Repressor to activator switch by mutations in the first Zn finger of the glucocorticoid receptor: is direct DNA binding necessary?
Transfection of HeLa cells with cDNA vectors expressing the wild-type human glucocorticoid receptor (GR) enabled dexamethasone to strongly repress cytokine- and second messenger-induced expression of cotransfected chimeric reporter genes containing transcription regulatory DNA elements from the human interleukin 6 (IL-6) promoter. Deletion of the DNA-binding domain or of the second Zn finger or a point mutation in the Zn catenation site in the second finger blocked the ability of GR to mediate repression of the IL-6 promoter. Unexpectedly, deletion of the first Zn finger, a point mutation in the Zn-catenation site in the first finger, or one in the steroid-specificity domain at the base of the first finger converted GR into a dexamethasone-responsive activator that enhanced basal and interleukin 1-induced IL-6 promoter function. These first-finger mutants of GR also mediated dexamethasone-responsive enhancement of expression of the herpesvirus thymidine kinase-chloramphenicol acetyltransferase (TK-105-CAT and TK-80-CAT) reporter genes but not of the murine mammary tumor virus long terminal repeat-CAT or the c-fos-CAT (pFC700) reporter genes. Wild-type GR was able to specifically bind to DNA fragments containing glucocorticoid response element sequences in both the murine mammary tumor virus and IL-6 promoters, albeit weakly to the latter, in a sequential DNA-binding immunoprecipitation assay. The first-finger mutants of GR, however, were inactive in this assay. Thus, mutations in the first Zn finger unmask unusual promoter-specific activation properties of GR that may not require direct high-affinity binding of the mutant GR to target DNA.
Ray
A
A
Rockefeller University, New York, NY 10021.
LaForge
K S
KS
Sehgal
P B
PB
D000595:Amino Acid Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D002872:Chromosome Deletion; D016207:Cytokines / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology*; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics*; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011487:Protein Conformation; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*; D015290:Second Messenger Systems / Q000187:drug effects; D014162:Transfection; D016335:Zinc Fingers / Q000235:genetics* / Q000502:physiology
D016207:Cytokines; D004268:DNA-Binding Proteins; D015850:Interleukin-6; D011965:Receptors, Glucocorticoid; D011994:Recombinant Proteins; D012097:Repressor Proteins; D003907:Dexamethasone; D015500:Chloramphenicol O-Acetyltransferase