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 rat liver nuclear extracts identified 7 major protein-binding domains termed P1 through P7 in a 796 base pair DNA fragment (base pairs -763 to +33). P1 through P4 are clustered in the -221/+33 region. These protein-binding domains contain several known consensus sequences such as a TATA box, CAAT box, Sp1, and CRE, which all have previously been implicated in the constitutive transcription of several genes. Oligonucleotide 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 well-characterized and purified transactivators (c-Fos, c-Jun, C/EBP, AP-2, and Sp1) have been shown to bind to the -221/+33 region. Other elements located upstream of the -221/+33 region, which includes nuclease protection domains P5-P7, are required for enhanced promoter activity of the 796 bp sequence. Promoter activity was measured by transient expression of a chloramphenicol acetyltransferase gene ligated to deletion fragments of the 5'-flanking region. Crucial element(s) for promoter activity and complex DNA-nuclear protein interactions were confined within a region spanning -221/+33. This region also retained more than 75% of the promoter activity of the 796 bp sequence. Additionally, this promoter region shows characteristics of both facultative and housekeeping gene promoters, suggesting complex transcriptional regulation.
Chang
M
M
Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
Naik
S
S
Johanning
G L
GL
Ho
L
L
Patel
M S
MS
D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D003850:Deoxyribonuclease I; D005347:Fibroblasts / Q000201:enzymology; D005809:Genes, Regulator; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D012867:Skin / Q000201:enzymology; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011768:Pyruvate Dehydrogenase Complex; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D003850:Deoxyribonuclease I