TRANSCRIPTIONAL MECHANISMS REGULATING ACTIVITY DEPENDENT GENE EXPRESSION

神经系统通过经验重塑的能力，以及骨骼肌适应不同环境要求的能力，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。这些依赖活动的过程通常需要突触信号与基因表达的选择性变化相耦合。我们使用小脑中NMDA受体和编码肌肉肌钙蛋白的收缩基因的调节作为模型系统，以确定在这些组织中介导活动-转录耦合的分子机制。NMDA受体(NR)动力学中最明显的发育变化发生在小脑，这是由于颗粒细胞被苔藓纤维输入支配而导致受体异构体亚单位组成的变化。我们发现，神经活动通过基本启动子两侧的150bp序列下调NR2B的转录。为了了解苔藓纤维/颗粒细胞突触成熟过程中NR2C亚基上调的细胞和分子机制，并评估该亚基的功能，我们利用同源重组技术设计了一只敲入小鼠，其中NR2C基因被Lac Z报告基因取代。通过桥脑神经元与杂合子NR2C/β-Gal敲入小鼠小脑片的共培养实验，我们发现苔藓纤维的输入是激活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是触发NR2C表达所必需的，谷氨酸是从突触前苔藓纤维终末释放出来的，激活ErbB和NMDA受体。我们还表明，通过这两种受体的共同信号可能发生在突触，因为NMDA和ErbB受体在突触后密度(PSD)时富含，在那里它们与含有PDZ蛋白-蛋白相互作用域的相同蛋白质相互作用。PDZ结构域蛋白很重要，因为它们将受体和通道搭建到信号分子上，从而将突触活动耦合到突触后神经元的信号级联反应中。肌肉的慢抽动和快抽动特性在很大程度上是由不同的去极化模式决定的，去极化模式选择性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，使用转基因小鼠，我们分离了已知的最短的增强子，这些增强子可以指导慢或快抽动肌肉中肌钙蛋白I基因的纤维类型特异性转录。我们已经在这两个增强子中确定了4个保守的顺式作用元件：其中3个已知基序(E box、MEF-2 Site和CACC)是在所有肌肉中指导肌肉特异性转录所必需的，但第四个基序是新的，对于指导纤维类型特异性是必要的。鉴定结合这个位点的转录因子的实验正在进行中。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在进化上的非凡保守性，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塑料细胞类型的成熟过程中，将活性与转录耦合的分子机制是否也是保守的。

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.