We are investigating the biological functions of the transcription factors, NGFI-A and NGF-C, which are members of a gene family that includes Krox20 and Egr3. All of these proteins are encoded by immediate-early genes (IEGs) and appear to antagonize the transcriptional repressor WT1, the product of the Wilms tumor gene. We are also studying the orphan nuclear receptor NGFI-B, which is also encoded by an IEG. These IEGs are rapidly activated in response to a variety of extracellular stimuli and their expression has been correlated with a number of cellular processes including differentiation, proliferation and apoptosis. In this proposal, we have outlined experiments which will lead to the identification of a factor(s) that interacts with and inhibits the activity of NGFI-A and possibly its family members in a manner reminiscent of the actions of the tumor suppressors pRb and p53. This will be accomplished by identifying the site of interaction of this factor by mutagenesis, followed by the identification of the factor using interactive cloning methodologies. The uninhibited, superactive NGFI-A mutant generated in these studies will be expressed in transgenic mice to test whether it promotes a phenotype similar to that resulting from a the lack of its repressive antagonist WT1. We have also outlined two series of experiments aimed at directly testing in vivo the role of NGFI-A and NGFI-B in regulating two critical biological systems where their importance has been detailed in in vitro studies. NGFI-A appears to be a critical determinant in the differentiation of progenitors into macrophages. Macrophage differentiation and function will be extensively examined in cells derived from NGFI-A/- mice that we have already produced by gene targeting. Functional differences between NGFI-A and its family members will be sought, and the domains responsible for these differences will be identified, using macrophage differentiation as a phenotypic assay. As macrophages influence a wide variety of processes, including resistance to infection and tumor surveillance, understanding how their differentiation is controlled will be an important advance. The nuclear receptors NGFI-B and SFI/ELP have been shown to regulate the gene encoding the steroidogenic enzyme 21-OHase in YI adrenocortical cells. A number of parameters of adrenal function will be examined in the NGFI-B -/- mice we have generated by gene targeting and in the SFI/ELP-/- mice we propose to produce. As these two factors can bind to the same crucial element in the 21 -OHase promoter, they provide an excellent system to study how competitive interactions modulate promoter activity, an emerging theme in gene regulation. This will be studied in vivo by producing transgenic mice containing mutant 21-OHase promoter/reporter constructs and monitoring their expression in normal and mutant mice under normal an stressful conditions. A truncated, constitutively active NGFI-B will be expressed in transgenic mice using its own promoter and a constitutive adrenal promoter (SCC) to test whether this orphan receptor is normally regulated by a hormone ligand. The importance of these studies resides in the fact that they will provide information about how these IEG products influence cellular processes in vivo.
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