Haem repression of the housekeeping 5-aminolaevulinic acid synthase gene in the hepatoma cell line LMH.
Haem is essential for the health and function of nearly all cells. 5-Aminolaevulinic acid synthase-1 (ALAS-1) catalyses the first and rate-controlling step of haem biosynthesis. ALAS-1 is repressed by haem and is induced strongly by lipophilic drugs that also induce CYP (cytochrome P450) proteins. We investigated the effects on the avian ALAS-1 gene promoter of a phenobarbital-like chemical, Glut (glutethimide), and a haem synthesis inhibitor, DHA (4,6-dioxoheptanoic acid), using a reporter gene assay in transiently transfected LMH (Leghorn male hepatoma) hepatoma cells. A 9.1 kb cALAS-1 (chicken ALAS-1) promoter-luciferase-reporter construct, was poorly induced by Glut and not by DHA alone, but was synergistically induced by the combination. In contrast, a 3.5 kb promoter ALAS-1 construct was induced by Glut alone, without any further effect of DHA. In addition, exogenous haem (20 microM) repressed the basal and Glut- and DHA-induced activity of luciferase reporter constructs containing 9.1 and 6.3 kb of ALAS-1 5'-flanking region but not the construct containing the first 3.5 kb of promoter sequence. This effect of haem was subsequently shown to be dependent on the -6.3 to -3.5 kb region of the 5'-flanking region of cALAS-1 and requires the native orientation of the region. Two deletion constructs of this approx. 2.8 kb haem-repressive region (1.7 and 1.1 kb constructs) retained haem-dependent repression of basal and drug inductions, suggesting that more than one cis-acting elements are responsible for this haem-dependent repression of ALAS-1. These results demonstrate that there are regulatory regions in the 5'-flanking region of the cALAS-1 gene that respond to haem and provide a basis for further investigations of the molecular mechanisms by which haem down-regulates expression of the ALAS-1 gene.
Kolluri
Sridevi
S
Department of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center, Farmington, CT 06030, USA.
Sadlon
Timothy J
TJ
May
Brian K
BK
Bonkovsky
Herbert L
HL
D000624:5-Aminolevulinate Synthetase / Q000235:genetics*; D000818:Animals; D045744:Cell Line, Tumor; D002645:Chickens; D015536:Down-Regulation / Q000187:drug effects*; D004357:Drug Synergism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005984:Glutethimide / Q000494:pharmacology; D006418:Heme / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D006537:Heptanoates / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics; D015854:Up-Regulation / Q000187:drug effects
D006537:Heptanoates; D006418:Heme; C020804:succinylacetone; D005984:Glutethimide; D000624:5-Aminolevulinate Synthetase