The Cre/loxP system--a versatile tool to study glucocorticoid signalling in mice.
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Alleles↗
Biomedical subjects
H M Reichardt
Publications and source records attributed to H M Reichardt.
Glucocorticoid signalling--multiple variations of a common theme.
Recent advances in molecular genetics have brought us closer to answering the question as to which mechanisms are used by steroid hormone receptors to control transcription. While binding to specific response elements in the promoter and enhancer regions of many genes has for a long time been considered as the major mode of action, a growing number of alternative transcriptional control mechanisms have been identified in the last couple of years. With the recent finding that DNA binding of the glucocorticoid receptor is not essential for survival, mechanisms depending on cross-talk with other transcription factors through protein-protein interactions have gained attention. In this mini-review, we will discuss the roles of DNA binding-dependent and -independent transcriptional modes of action in development and physiology as exemplified by the analysis of glucocorticoid receptor function.
Adrenal Glands↗
DNA binding of the glucocorticoid receptor is not essential for survival.
Transcriptional regulation by the glucocorticoid receptor (GR) is essential for survival. Since the GR can influence transcription both through DNA-binding-dependent and -independent mechanisms, we attempted to assess their relative importance in vivo. In order to separate these modes of action, we introduced the point mutation A458T into the GR by gene targeting using the Cre/loxP system. This mutation impairs dimerization and therefore GRE-dependent transactivation while functions that require cross-talk with other transcription factors, such as transrepression of AP-1-driven genes, remain intact. In contrast to GR-/- mice, these mutants termed GRdim are viable, revealing the in vivo relevance of DNA-binding-independent activities of the GR.
Amino Acid Sequence↗
Genetic dissection of glucocorticoid receptor function in mice.
Upon hormone binding, the activated glucocorticoid receptor (GR) functions as a transcription factor via different modes of action to control gene expression. Recent gene-targeting studies in mice provide new insight into the role of GR in vivo and are helping decipher the molecular mechanisms underlying its actions.
Animals↗
Analysis of glucocorticoid signalling by gene targeting.
Glucocorticoids are involved in the regulation of numerous physiological processes. The majority of these effects are thought to be mediated by the glucocorticoid receptor (GR) via activation and repression of gene expression. In most cases activation requires binding of a receptor-dimer to DNA while repression is mediated by protein-protein-interaction of GR-monomers with other transcription factors. To analyse the molecular mechanisms that underlie glucocorticoid effects, mouse mutations in the GR gene were generated and analysed. In order to address the role of glucocorticoid receptor signalling during development and in physiology, the gene was disrupted by gene targeting. Most of the mice homozygous for the mutation die shortly after birth due to severe lung atelectasis. Additional defects were found in the adrenals, liver, brain, bone marrow and thymus as well as in the feedback-regulation of the HPA-axis. To approach the question which functions of the GR are regulated by DNA-binding and which by protein-protein-interaction, a point mutation was introduced into the dimerization domain of the GR which is located in the DNA-binding domain. By homologous recombination in ES-cells using the Cre/loxP-system, mice carrying this mutation were generated [GR(dim) mice]. The mice are fully viable although they show impaired inducibility of gluconeogenetic enzymes in liver, defects in longterm renewal of erythroid progenitors and increased expression of POMC and ACTH in the pituitary. However neither in the lung nor the adrenals were any histological abnormalities found. In conclusion GR(dim)-mice represent a valuable tool to further analyse mechanisms of physiological effects of the GR.
Animals↗
Absence of glucocorticoid receptor-beta in mice.
Two human glucocorticoid receptor (GR) isoforms, GRalpha and GRbeta, are derived from the same gene by alternative splicing involving exon 9 of the GR locus. The non-ligand binding isoform GRbeta was proposed to act as a transdominant negative inhibitor of GRalpha, thus modulating glucocorticoid responsiveness of target tissues. To study GRbeta in mice we characterized the genomic region around exon 9 of the murine GR gene. Sequence analysis revealed that the presumed exon 9beta contained an open reading frame of 59 amino acids. In contrast, human exon 9beta encoded only 15 amino acids. Using reverse transcriptase polymerase chain reaction the absence of GRbeta mRNA was demonstrated in all adult mouse tissues examined. To exclude the possibility that the polymerase chain reaction conditions employed were not suitable for the amplification of GRbeta mRNA, we synthesized an artificial template corresponding to the presumed GRbeta mRNA spanning exons 7, 8, and 9beta. Various amounts of this template were added to brain cDNA preparations and as little as 25 molecules were detectable under the polymerase chain reaction conditions chosen. Since GRbeta is not conserved across species its physiological significance in humans appears questionable.
