PHYLOGENETIC FOOTPRINTING OF THE HUMAN CYTOCHROME-C-OXIDASE SUBUNIT VB PROMOTER

The human COX5B gene encodes subunit Vb of cytochrome c oxidase (COX), COX Vb is 1 of the 10 subunits of the mitochondrial COX complex encod ed by a nuclear gene We have defined a region in the human COX5B promo ter essential for gene expression and shown by phylogenetic footprinti ng. of 11 primate COX5B promoters that many cis-regulatory elements in this region are evolutionarily conserved, The transcription start sit e of human COX5B was 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 human COX5B ene was shown to function as a promoter for the chloramphenicol acetyl transferase (CAT) gene iu . expression vectors when transfected into HeLa cells, The human COX5B gene is located in a CpG island and contains several potential bindin g sites for the transcription factor Spl, hut no consensus TATA box el ement, Several sequence elements associated with tile 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 re peats containing binding sites for ets-domain proteins, such as NRF-2/ GABP, Many features of the human COX5B promoter are conserved in the C OX5B promoters of primates, in particular, the presence of a single bi nding site for NRF-1 aid multiple sites for Sp1 and NRF-S/GABP, Electr ophoretic mobility shift assays demonstrate that the conserved NRF-1 s ite in primate COX5B promoters is specifically recognized by a factor present in HeLa nuclear extracts, Phylogenetic footprinting has identi fied additional conserved elements that may also function as binding s ites for regulatory factors. (C) 1996 Academic Press, Inc.