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

被引:17
作者
CHANG, M
NAIK, S
JOHANNING, GL
HO, L
PATEL, MS
机构
[1] CASE WESTERN RESERVE UNIV, SCH MED, CTR INHERITED DISORDERS ENERGY METAB, DEPT BIOCHEM, CLEVELAND, OH 44106 USA
[2] CASE WESTERN RESERVE UNIV, SCH MED, PEW CTR MOLEC NUTR, CLEVELAND, OH 44106 USA
关键词
D O I
10.1021/bi00067a014
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 with in 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.
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页码:4263 / 4269
页数:7
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