Functional analysis of the promoter and chromosomal localization for human LEP503, a novel lens epithelium gene.
LEP503 is a novel gene product isolated from lens epithelial cells by a subtractive cDNA cloning strategy. It is highly conserved in different vertebrate species and developmentally regulated in postnatal rat lens, suggesting that LEP503 may be an important lens epithelium gene involved in the processes of lens epithelial cell differentiation. The expression of LEP503 is highly restricted to lens epithelial cells in vivo. To investigate the molecular mechanisms regulating the promoter of the human LEP503, we cloned and characterized the promoter of the human LEP503 gene. The transcription start site was localized to a nucleotide C 22 base pairs (bp) 5' of the initiation methionine codon. By reporter gene transfection experiments, we found that approximately 2.5-kb of LEP503 5'-flanking sequence directed high level luciferase activity in human lens epithelial cells; further deletion analysis revealed positive regulatory element between bp -401 and +22. Mutation analysis in each of the seven potential binding sites for transcription factors within the region between -401 and +22 showed that the AP-1 element at -131 and the Sp1 element at -48 are the most important sites for the tissue-specific expression of LEP503. Consistent with lens epithelial cell-restricted expression of LEP503 mRNA, we found that the approximately 2.5-kb 5'-flanking sequence directed high-level promoter activity in lens epithelial cells but not in other cell types. Understanding the LEP503 promoter will allow us to investigate lens epithelial cell-specific gene regulation and to uncover methods for targeting gene delivery specifically to lens epithelial cells. The LEP503 gene is mapped to human chromosome 1q22, the same location to which zonular pulverulent cataract was previously mapped.
Wen
Y
Y
The Membrane Biology Laboratory, Department of Physiology, School of Medicine, UCLA, Los Angeles, CA 90073, USA. ywen@ucla.edu
Ibaraki
N
N
Reddy
V N
VN
Sachs
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D002460:Cell Line; D002874:Chromosome Mapping; D002878:Chromosomes, Human, Pair 1; D006224:Cricetinae; D003459:Crystallins / Q000096:biosynthesis / Q000235:genetics*; D004268:DNA-Binding Proteins; D006801:Humans; D007908:Lens, Crystalline; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D051381:Rats; D014158:Transcription, Genetic
D003459:Crystallins; D004268:DNA-Binding Proteins; C431569:LENEP protein, human