Binding of the 97 kDa glucocorticoid receptor to the 5' upstream flanking region of the mouse c-Ha-ras oncogene.
Glucocorticoids regulate transcription of specific genes through the interaction of glucocorticoid hormone receptor complexes with DNA binding sites called glucocorticoid response elements (GREs). The GRE consensus sequence has been defined to be the imperfect palindromic sequence 5'-GGTACANNNTGTTCT-3', the most highly conserved portion being the 5'-TGTTCT-3' hexamer. We have identified 5 potential GREs in the 5' upstream noncoding region of the mouse c-Ha-ras oncogene, two with the same hexanucleotide sequence and three with a similar sequence. When subcloned fragments of the mouse c-Ha-ras 5' upstream region (containing the 2 hexamer GREs of exact homology) were fused to the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into HeLa cells, CAT expression driven from the ras promoter was induced up to 3-fold in the presence of dexamethasone. To determine whether the 5' upstream region of the mouse Ha-ras gene was capable of specifically interacting with the glucocorticoid receptor complex, we performed Southwestern blot analysis showing that cloned DNA fragments from the 5' upstream region of the mouse c-Ha-ras gene were able to bind a 97 kDa protein in whole cell extracts from both primary SENCAR mouse epidermal cells and HeLa G cells. Immunodepletion of the epidermal cell extract with a monoclonal antibody to the glucocorticoid receptor verified that the 97 kDa protein bound by the Ha-ras 5' region was indeed the glucocorticoid receptor protein. Our results demonstrate that the upstream noncoding region of the mouse c-Ha-ras gene binds the glucocorticoid receptor. Furthermore, the presence of glucocorticoids enhances the transcription of the mouse Ha-ras promoter region in a transient gene expression assay.
Strawhecker
J M
JM
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha 68105-1065.
Betz
N A
NA
Neades
R Y
RY
Houser
W
W
Pelling
J C
JC
D000818:Animals; D001483:Base Sequence; D002471:Cell Transformation, Neoplastic; D015342:DNA Probes; D004817:Epidermis / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D011905:Genes, ras; D005822:Genetic Vectors; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011485:Protein Binding; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D014162:Transfection
D015342:DNA Probes; D011965:Receptors, Glucocorticoid