Project description:The early growth response (Egr) family of transcriptional regulators consists of four closely related molecules (Egr1-4) that regulate target genes involved in cellular growth and differentiation. In the brain, Egr transcription factors have a critical role in learning and memory processing, presumably by regulating effector target genes that alter synaptic efficacy or mediate structural changes in neurons. Previous work suggests that Egr1 and Egr3 are the most important synaptic activity induced Egr molecules in the brain and they appear to have redundant regulatory function. How Egr transcriptional regulators influence learning and memory processing in the brain is unknown because target genes regulated by them have not been identified. Using Affymetrix microarray analysis and Egr loss-of-function mice, we will begin to characterize the gene regulatory networks modulated by Egr transcription factors in the brain. We anticipate that basic mechanisms related to transcriptional control of learning and memory related plasticity and the identification of plasticity effector molecules that may be involved in synaptic dysfunction associated with degenerative diseases or brain injury will result from these studies. To identify Egr transcription factor target gene regulation in brain: Target genes regulated by Egr transcription factors have not been identified in the brain, yet the transcription factors are essential for normal learning and memory processes. Using Egr1/3 double knockout and wild type littermate mice, we will compare gene expression profiles from somatosensory cortex to identify genes that are deregulated in Egr1/3 dKO brains. Egr1 and Egr3 gene expression is coupled to synaptic N-methyl D-aspartate (NMDA) receptor activation, mitogen activated protein kinase (MAPK) signaling engaged by NMDA receptor activation and long term synaptic potentiation (LTP). Previous studies have demonstrated defects in late phase LTP, long-term memory in hippocampal dependent tasks and reconsolidation of memories in Egr1-deficient mice, but the target effector molecules regulated by Egr transcription factors are not known. We hypothesize that it will be possible to identify Egr dependent target genes by using Affymetrix microarray analysis to compare gene expression from wild type cerebral cortex that has high levels of Egr protein expression with gene expression in cortex from Egr1/3 double knockout mice. Egr1 and Egr3 are highly expressed in mouse cortex and hippocampus twenty one days after birth because of the large amount of maternal stimulation they receive prior to weaning. We will compare the gene expression profile in somatosensory cortex from P21 wild type mice to that of P21 Egr1/3 dKO mice. We will perform microarray analysis using the Mouse 430 2.0 gene array with RNA samples from 3 wild type and 3 1/3 dKO brains (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 brains. Our main goal is to identify genes that are directly regulated by Egr3. 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.

Project description:The early growth response (Egr) family of transcriptional regulators consists of four closely related molecules (Egr1-4) that regulate target genes involved in cellular growth and differentiation. In the brain, Egr transcription factors have a critical role in learning and memory processing, presumably by regulating effector target genes that alter synaptic efficacy or mediate structural changes in neurons. Previous work suggests that Egr1 and Egr3 are the most important synaptic activity induced Egr molecules in the brain and they appear to have redundant regulatory function. How Egr transcriptional regulators influence learning and memory processing in the brain is unknown because target genes regulated by them have not been identified. Using Affymetrix microarray analysis and Egr loss-of-function mice, we will begin to characterize the gene regulatory networks modulated by Egr transcription factors in the brain. We anticipate that basic mechanisms related to transcriptional control of learning and memory related plasticity and the identification of plasticity effector molecules that may be involved in synaptic dysfunction associated with degenerative diseases or brain injury will result from these studies. To identify Egr transcription factor target gene regulation in brain: Target genes regulated by Egr transcription factors have not been identified in the brain, yet the transcription factors are essential for normal learning and memory processes. Using Egr1/3 double knockout and wild type littermate mice, we will compare gene expression profiles from somatosensory cortex to identify genes that are deregulated in Egr1/3 dKO brains. Egr1 and Egr3 gene expression is coupled to synaptic N-methyl D-aspartate (NMDA) receptor activation, mitogen activated protein kinase (MAPK) signaling engaged by NMDA receptor activation and long term synaptic potentiation (LTP). Previous studies have demonstrated defects in late phase LTP, long-term memory in hippocampal dependent tasks and reconsolidation of memories in Egr1-deficient mice, but the target effector molecules regulated by Egr transcription factors are not known. We hypothesize that it will be possible to identify Egr dependent target genes by using Affymetrix microarray analysis to compare gene expression from wild type cerebral cortex that has high levels of Egr protein expression with gene expression in cortex from Egr1/3 double knockout mice. Egr1 and Egr3 are highly expressed in mouse cortex and hippocampus twenty one days after birth because of the large amount of maternal stimulation they receive prior to weaning. We will compare the gene expression profile in somatosensory cortex from P21 wild type mice to that of P21 Egr1/3 dKO mice. We will perform microarray analysis using the Mouse 430 2.0 gene array with RNA samples from 3 wild type and 3 1/3 dKO brains (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 brains. Our main goal is to identify genes that are directly regulated by Egr3. 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

Project description:Sivakumar2011 - EGF Receptor Signaling Pathway EGFR belongs to the human epidermal receptor (HER) family of receptor tyrosine kinases, which consists of four closely related receptors (EGFR (HER1, erbB1), HER2 (neu, erbB2), HER3 (erbB3), and HER4 (erbB4)) that mediate cellular signaling pathways involved in growth and proliferation in response to the binding of a variety of growth factor ligands. There are currently six known endogenous ligands for EGFR: EGF, transforming growth factor- (TGF-), amphiregulin, betacellulin, heparin-binding EGF (HB-EGF), and epiregulin.Upon ligand binding, the EGFR forms homo- or heterodimeric complexes (usually with HER2), which leads to activation of the receptor tyrosine kinase, via autophosphorylation. References: The EGF receptor family--multiple roles in proliferation, differentiation, and neoplasia with an emphasis on HER4. An open-and-shut case? Recent insights into the activation of EGF/ErbB receptors. EGF receptor signaling: putting a new spin on eye development. Epidermal growth factor receptor: a promising target in solid tumours. This model is described in the article: A systems biology approach to model neural stem cell regulation by notch, shh, wnt, and EGF signaling pathways. Sivakumar KC, Dhanesh SB, Shobana S, James J, Mundayoor S. Omics: a Journal of Integrative Biology. 2011; 15(10):729-737 Abstract: The Notch, Sonic Hedgehog (Shh), Wnt, and EGF pathways have long been known to influence cell fate specification in the developing nervous system. Here we attempted to evaluate the contemporary knowledge about neural stem cell differentiation promoted by various drug-based regulations through a systems biology approach. Our model showed the phenomenon of DAPT-mediated antagonism of Enhancer of split [E(spl)] genes and enhancement of Shh target genes by a SAG agonist that were effectively demonstrated computationally and were consistent with experimental studies. However, in the case of model simulation of Wnt and EGF pathways, the model network did not supply any concurrent results with experimental data despite the fact that drugs were added at the appropriate positions. This paves insight into the potential of crosstalks between pathways considered in our study. Therefore, we manually developed a map of signaling crosstalk, which included the species connected by representatives from Notch, Shh, Wnt, and EGF pathways and highlighted the regulation of a single target gene, Hes-1, based on drug-induced simulations. These simulations provided results that matched with experimental studies. Therefore, these signaling crosstalk models complement as a tool toward the discovery of novel regulatory processes involved in neural stem cell maintenance, proliferation, and differentiation during mammalian central nervous system development. To our knowledge, this is the first report of a simple crosstalk map that highlights the differential regulation of neural stem cell differentiation and underscores the flow of positive and negative regulatory signals modulated by drugs. This model is hosted on BioModels Database and identified by: BIOMD0000000394. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

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