Alternative Splicing↗
Feedback control of glucocorticoid production is established during fetal development.
BACKGROUND: Glucocorticoids are involved in the regulation of metabolic, immunological, and developmental processes. Their synthesis is tightly controlled by feedback regulation through the hypothalamus-pituitary-adrenal (HPA) axis, allowing the organism to respond to stress in an adequate manner and to adapt to new situations. Disturbance of these regulatory mechanisms leads to major human diseases. By generating mice with a targeted mutation in the glucocorticoid receptor (GR) locus, it was possible to analyze the mechanism by which glucocorticoids control the HPA axis, under conditions where at least part of the feedback control was absent early in development. MATERIALS AND METHODS: RNase-protection and in situ hybridization assays were used to compare messenger RNA (mRNA) levels of genes involved in the control of the HPA axis in both GR-mutant and wild-type animals. RESULTS: Negative feedback regulation of the HPA axis by glucocorticoids, which is established around Day E16.5 of embryonic development in wild-type mice, does not occur in GR-mutants, resulting in an increased expression of proopiomelanocortin mRNA in the anterior lobe of the pituitary and of corticotropin-releasing hormone mRNA in the paraventricular nucleus of the hypothalamus. However, the expression of both arginine vasopressin and mineralocorticoid receptor in the brain is not affected. In the neurointermediate lobe of the pituitary, expression of the proopiomelanocortin gene was inversely regulated, compared with its expression in the anterior lobe. CONCLUSIONS: GR-dependent regulation of the HPA axis is established during fetal development, suggesting that maternal factors have an important role in influencing the HPA axis of the adult offspring.
Animals↗
Mutagenesis of the glucocorticoid receptor in mice.
The glucocorticoid receptor is an ubiquitously expressed transcription factor involved in the regulation of many different physiological processes. Activated by glucocorticoids the receptor regulates transcription positively or negatively either by direct binding to DNA or by protein protein interactions. In order to define the role of the receptor during development and in physiology several mutations have been generated in the mouse. Mice with a disrupted glucocorticoid receptor gene die shortly after birth due to respiratory failure indicating an important role of the receptor in lung function. Transcription of genes encoding gluconeogenic enzymes in the liver is decreased, proliferation of erythroid progenitors is impaired and the HPA axis is strongly upregulated. To analyze molecular mechanisms of glucocorticoid receptor action in vivo a point mutation has been introduced into the mouse genome which allows to separate DNA-binding-dependent from DNA-binding-independent actions of the receptor. Mice homozygous for the point mutation survive indicating that DNA-binding of the receptor is not required for survival. Induction of glucoconegenic enzymes and proliferation of erythroid progenitors however is impaired. Interestingly, repression of corticotropin releasing factor (CRF) synthesis is maintained, whereas proopiomelanocortin (POMC) expression is upregulated. Since mice with a disrupted glucocorticoid receptor gene die shortly after birth attempts using the Cre/loxP-recombination system are made to bypass early lethality and to study the function of the receptor in defined cell types of adult animals.
Animals↗
Molecular genetic analysis of glucocorticoid signaling using the Cre/loxP system.
Glucocorticoids (GC) are involved in a plethora of physiological processes that range from the regulation of the stress response and the control of the immune system to modulation of behavior. Most GC effects are mediated by the glucocorticoid receptor (GR) via activation and repression of gene expression. Whereas in most cases activation requires DNA binding of the receptor, repression is usually mediated by protein-protein interaction with other transcription factors. To decipher the molecular mode of action of GR, mice were generated by gene targeting carrying a point mutation in one of the dimerization domains, thus abrogating DNA binding by GR. Analysis of these mice demonstrated that thymocyte apoptosis and stress erythropoiesis require the DNA binding-dependent function of GR, whereas lung development and the anti-inflammatory activity of GR are mediated by protein-protein interaction. Furthermore, to study the role of GC in the brain, mice were generated specifically lacking GR function in the nervous system. Using these mice we demonstrated that GR is essential for the regulation of the HPA-axis and the stress response, as well as for the control of emotional behavior. Taken together, gene targeting using the Cre/loxP system proved to be highly valuable for the analysis of both molecular mechanism and tissue-specific functions of the GR.
Animals↗