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.
Highlights
HeLa, human placenta, and human breast tumor (MCF-7) cells
We have examined the importance of the 5Ј-regulatory sequences in the promoter region of the hGR gene in an effort to identify and to characterize factors involved in the regulatory pathway (Fig. 1)
Identification of DNA Sequences Directing the Tissue-specific Expression of GR Gene—To identify sequences involved in the regulation of hGR gene, MCF-7, HeLa, and human placenta cells were cotransfected with hGR gene deletion mutants fused to the CAT reporter gene
Summary
HeLa, human placenta, and human breast tumor (MCF-7) cells. The chimeric gene construct ؊892 5GAAGTGACACACTTC3 ؊878-CAT was sufficient for high level of expression in HeLa and placenta cells in culture. 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 the actions of glucocorticoids by direct interactions and regulates the transcription of glucocorticoid responsive genes [5,6,7,8,9]. Considering that the glucocorticoid effects are mediated by GR, we have constructed a series of 5Ј deletion mutants containing the promoter region of the hGR gene fused to chloramphenicol acetylreceptor enhancing factors Both Ku80 and Ku70 transferase (CAT) chimeric gene and introduced them into polypeptides were present in high concentrations in hu- human placenta cells in culture.
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