CHARACTERIZATION OF THE PROMOTER REGULATORY REGION OF THE HUMAN PYRUVATE-DEHYDROGENASE BETA-GENE

A genomic clone (19 kb) harboring the intron-exon sequences and the promoter-regulatory region of the E1 beta gene of human pyruvate dehydrogenase complex was isolated by screening a placental genomic library. The nucleotide sequence of the promoter region (1245 bp) showed 18 differences (including mismatches, insertions, and deletions) as compared to the published sequence [Koike et al, (1990) Proc. Natl. Acad. Sci. U.S.A, 87, 5594-5597]. The E1 beta promoter lacked a TATA box homology but contained initiator sequences (two) and Spl sites (three) which are frequently found in TATA-less promoters. The DNase I footprinting pattern of the promoter region with crude rat liver nuclear extracts showed at least seven regions of protein binding and nuclease protection (P1-P7). The DNase I protected regions contained consensus nucleotide sequences recognized by GATA-1, Sp1, IgNF-A, Lva, bicoid Q9, NF-kB, HNF-5, H4TF-1, WAP5, and ADH transcription factors. Transient expression of chloramphenicol acetyltransferase (CAT) suggested the possible presence of negative elements located within the sequence from -2316 to -930, whereas deletion constructs containing -929 to +32 and -98 to +32 DNA sequences showed approximately 7- and 20-fold increases in CAT activity over the basal CAT activity. Additional studies indicated the presence of an orientation-dependent cis element (or elements) within the region from -282 to -397 that acts as an enhancer or a repressor upon a heterologous thymidine kinase promoter. The results show that the human E1 beta promoter represents an unusual variation in a housekeeping gene promoter with a unique combination of initiator sequences and Sp1 sites together with a protein-binding site (or sites) within the first 100 bp upstream of the transcription start site.