Glucocorticoid-stimulated gene expression knocked out by knock-in mutation.

Glucocorticoid receptors (GRs) are ligand-dependent transcription modulators. They belong to a superfamily of nuclear hormone receptors including receptors for steroid and thyroid hormones, retinoids, vitamin D and peroxisomal activators among others (1). The receptor can be divided into three functional domains: (1) an N-terminal activation domain (AF1 or t1); (2) a central DNA-binding domain; and (3) a liganddependent activation domain (AF2 or t2), which is a part of the ligand-binding domain in the C-terminus. The GR is essential for survival. ‘Knock-out’ of the receptor in mice resulted in impaired lung development, and the newborn mice died shortly after birth because of respiratory failure (2). Several other abnormalities associated with a loss of glucocorticoid stimulation were observed in these mice including a reduced level of gluconeogenic enzymes in the liver, increased serum levels of corticotrophin-releasing hormone (CRH), adrenocorticotrophic hormone (ACTH) and corticosterone, hyperplasia of the adrenal cortex, loss of glucocorticoid-dependent thymocyte apoptosis, and impaired proliferation of erythroid progenitor cells. Activation of GRs may stimulate or repress gene transcription (3). The glucocorticoid-induced transcription of target genes is mediated by glucocorticoidresponsive elements (GREs), which are palindromic sequences in the regulatory region of these genes. A GRE is a binding site for the activated GR, and transactivation depends on the dimerization of two GRs on the GRE. There are probably three ways in which GRs can repress gene transcription. The proopiomelanocortin (POMC) gene is negatively regulated by a direct interaction of GR with its promoter (4). Secondly, it may interact with the DNA-binding domain together with another transcription factor, and, finally, glucocorticoid-induced repression of gene transcription can be independent of direct binding of GR to DNA. As examples, the activated receptor has been shown to interfere with signalling via the cAMP-responsive transcription factor (CREB) or transcription factors using AP-1 elements by protein‐protein interactions (5‐7). Furthermore, some of the immunosuppressive effects of glucocorticoids have been associated with a repression of the activity of the transcription factor NF-kB by a direct interaction between GRs and NF-kB, even though this regulation may be even more complex (8). In cell culture, the consequences of a defect in the dimerization of GRs have been studied by introducing a point mutation in the region of the GR gene coding for the D loop of the receptor protein (9, 10). This loop consists of five amino acids critical for receptor dimerization. Changing the amino acid alanine in position 458 in the mouse GR to threonine (A458T) abolished the formation of receptor dimers and activation of gene transcription via GREs, whereas effects on transcriptional repression were almost unaffected. The consequences of this dimerization defect have now been studied in vivo. Reichardt et al. (11) (reviewed by Karin (12)) created a strain of mice with alanine 458 replaced by threonine in the GR protein. In contrast with GR knock-out mice, there were no signs of impaired lung development and both sexes were fertile. An unexpected almost Mendelian ratio of the three genotypes with 21% homozygous mutants (denoted GR dim/dim ) was found among the offspring in contrast with the lethal effect of homozygous GR knock-out in mice. As expected a series of experiments demonstrated that the mutation specifically affected DNA-binding-dependent transcriptional activation by the GR. Immortalized embryonic fibroblasts from GR dim/dim and control mice were incubated in the absence or presence of dexamethasone and two reporter genes with different GREs. Only minimal activation of the reporter genes was observed in GR dim/dim cells in contrast with the activation seen in control cells. Dexamethasoneinduced transactivation of gluconeogenic enzymes in the liver was also lost in the A458T mutants, indicating the need for dimerization and GRE binding for these effects. However, the protein‐protein interactions were preserved, as the expression of collagenase 3 and gelatinase B, which is transcriptionally activated by phorbol esters via an AP-1 element, was almost completely abolished by dexamethasone in control but