TRANSCRIPTIONAL MECHANISMS REGULATING ACTIVITY DEPENDENT GENE EXPRESSION

调节活性依赖性基因表达的转录机制

• 批准号: 6290193
• 负责人: ANDRES BUONANNO
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6290193
• 关键词: animal genetic material tag; developmental genetics; developmental neurobiology; electrophysiology; electrostimulus; gene expression; gene induction /repression; genetic regulatory element; genetic transcription; genetically modified animals; laboratory rat; motor neurons; myofibrils; myogenesis; neurophysiology; nucleic acid sequence; regulatory gene; striated muscles; troponin

The ability of the nervous system to be remodeled with experience, and of skeletal muscles to adapt to different environmental demands, results from the phenotypic changes of these cells in response to neural activity during maturation. These activity-dependent processes often require the coupling of synaptic signals to selective changes in gene expression. We have used the regulation of NMDA receptors in the cerebellum and of the contractile genes encoding muscle troponins, as model systems to identify the molecular mechanisms that mediate activity-transcription coupling in these tissues. The most obvious developmental change in NMDA receptor (NR) kinetics occurs in the cerebellum, which results from a switch in the heteromeric subunit composition of the receptor as granule cells are innervated by mossy fiber inputs. We found that neural activity down-regulates NR2B transcription through a 150 bp sequence flanking the basal promoter. In order to understand the cellular and molecular mechanisms that upregulate the NR2C subunit during the maturation of mossy fiber/granule cell synapses and to assess the function of this subunit, we engineered a knock-in mouse where the NR2C gene was replaced by the Lac Z reporter using homologous recombination. Using co-culture experiments of pontine neurons with cerebellar slices from heterozygote NR2C/beta-gal knock-in mice, we found that the mossy fiber inputs are necessary to activation expression of the NR2C gene. This is consistent with our previous data showing that the factor Nrg-1 plus the neurotransmitter glutamate, which are released from the presynaptic mossy fiber terminals and activate ErbB and NMDA receptors, are necessary to trigger NR2C expression. We have also shown that the co-signaling via these 2 receptors may occur at synapses because NMDA and ErbB receptors are enriched at postsynaptic densities (PSD) where they interact with the same proteins harboring PDZ protein- protein interaction domains. The PDZ-domain proteins are important because they scaffold receptors and channels to signaling molecules, thus coupling synaptic activity to signaling cascades in the postsynaptic neurons. The slow- and fast-twitch properties of muscles are largely determined by distinct patterns of depolarization, which selectively regulate the transcription of contractile genes. To identify the pathways that couple specific patterns of activity to selective changes in gene expression, using trangenic mice we have isolated the shortest enhancers known to direct fiber- type-specific transcription of troponin I genes in either slow- or fast-twitch muscles. We have identified 4 conserved cis-acting elements in both enhancers: 3 of these previously known motifs (E box, MEF-2 site and CACC) are necessary to direct muscle-specific transcription in all muscles, but the fourth motif, which is novel, is necessary to direct fiber- type-specificity. Experiments are in progress to identify the transcription factors binding this site. Considering the extraordinary evolutionary conservation of transcription factor pathways used to execute epigenetic and genetic regulatory programs, as exemplified by the basic/helix-loop- helix (b/HLH) factors in neural and muscle commitment, our long-term goal is to determine if the molecular mechanisms that couple activity to transcription are also conserved during maturation of these two plastic cell types.

神经系统随着经验而重塑的能力，以及骨骼肌适应不同环境需求的能力，是这些细胞在成熟过程中响应神经活动的表型变化的结果。这些活动依赖性过程通常需要突触信号与基因表达的选择性变化的耦合。我们已经使用的NMDA受体在小脑和编码肌肉肌钙蛋白的收缩基因的调节，作为模型系统，以确定介导这些组织中的活性-转录偶联的分子机制。NMDA受体（NR）动力学中最明显的发育变化发生在小脑中，这是由于颗粒细胞受苔藓纤维输入的神经支配时受体的异聚体亚基组成发生了变化。我们发现，神经活动下调NR 2B转录通过侧翼的基础启动子的150 bp序列。为了了解在苔藓纤维/颗粒细胞突触成熟过程中上调NR 2C亚基的细胞和分子机制，并评估该亚基的功能，我们设计了一种敲入小鼠，其中NR 2C基因使用同源重组被Lac Z报告基因取代。使用脑桥神经元与来自杂合子NR 2C/β-gal敲入小鼠的小脑切片的共培养实验，我们发现苔藓纤维输入对于激活NR 2C基因的表达是必要的。这与我们以前的数据一致，这些数据表明，Nrg-1因子加上神经递质谷氨酸，从突触前苔藓纤维末端释放并激活ErbB和NMDA受体，是触发NR 2C表达所必需的。我们还表明，通过这2种受体的共信号传导可能发生在突触处，因为NMDA和ErbB受体在突触后密度（PSD）处富集，在那里它们与携带PDZ蛋白-蛋白相互作用结构域的相同蛋白相互作用。PDZ结构域蛋白是重要的，因为它们支架受体和通道的信号分子，从而耦合突触活动的信号级联在突触后神经元。肌肉的慢颤和快颤特性在很大程度上由去极化的不同模式决定，去极化选择性地调节收缩基因的转录。为了鉴定将特定的活性模式与基因表达的选择性变化相结合的途径，使用转基因小鼠，我们分离出了已知在慢肌或快肌中指导肌钙蛋白I基因的纤维类型特异性转录的最短增强子。我们已经在两种增强子中鉴定了4个保守的顺式作用元件：这些先前已知的基序中的3个（E盒、MEF-2位点和CACC）是在所有肌肉中指导肌肉特异性转录所必需的，但是第四个基序是新颖的，是指导纤维类型特异性所必需的。实验正在进行中，以确定结合该网站的转录因子。考虑到用于执行表观遗传和遗传调控程序的转录因子途径的非凡进化保守性，例如神经和肌肉定型中的基本/螺旋-环-螺旋（B/HLH）因子，我们的长期目标是确定将活性与转录偶联的分子机制在这两种塑性细胞类型的成熟过程中是否也是保守的。