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 co mplexes 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 footprint ing analysis of the promoter region (-322 to +47 bp) revealed four major pr otein-binding domains (termed P1-P4). Nested deletions and site-specific mu tations of approximate to 100 bp proximal promoter region identified two el ements, TACGAC direct repeat sequence and cAMP-response element (CRE)-like site, which are localized in the P2 and P1 domains, respectively, and media te basal transcription of the DLD gene. Electrophoretic mobility supershift assays showed that the CRE-like site is associated with CRE binding protei n. Interestingly, when DLD promoter constructs (-1.8 kb to +47 bp and -78 t o +47 bp) fused with the chloramphenicol acetyltransferase (CAT) reporter g ene were transiently transfected into human HepG2 cells either in the prese nce or absence of 0.5 mM 8-Br-cAMP, the levels of CAT expression remained u naffected. In addition, endogenous DLD mRNA levels in HepG2 cells also rema ined 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 hu man DLD promoter, but does not confer cAMP-dependent gene regulation. (C) 2 001 Elsevier Science Ltd. All rights reserved.