Cloning and characterization of the promoter region of the bovine membrane tethering protein p115 gene and its regulation in mammary epithelial cells

Abstract

To elucidate the molecular basis of the expression of the membrane tethering protein p115, a genomic DNA clone including the 5′-flanking region, first exon, and part of the first intron of the p115 gene was isolated from a bovine genomic DNA library in λFIX II. Nine transcriptional initiation sites (at −189, −183, −178, −177, −164, −151, −149, −133 and −129 from the translation initiation site) were determined by the CapSite® Hunting method. The 5′-flanking region (a 2.6-kb fragment) of the bovine p115 gene ligated to a luciferase reporter gene displayed promoter activity in primary cultured bovine mammary epithelial (BME) cells and in human MCF-7 breast carcinoma cells. The luciferase reporter gene assays of the promoter deletion constructs suggested the region required for promoter activity of the bovine p115 gene. The region that retained promoter activity contained a potential nuclear respiratory factor-1 (NRF-1) binding site. In both the BME and MCF-7 cells, mutagenesis to impair the NRF-1 consensus sequence in the p115 promoter gave substantially lowered levels of luciferase expression. An electrophoretic mobility shift assay (EMSA) showed that the NRF-1 consensus sequence and nuclear protein formed a complex that was abolished by oligonucleotides containing the authentic NRF-1 binding site, and was also supershifted with an antibody to NRF-1. In luciferase reporter gene assays of the p115 promoter constructs, treatment of the MCF-7 cells with estradiol-17β (E 2), insulin, or both stimulated the p115 promoter activity correlating with the cell proliferation rate. These results indicate that the NRF-1 response element in the p115 promoter is important for promoter function, and that it involves the binding of NRF-1. Furthermore, the results suggested that p115 gene transcription is activated by E 2, by insulin, or by both in association with the stimulation of mammary epithelial cell proliferation.