TRANSCRIPTIONAL MECHANISMS REGULATING ACTIVITY DEPENDENT GENE EXPRESSION

神经诱发的电活动对于形成 突触发育和成熟过程中的神经回路 连接.这些依赖于活动的过程需要耦合 突触信号对基因表达的选择性变化。我们已经使用 小脑颗粒细胞和骨骼肌作为模型系统， 介导活性-转录偶联的因子。的 NMDA受体（NR）的电生理学性质被一种 当颗粒细胞受到神经支配时， 神经元能苔藓纤维，表明活动和/或 神经源性因子调节NR 2B表达的抑制， NR 2C亚基基因的激活。使用转基因小鼠，我们发现， NR 2B基因的阻遏是由1.8 kb的5 ′-侧翼序列赋予的， 顺序在转染的细胞中进一步描绘了调控元件。 分离的颗粒神经元的培养物;这些细胞发育功能 突触从4-10天的培养和抑制NR 2B的表达， 对活动的回应。最小NR 2B启动子构建体（-135/+15），其 赋予神经特异性和活性依赖性抑制， 鉴定该元件被用于识别转录因子 介导NR 2B基因的活性依赖性抑制。相比之下 NR 2B亚单位基因，NR 2C表达增加后，颗粒细胞 神经支配我们发现NR 2C基因的激活需要2 会聚信号：神经调节蛋白（Nrg）和通过NMDA受体的活性。 Nrg（也称为ARIA）是一种神经因子，在神经细胞中积累， 海马能苔藓纤维/颗粒细胞突触。我们发现Nrg 在小脑切片培养物中刺激NR 2C表达>100倍。 添加活性抑制剂TTX（钠通道阻滞剂）或AP-5 (NR阻断剂）的切片取消了Nrg依赖的刺激， NR 2C; DNQX（AMPA受体阻断剂）无影响。符合 这些发现，我们发现神经调节素受体（erbB受体）， 酪氨酸激酶）在NR 2B/NR 2C之前由颗粒细胞表达 子单元开关这些结果表明，类似的机制和 因素（即。Nrg）用于调节肌肉中的受体组成 和中枢神经系统之间的联系 为了了解特定的活动模式如何调节基因表达，我们 已经研究了运动神经元引起的不同去极化模式 差异调节慢或快收缩的转录， 骨骼肌中的蛋白质基因。我们对肌钙蛋白I的研究 慢（TnIs）和快（TnIs）基因，它们被 不同的去极化频率（分别为10和100 Hz）， 重点是鉴定顺式和反式作用因子， 调节对活动模式的特定反应。我们已经确定 一个128 bp的慢上游调控元件（SURE）和一个144 bp的快上游调控元件（SURE）， 内含子调节元件（FIRE），其引导慢或 转基因小鼠的快速肌肉特异性转录。 有趣的是， TnI SURE和FIRE具有4个共同顺式作用元件：富含A/T的 序列（结合MEF 2），E盒（结合MyoD相关因子），CACC盒， 和一个新的基序（GCAGGCA），我们将其命名为CAGG盒。电泳 用肌核提取物进行的迁移率变化测定表明， 与这些图案结合。在培养的 肌细胞和转基因小鼠表明， 蛋白质-DNA复合物是增强子功能所必需的。 实验 正在确定这些要素中的哪些要素以及相应的 转录因子介导TnI的频率特异性反应 基因，也可能参与频率依赖性调节， 神经基因

Nerve-elicited electrical activity is important for the formation of neural circuits during development and maturation of synaptic connections. These activity-dependent processes require the coupling of synaptic signals to selective changes in gene expression. We have used cerebellar granule cells and skeletal muscle as model systems to identify factors that mediate activity-transcription coupling. The electrophysiological properties of NMDA receptors (NR) are modified by a subunit switch that occurs when granule cells are innervated by glutamatergic mossy fibers, suggesting that activity and/or neurally-derived factors regulate the repression of NR2B expression and activation of the NR2C subunit gene. Using transgenic mice, we found that repression of the NR2B gene is conferred by 1.8 kb of 5'-flanking sequence. Regulatory elements were delineated further in transfected cultures of dissociated granule neurons; these cells develop functional synapses from 4-10 days in culture and repress NR2B expression in response to activity. A minimal NR2B promoter construct (-135/+15) that confers neural-specificity and activity-dependent repression was identified. This element is being used to identify transcription factors mediating the activity-dependent repression of the NR2B gene. In contrast to the NR2B subunit gene, NR2C expression increases after granule cell innervation. We found that activation of the NR2C gene requires 2 converging signals: neuregulin (Nrg) and activity through NMDA receptors. Nrg (also known as ARIA) is a neural factor that accumulates at the glutamatergic mossy fiber/granule cell synapse. We found that Nrg stimulates NR2C expression by >100-fold in cerebellar slice cultures. Addition of the activity inhibitors TTX (sodium channel blocker) or AP-5 (NR blocker) to the slices abolished the Nrg-dependent stimulation of NR2C; DNQX (an AMPA receptor blocker) had no effect. Consistent with these findings, we found that neuregulin receptors (erbB receptor tyrosine kinases) are expressed by granule cells prior to the NR2B/NR2C subunit switch. These results demonstrate that similar mechanisms and factors (i.e.. Nrg) are used to regulate receptor composition in muscle and the CNS during synaptogenesis. To understand how specific activity patterns regulate gene expression, we have studied how distinct depolarization patterns elicited by motoneurons differentially regulate transcription of either slow-or fast-contractile protein genes in skeletal muscle. Our studies on the muscle troponin I slow (TnIs) and fast (TnIf) genes, which are differentially stimulated by distinct depolarization frequencies (10 vs. 100 Hz, respectively), have focused on the identification of cis- and trans-acting factors that mediate the specific responses to activity patterns. We have identified a 128 bp slow upstream regulatory element (SURE) and a 144 bp fast intronic regulatory element (FIRE) that direct either slow- or fast-muscle-specific transcription in transgenic mice. Interestingly, the TnI SURE and FIRE have 4 common cis-acting elements: an A/T-rich sequence (binds MEF2), an E box (binds MyoD-related factors), a CACC box, and a novel motif (GCAGGCA) that we denoted the CAGG box. Electrophoretic mobility shift assays with muscle nuclear extracts demonstrate specific binding to these motifs. Functional studies performed in cultured myocytes and transgenic mice demonstrate that interaction of multiple protein-DNA complexes are necessary for enhancer function. Experiments are in progress to identify which of these elements and corresponding transcription factors mediate the frequency-specific response of the TnI genes, and may also participate in frequency-dependent regulation of neural genes.