Activation of human insulin-like growth factor binding protein-1 gene promoter by a distal regulatory sequence in a human endometrial adenocarcinoma cell line.
Insulin-like growth factor-binding protein-1 (IG-FBP-1) is the major secretory protein of decidualized human endometrium. To understand IGFBP-1 gene regulation in human endometrium, we studied the IGFBP-1 gene promoter activity in human endometrial adenocarcinoma cell line HEC-1B. Previously, we have reported that a 105-base pair (bp) ClaI/RsaI fragment, from -2732 to -2628, of IGFBP-1 promoter enhances promoter activity by 10-fold in HEC-1B cells. In this study we have characterized the activation of IGFBP-1 promoter by this distal regulatory sequence. Transient transfection assays with deletion constructs demonstrated that the activating cis-elements were located in a 59-bp fragment, from -2686 to -2628, which enhanced promoter activity 50-fold. Transient transfections and gel mobility shift assays with oligo-directed mutants revealed three cis-elements within this 59-bp region: I) ATGGGTGGGA (-2675 to -2666), II) GCTGAGCAAGTGCACAACTATCC (-2660 to -2638), and III) AGGGCGGAGT (-2637 to -2628). In nuclear extracts of HEC-1B cells, at least two proteins bound to cis-element III, one of which was transcription factor Sp1 since antibody against Sp1 caused a supershift in a gel mobility shift assay. A protein with a molecular mass of approximately 100 kilodaltons bound to cis-element I as revealed by Southwestern blotting. An unidentified protein bound to cis-element II. Mutations in cis-element I, II, and III reduced promoter activity by 37%, 86%, and 88%, respectively, indicating that there was a synergistic function among these three cis-elements.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Obstetrics and Gynecology, State University of New York at Stony Brook 11794, USA.
J
J
Gao
J
J
Mazella
L
L
Tseng
D000230:Adenocarcinoma; D001483:Base Sequence; D016889:Endometrial Neoplasms; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011372:Progestins / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D012065:Relaxin / Q000378:metabolism; D014407:Tumor Cells, Cultured
D018970:Insulin-Like Growth Factor Binding Protein 1; D011372:Progestins; D012065:Relaxin