Phylogenetic Footprinting of the Human Cytochrome c Oxidase Subunit Vb Promoter

The humanCOX5Bgene encodes subunit Vb of cytochromecoxidase (COX). COX Vb is 1 of the 10 subunits of the mitochondrial COX complex encoded by a nuclear gene. We have defined a region in the humanCOX5Bpromoter essential for gene expression and shown by phylogenetic footprinting of 11 primateCOX5Bpromoters that manycis-regulatory elements in this region are evolutionarily conserved. The transcription start site of humanCOX5Bwas mapped 58 bp upstream of the initiation Met codon by primer extension using a thermostable reverse transcriptase. A 475-bp region (−456 to +20) of the humanCOX5Bgene was shown to function as a promoter for the chloramphenicol acetyl transferase (CAT) gene in expression vectors when transfected into HeLa cells. The humanCOX5Bgene is located in a CpG island and contains several potential binding sites for the transcription factor Sp1, but no consensus TATA box element. Several sequence elements associated with the transcriptional regulation of respiratory genes were also found in the promoter and 5′ flanking region, including a single NRF-1 site and two 9-bp direct repeats containing binding sites forets-domain proteins, such as NRF-2/GABP. Many features of the humanCOX5Bpromoter are conserved in theCOX5Bpromoters of primates, in particular, the presence of a single binding site for NRF-1 and multiple sites for Sp1 and NRF-2/GABP. Electrophoretic mobility shift assays demonstrate that the conserved NRF-1 site in primateCOX5Bpromoters is specifically recognized by a factor present in HeLa nuclear extracts. Phylogenetic footprinting has identified additional conserved elements that may also function as binding sites for regulatory factors.