title: Expression of human glucocorticoid receptor gene and interaction of nuclear proteins with the transcriptional control element.
abstract: We have identified sequences responsible for the expression of the human glucocorticoid receptor gene (GR gene) using a set of 5' promoter deletion mutants in HeLa, human placenta, and human breast tumor (MCF-7) cells. The chimeric gene construct -892 5'-GAAGTGACACACTTC3' -878-CAT was sufficient for high level of expression in HeLa and placenta cells in culture. Deletion of palindromic sequences decreased levels of GR expression in these cells. By oligonucleotide-affinity chromatography with the palindromic glucocorticoid receptor enhancing factor-binding element (GREFE), we have isolated from human placenta nuclear extract two novel proteins glucocorticoid receptor enhancing factors 1 and 2 (GREF1 and GREF2), with apparent molecular masses of 80 and 62 kDa, respectively. These proteins, similar to the DNA-binding autoantigen Ku are, like Ku, heterodimers of polypeptide subunits p80 and p62, immunologically related to factors binding to proximal sequence element 1 in the promoter of small nuclear RNA (PSE1) and transferrin receptor enhancing factors. Both Ku80 and Ku70 polypeptides were present in high concentrations in human placenta and HeLa cells. In MCF-7 cells, however, only a high level of p62 was detected. While cotransfection of pcDNA-Ku80 with pHGR(-892 to -878)-CAT potentiated the expression of CAT, introduction of pcDNA-Ku70 did not affect the expression of CAT in transfected MCF-7 cells. UV cross-linking analysis showed that only GREF1 contacted DNA directly. Supershift assays with monoclonal antibodies Ab 111 (Ku80) or Ab N3H10 (Ku70) showed a direct interaction of GREF1 and GREF2 heterodimers with the palindrome. Partial peptide fingerprinting of GREF1 and GREF2 using alpha-chymotrypsin and immunoblotting with Ab 111 and Ab N3H10 confirmed their identities as Ku80 and Ku70, respectively.
authors.affiliation: Department of Molecular Endocrinology, Laval University Hospital Center, 2705 Boulevard Laurier, Sainte Foy, G1V 4G2, Québec, Canada.
authors.forename: N
authors.initials: N
authors.lastname: Warriar
authors.forename: N
authors.initials: N
authors.lastname: Pagé
authors.forename: M V
authors.initials: MV
authors.lastname: Govindan
mesh_terms: D000911:Antibodies, Monoclonal; D034961:Antigens, Nuclear; D001483:Base Sequence; D002460:Cell Line; D002678:Chimera / Q000235:genetics; D003432:Cross-Linking Reagents; D004247:DNA / Q000235:genetics; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000235:genetics / Q000276:immunology; D005260:Female; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D007120:Immunochemistry; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D010449:Peptide Mapping; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D011965:Receptors, Glucocorticoid / Q000235:genetics*; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D014466:Ultraviolet Rays
chemical_list: D000911:Antibodies, Monoclonal; D034961:Antigens, Nuclear; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D011965:Receptors, Glucocorticoid; D004247:DNA; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen