Transcription profiling of mouse cerebral cortex from Egr1/3 double knockout vs. wild type

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.早期生长反应（Egr）转录调控因子家族由4个紧密相关的分子（Egr1-4）组成，它们可调控参与细胞生长与分化的靶基因。在大脑中，Egr转录因子对学习与记忆加工具有关键作用，推测其通过调控可改变突触效能或介导神经元结构改变的效应靶基因来实现这一功能。既往研究表明，Egr1与Egr3是大脑中受突触活动诱导的最主要Egr分子，二者似乎存在冗余的调控功能。目前尚不明确Egr转录调控因子如何影响大脑的学习与记忆加工，因为尚未鉴定出其调控的靶基因。 本研究将采用Affymetrix基因芯片分析技术与Egr功能缺失小鼠模型，着手解析大脑中受Egr转录因子调控的基因调控网络。我们预期，本研究将阐明学习记忆相关可塑性的转录调控基础机制，并鉴定出可能参与退行性疾病或脑损伤相关突触功能异常的可塑性效应分子。 为鉴定大脑中Egr转录因子的靶基因调控机制：尽管Egr转录因子对正常学习与记忆过程至关重要，但目前尚未在大脑中鉴定出其调控的靶基因。我们将采用Egr1/3双基因敲除（double knockout, dKO）小鼠及其野生型同窝对照小鼠，比较二者躯体感觉皮层的基因表达谱，以鉴定出Egr1/3 dKO小鼠大脑中表达失调的基因。 Egr1与Egr3的基因表达与突触N-甲基-D-天冬氨酸（NMDA）受体激活、NMDA受体激活所介导的丝裂原活化蛋白激酶（MAPK）信号通路以及长时程突触增强（LTP）紧密偶联。既往研究已证实，Egr1基因缺失小鼠存在晚时相LTP缺陷、海马依赖型任务的长时程记忆缺陷以及记忆再巩固缺陷，但目前仍不清楚Egr转录因子所调控的效应靶分子。 我们提出假说：通过采用Affymetrix基因芯片分析技术，比较高表达Egr蛋白的野生型大脑皮层与Egr1/3双基因敲除小鼠皮层的基因表达谱，即可鉴定出Egr依赖型靶基因。出生后第21天（P21）的小鼠皮层与海马体中Egr1与Egr3呈高表达，这是因为它们在断奶前受到了大量的母源性刺激。我们将比较出生后第21天（P21）野生型小鼠与P21 Egr1/3 dKO小鼠躯体感觉皮层的基因表达谱。 我们将采用小鼠430 2.0基因芯片，对3只野生型小鼠与3只Egr1/3 dKO小鼠的大脑RNA样本进行芯片分析（共计6张芯片）。我们将通过配对t检验，筛选出表达变化幅度≥2倍的显著差异表达基因（包括上调与下调基因）。我们将采用实时荧光定量PCR（real-time PCR）对候选差异基因在野生型与Egr1/3 dKO小鼠大脑中进行验证。 本研究的主要目标是鉴定出受Egr3直接调控的基因。鉴于差异表达基因列表中可能同时包含直接与间接靶基因，我们将对经实时荧光定量PCR验证的靶基因进一步采用染色质免疫沉淀联合PCR（ChIP-PCR）进行筛选，以确定Egr1和/或Egr3是否结合于候选靶基因的潜在调控区域。