title: SP1 is a transcriptional regulator of URG-4/URGCP gene in hepatocytes.
abstract: URG-4/URGCP gene was implicated as an oncogene that contributes hepatocarcinogenesis regulated by Hepatitis-B-virus-encoded X antigen. However, the mechanism of transcriptional regulation of this gene remains largely unknown. For this reason, we focused on the functional analyses of URG4/URGCP promoter site. First, 545 bp of URG-4/URGCP, -482/+63, and three different 5'-truncated constructs, -109/+63, -261/+63, -344/+63 were cloned by PCR-based approach into pMetLuc luciferase reporter vector. Transient transfection assay showed that, -109/+63 construct has the highest activity. The promoter of URG-4/URGCP gene contained a CpG island region spanning 400 bp from translation start site. Many SP1/GC boxes, named GC-1 to GC-10 are present in 545 bp of URG-4/URGCP promoter. Because of presence of multiple SP1/GC boxes, promoter constructs were transiently co-transfected with SP1 expression vector to determine the effect of SP1 on URG-4/URGCP promoter activity. Co-transfection analyses induced the basal activity of -268/+63, -344/+63 and -482/+63 constructs. EMSA analysis of GC-4, GC-5, GC-6 and GC-7 binding sites located in -128/-148 bases, showed two DNA-protein binding complexes. Competition assay and super-shifted complexes indicated these complexes are resulted from SP1 binding. Also, site-directed mutagenesis of potential SP1 binding sites diminished both DNA-protein complexes and SP1-mediated upregulation of URG-4 promoter activity. These findings are valuable for understanding transcriptional regulation of URG4/URGCP that has a pivotal role in cancer progression.
authors.affiliation: Department of Molecular Biology and Genetics, Faculty of Science and Literature, Balikesir University, Balıkesir, Turkey.
authors.forename: Esra
authors.initials: E
authors.lastname: Tokay
authors.affiliation: Department of Molecular Biology and Genetics, Faculty of Science and Literature, Balikesir University, Balıkesir, Turkey. fkockar@balikesir.edu.tr.
authors.forename: Feray
authors.initials: F
authors.lastname: Kockar
mesh_terms: D045744:Cell Line, Tumor; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements / Q000502:physiology*; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology*; D015854:Up-Regulation / Q000502:physiology*
chemical_list: D009363:Neoplasm Proteins; D016329:Sp1 Transcription Factor; C108209:SP1 protein, human; C470990:URGCP protein, human
keywords: Promoter; SP1; Transcriptional regulation; URG4/URGCP