Human dihydrolipoamide dehydrogenase gene transcription is mediated by cAMP-response element-like site and TACGAC direct repeat.
Dihydrolipoamide dehydrogenase is a common component of four multienzyme complexes which are involved in oxidation of carbohydrates, lipids and amino acids. To better understand the regulation of human DLD gene expression, we have analyzed the proximal promoter region of this gene. DNase I footprinting analysis of the promoter region (-322 to +47 bp) revealed four major protein-binding domains (termed P1-P4). Nested deletions and site-specific mutations of approximately 100 bp proximal promoter region identified two elements, TACGAC direct repeat sequence and cAMP-response element (CRE)-like site, which are localized in the P2 and P1 domains, respectively, and mediate basal transcription of the DLD gene. Electrophoretic mobility supershift assays showed that the CRE-like site is associated with CRE binding protein. Interestingly, when DLD promoter constructs (-1.8 kb to +47 bp and -78 to +47 bp) fused with the chloramphenicol acetyltransferase (CAT) reporter gene were transiently transfected into human HepG2 cells either in the presence or absence of 0.5 mM 8-Br-cAMP, the levels of CAT expression remained unaffected. In addition, endogenous DLD mRNA levels in HepG2 cells also remained unaffected by treatment with 0.5 mM 8-Br-cAMP. These results indicate that the CRE binding protein is essential for basal transcription of the human DLD promoter, but does not confer cAMP-dependent gene regulation.
Yang
H S
HS
Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, 140 Farber Hall, 3435 Main Street, Buffalo, NY 14214, USA.
Morris
J I
JI
Wang
Q
Q
Korotchkina
L G
LG
Kwon
M
M
Patel
M S
MS
D020121:5' Untranslated Regions / Q000235:genetics; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D006528:Carcinoma, Hepatocellular; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D000242:Cyclic AMP / Q000378:metabolism; D004247:DNA / Q000235:genetics*; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I; D008058:Dihydrolipoamide Dehydrogenase / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D008113:Liver Neoplasms; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D016415:Sequence Alignment; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D020121:5' Untranslated Regions; D004268:DNA-Binding Proteins; D015124:8-Bromo Cyclic Adenosine Monophosphate; D004247:DNA; D000242:Cyclic AMP; D008058:Dihydrolipoamide Dehydrogenase; D015500:Chloramphenicol O-Acetyltransferase; D003850:Deoxyribonuclease I