TRANSCRIPTIONAL MECHANISMS REGULATING ACTIVITY DEPENDENT GENE EXPRESSION

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，都是这些细胞在成熟过程中响应神经活动而发生表型变化的结果。这些活动依赖的过程通常需要突触信号耦合到基因表达的选择性变化。我们利用小脑中NMDA受体和编码肌钙蛋白的收缩基因的调节，作为模型系统来确定这些组织中介导活性-转录偶联的分子机制。NMDA受体（NR）动力学最明显的发育变化发生在小脑，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质亚基组成发生了变化。我们发现神经活动通过基础启动子侧的150bp序列下调NR2B转录。为了了解苔藓纤维/颗粒细胞突触成熟过程中上调NR2C亚基的细胞和分子机制，并评估该亚基的功能，我们设计了一个敲入小鼠，其中NR2C基因通过同源重组被Lac Z报告基因取代。通过NR2C/ β -gal敲入小鼠杂合子小脑切片与脑桥神经元的共培养实验，我们发现苔藓纤维输入是激活NR2C基因表达所必需的。这与我们之前的数据一致，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NR2C表达所必需的。我们还表明，通过这两种受体的共同信号传导可能发生在突触上，因为NMDA和ErbB受体在突触后密度（PSD）富集，它们与含有PDZ蛋白-蛋白相互作用域的相同蛋白相互作用。pdz结构域蛋白很重要，因为它们支撑受体和信号分子通道，从而将突触活动与突触后神经元的信号级联结合起来。肌肉的慢速和快速抽搐特性在很大程度上是由不同的去极化模式决定的，去极化模式有选择地调节收缩基因的转录。为了确定将特定的活动模式与基因表达的选择性变化相结合的途径，我们使用转基因小鼠分离出已知的最短增强子，以指导肌钙蛋白I基因在慢肌或快肌收缩中的纤维类型特异性转录。我们已经在这两种增强子中确定了4个保守的顺式作用元件：其中3个已知的基序（E box， MEF-2位点和CACC）是指导所有肌肉特异性转录所必需的，但第四个基序是新的，是指导纤维类型特异性所必需的。实验正在进行中，以确定结合该位点的转录因子。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，例如神经和肌肉承诺中的基本/螺旋-环-螺旋（b/HLH）因子，我们的长期目标是确定在这两种可塑性细胞类型的成熟过程中，将活性与转录结合的分子机制是否也保守。

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.