Klaus H. Kaestner

TARGETING OF THE CREB GENE LEADS TO UP-REGULATION OF A NOVEL CREB MRNA ISOFORM

To define the role of cAMP signaling in gene control, we have generated mice with a mutation in the cAMP response element binding protein (CREB) gene. Mice carrying this mutation are viable but show an impairment in memory consolidation. In further analysis of these mice, we have found an up‐regulation of a CREB isoform that has not been described previously . The new isoform, termed CREB beta, has nearly the same transactivation potential as the other CREB isoforms and is expressed ubiquitously. The up‐regulation appears to be due to an increase in alternative splicing or mRNA stability, but not to an increase in transcriptional rate. Due to the relatively low levels of expression in all tissues, the role of this isoform is likely to be minor in the wild‐type mouse. However, its dramatic up‐regulation in the mutant mouse, together with the specific deficiencies recently observed in these mice, suggest that it has a very specific role in compensating for CREB alpha and delta in some, but not all, areas where CREB function has been implicated. Together with the up‐regulation of the cAMP response element modulator protein (CREM) mRNA and protein levels demonstrated previously in CREB mutant mice, we suggest that the up‐regulation of CREB beta may also contribute to compensation within the CREB/ATF family of transcription factors, when CREB delta and CREB alpha are absent.

The mouse homolog of the region specific homeotic gene spalt of Drosophila is expressed in the developing nervous system and in mesoderm-derived structures

The region specific homeotic gene spalt (sal) of Drosophila determines the specification of terminal segments. Its mutation leads to an incomplete transformation of terminal segments into trunk-like segments. The gene product is a zinc finger protein with a novel structure. We have isolated the mouse homolog of the Drosophila spalt gene (msal). The msal cDNA sequence is similar to its Drosophila counterpart in that it contains seven C2H2-type zinc finger motifs grouped into three pairs plus a single zinc finger closely linked to the middle pair. The two genes exhibit high sequence similarity in the zinc finger regions and to a lower extent in the putative transactivation domains. We have analysed the expression pattern of msal and show that it is expressed in the developing neuroectoderm of the brain, the inner ear and the spinal cord and in urogenital ridge-derived structures such as testis, ovaries and kidneys. A weaker and transient expression is seen in early embryos in the branchial arches and in tissues like the notochord, the limb buds and the heart. Given its role in Drosophila melanogaster and its strong sequence conservation, this expression pattern suggests an important role for msal in the development of the nervous system.

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.

Universal β-galactosidase cloning vectors for promoter analysis and gene targeting

Two new plasmid vectors suitable for generating fusions with the lacZ gene have been developed and tested. The vectors can be applied in the analysis of regulatory elements of eukaryotic genes in both transient and stable transfection experiments. In addition, they can be utilized as the backbone of gene targeting vectors, allowing the assessment of the expression pattern of the targeted gene by staining for beta-galactosidase activity.