Homodimerization of the glucocorticoid receptor is not essential for response element binding: activation of the phenylethanolamine N-methyltransferase gene by dimerization-defective mutants.
We have tested the commonly held hypothesis that glucocorticoid receptors (GRs) must dimerize via their DNA binding domain (DBD) to bind to glucocorticoid response elements (GRE) and induce gene expression. Guided by the GR dimerization-deficient dim/dim knock-in mouse, which expresses normal mRNA levels of the strictly GR-dependent phenylethanolamine N-methyltransferase (PNMT) gene, we analyzed in detail the regulation of the PNMT 5'-flanking region using wild-type GR (GRwt) and GR dimer mutants (GRdms). We demonstrated that mouse and rat PNMT 5'-regulatory fragments are more strongly induced by GRdms than by GRwt. Footprinting analysis revealed five regions where a GR-DBD peptide could bind. We delineated a 105-bp region containing two footprints with near-consensus glucocorticoid response elements and multiple half-sites that was sufficient for transactivation via both GRwt and GRdms. Finally, we demonstrated direct binding of GRdms proteins to this responsive region using EMSA. We propose that on a subset of GR-responsive promoters, exemplified by the PNMT gene, GRs can form concerted multimers in a manner that is independent of the DBD-dimer interface. We further suggest that protein-DNA and protein-protein interactions that support such complexes are essential for activation of this type of gene, and that DNA binding of GR might be essential to survival.
Adams
Melanie
M
Division of Nephrology, Department of Medicine, University of California, San Francisco, San Francisco, California 94143-1341, USA.
Meijer
Onno C
OC
Wang
Jian
J
Bhargava
Aditi
A
Pearce
David
D
D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D018983:DNA Footprinting; D003850:Deoxyribonuclease I; D019281:Dimerization; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D017930:Genes, Reporter; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D010625:Phenylethanolamine N-Methyltransferase / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion
D011965:Receptors, Glucocorticoid; D011994:Recombinant Proteins; D010625:Phenylethanolamine N-Methyltransferase; D003850:Deoxyribonuclease I