Characterization of the human SLC22A18 gene promoter and its regulation by the transcription factor Sp1.
SLC22A18, a poly-specific organic cation transporter, is paternally imprinted in humans and mice. It shows loss-of-heterozygosity in childhood and adult tumors, and gain-of-imprinting in hepatocarcinomas and breast cancers. Despite the importance of this gene, its transcriptional regulation has not been studied, and the promoter has not yet been characterized. We therefore set out to identify the potential cis-regulatory elements including the promoter of this gene. The luciferase reporter assay in human cells indicated that a region from -120 bp to +78 bp is required for the core promoter activity. No consensus TATA or CAAT boxes were found in this region, but two Sp1 binding sites were conserved in human, chimpanzee, mouse and rat. Mutational analysis of the two Sp1 sites suggested their requirement for the promoter activity. Chromatin-immunoprecipitation showed binding of Sp1 to the promoter region in vivo. Overexpression of Sp1 in Drosophila Sp1-null SL2 cells suggested that Sp1 is the transactivator of the promoter. The human core promoter was functional in mouse 3T3 and monkey COS7 cells. We found a CpG island which spanned the core promoter and exon 1. COBRA technique did not reveal promoter methylation in 10 normal oral tissues, 14 oral tumors, and two human cell lines HuH7 and A549. This study provides the first insight into the mechanism that controls expression of this imprinted tumor suppressor gene. A COBRA-based assay has been developed to look for promoter methylation in different cancers. The present data will help to understand the regulation of this gene and its role in tumorigenesis.
Ali
Abdullah Mahmood
AM
Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India.
Bajaj
Vineeta
V
Gopinath
K S
KS
Kumar
Arun
A
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D017124:Conserved Sequence; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation; D004330:Drosophila / Q000166:cytology; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D027701:Organic Cation Transport Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D013045:Species Specificity
D027701:Organic Cation Transport Proteins; C111653:SLC22A18 protein, human; D016329:Sp1 Transcription Factor