at closely located positions were mutated without loss of transcriptional activity. The dependence of transcriptional activation of cytochrome c oxidase VIaH on cis-elements similar to those found in contractile protein genes su ggests that the striated muscle-specific expression is coregulated by mechanisms that control the lineage-specific expression of several con tractile and cytosolic proteins.