Characterization of a carbohydrate response element regulating the gene for human galactose-1-phosphate uridyltransferase.
Human galactose-1-phosphate uridyltransferase (hGALT) is a central enzyme in the conserved pathway by which galactose is converted to energy, UDP-galactose and UDP-glucose. A natural mutation that deleted -119 to -116 bp (delGTCA) of the promoter decreased hGALT mRNA and enzyme activity and prompted analysis of hGALT gene regulation. Regulatory domains were identified by inspection and confirmed in a reporter system. Previous studies by others were confirmed that HepG2 cells grown in D-glucose increased hGALT enzyme activity and mRNA by 30%. We extended these observations by sequencing the promoter region and identifying a potential carbohydrate response element (ChoRE). The response to glucose rose to 190% when a plasmid construct containing a luciferase reporter and only the -165 bp region as a promoter was transfected into HepG2 and NIH:OVCAR-3. By contrast, fibroblasts transfected with the identical construct failed to respond to glucose. Within the -165 bp region there were two enhancer (E-box) motifs that encompassed the delGTCA mutation. The deletion diminished the positive regulatory response, but an additional GTCA repeat unexpectedly increased the response. Using this postulated ChoRE as a probe in electrophoretic mobility shift assays, multiple nuclear proteins bound and one was identified as a member of the basic/helix-loop-helix/leucine zipper enhancer-binding (b/HLH/LZ) family. Increased binding of proteins correlated with increased hGALT expression when the spacing between E-box motifs was enlarged but the carbohydrate response was dampened. When the 3(')E-box was mutated, b/HLH/LZ binding and gene expression were abolished. We conclude that the hGALT promoter region contains a ChoRE in which the spacing between and the sequence of its E-box motifs are critical. One nuclear protein of the b/HLH/LZ family is necessary, but not sufficient for the carbohydrate response.
Elsas
Louis J
LJ
Department of Pediatrics, Division of Medical Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA. lje@rw.ped.emory.edu
Webb
Aimee L
AL
Langley
Sharon D
SD
D001665:Binding Sites; D002460:Cell Line; D024721:E-Box Elements / Q000235:genetics / Q000502:physiology; D005786:Gene Expression Regulation; D005947:Glucose / Q000378:metabolism; D006801:Humans; D011401:Promoter Regions, Genetic / Q000502:physiology; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase / Q000235:genetics*
D005694:UTP-Hexose-1-Phosphate Uridylyltransferase; D005947:Glucose