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Volume 271, Number 31, Issue of August 2, 1996 pp. 18662-18671
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Expression of Human Glucocorticoid Receptor Gene and Interaction of Nuclear Proteins with the Transcriptional Control Element

(Received for publication, September 21, 1995, and in revised form, March 4, 1996)

From the Department of Molecular Endocrinology, Laval University Hospital Center, 2705 Boulevard Laurier, Sainte Foy, G1V 4G2, Québec, Canada

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 -chymotrypsin and immunoblotting with Ab 111 and Ab N3H10 confirmed their identities as Ku80 and Ku70, respectively.

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