MULTIPLE PROTEIN-BINDING DOMAINS AND FUNCTIONAL CIS-ELEMENTS IN THE 5'-FLANKING REGION OF THE HUMAN PYRUVATE-DEHYDROGENASE ALPHA-SUBUNIT GENE

We have characterized the 5'-flanking region of the alpha-subunit gene of the human pyruvate dehydrogenase (E1). DNase I footprinting with r at liver nuclear extracts identified 7 major protein-binding domains t ermed P1 through P7 in a 796 base pair DNA fragment (base pairs -763 t o +33). P1 through P4 are clustered in the -221/+33 region. These prot ein-binding domains contain several known consensus sequences such as a TATA box, CAAT box, Sp1, and CRE, which all have previously been imp licated in the constitutive transcription of several genes. Oligonucle otide competition studies indicate that oligonucleotides specific for CTF/NF-1 and Sp1 displaced the nuclear proteins bound to the CAAT box (within P3) and an Sp1 site (within P4), respectively. Several other w ell-characterized and purified transactivators (c-Fos, c-Jun, C/EBP, A P-2, and Sp1) have been shown to bind to the -221/+33 region. Other el ements located upstream of the -221/+33 region, which includes nucleas e protection domains P5-P7, are required for enhanced promoter activit y of the 796 bp sequence. Promoter activity was measured by transient expression of a chloramphenicol acetyltransferase gene ligated to dele tion fragments of the 5'-flanking region. Crucial element(s) for promo ter activity and complex DNA-nuclear protein interactions were confine d with in a region spanning -221/+33. This region also retained more t han 75% of the promoter activity of the 796 bp sequence. Additionally, this promoter region shows characteristics of both facultative and ho usekeeping gene promoters, suggesting complex transcriptional regulati on.