tourt-affy-arabi-307860

The Early Growth Response (Egr) family of transcription factors consists of 4 members (Egr1-4) that are expressed in a wide variety of cell types. A large body of evidence point to a role for Egr transcription factors in growth, survival, and differentiation. A major unanswered question is whether Egr transcription factors serve similar functions in diverse cell types by activating a common set of target genes. Signal transduction cascades in neurons and lymphocytes show striking parallels. Activation of either cell type activates the Ras-MAPK pathway and, in parallel, leads to increases in intracellular calcium stimulating the calcineurin-NFAT pathway. In both cell types, the strength of the activation signal affects the cellular outcomes and very strong stimuli lead to cell death. Notably both these pathways converge on the induction of Egr genes. We believe that downstream targets of Egr transcription factors in lymphocytes may also be activated by Egr factors in activated neurons. There is precedence for common target gene activation in these two cell types: apoptosis in both activated T cells and methamphetamine stimulated neurons occurs via FasL induction by NFAT transcription factors. We propose to use developing T lymphocytes (thymocytes) as a model system for discovery of Egr-dependent target genes for several reasons. First, we have observed a prominent survival defect in thymocytes from mice deficient in both Egr1 and Egr3 (1/3 DKO) and a partial differention block in the immature double negative (DN) stage. In addition, thymocytes are an easily manipulatable cell type, and the DN subpopulation affected in 1/3 DKO mice can be isolated to very high purity. We anticipate that 1/3 DKO thymocytes will provide an excellent experimental system that will provide insight into Egr-dependent transcription in neuronal development, activation, and death. To identify Egr transcription factor target genes in developing thymocytes. Egr1/3 DKO mice show a profound defect in thymocyte survival and differentiation, but the target genes downstream of Egr1 and Egr3 are unknown. Using Egr1/3 double knockout and wild type littermate mice, we will compare gene expression profiles from DN thymocytes to identify genes that are deregulated in Egr1/3 DKO cells. Egr1 and Egr3 expression is necessary for the proper survival and differentiation of developing thymocytes. We hypothesize that we will be able to identify crucial target genes of Egr1 and Egr3 by comparing gene expression profiles from wild type and 1/3 DKO thymocytes. Egr1 and Egr3 are first expressed in the immature double negative (DN) thymocytes. We will isolate DN thymocytes by FACS sorting which allows isolation of cell populations of >99% purity. We will perform microarray analysis using the Mouse 430 2.0 gene array with thymocyte RNA samples from 3 wild type and 3 1/3 DKO mice (6 arrays total). Differentially regulated genes (up-regulated and down-regulated) will be identified from the list of genes with significantly altered expression greater than or equal to 2-fold by paired T test. Interesting genes will be validated by real-time PCR in wild type and 1/3 DKO thymocytes. Recognizing that both direct and indirect target genes may be identified in the list of differentially expressed genes, real-time PCR validated target genes will be further screened using chromatin immunoprecipitation coupled with PCR (ChIP-PCR) to determine whether Egr1 and/or Egr3 are bound to potential regulatory regions of the putative target genes. Keywords: other