Transcription factor mediation of transcriptome changes and functional remodeling in osmotically stressed hypothalamic n

这个项目背后的驱动力是需要快速利用基因组信息以获得生理理解。我们现在知道哺乳动物大约有3万个基因。这些数据提出了两个问题，第一，这些基因在哪里以及什么时候表达，第二，这些基因有什么作用？我们已经在一个健壮的模型系统中解决了这些问题，即下丘脑的生理挑战的加压素(VP)神经元。当动物脱水时，多肽激素VP被释放，并通过血液流动到位于肾脏的特定受体靶点，在那里它减少水分的排泄，从而促进节水。伴随而来的是VP神经元的形态、电生理特性以及生物合成和分泌活动的大量变化。我们希望从基因差异表达的角度来理解这种功能可塑性及其生理后果。我们使用了微阵列技术，使我们能够在一次测试中查看数万个基因的表达。因此，我们编制了目录，代表了在下丘脑不同区域表达的RNA群体的全面描述。此外，我们已经确定了由于慢性脱水而上调或下调的转录本。我们现在选择了5个基因进行进一步研究，因为它们编码转录因子，转录因子是在细胞核中工作的蛋白质，控制基因表达的启动，从而共同控制细胞所有信使RNA的组成。这些基因可能是VP神经元可塑性的重要调节因子。为了验证这一假设，我们将：1.使用独立的方法检查阵列数据的正确性；2.使用一种称为染色质免疫沉淀的方法找出这些转录因子调节哪些基因；3.使用基因转移到整个生物体来确定目标基因产物的活性增加或减少在控制水平衡中的功能后果。这将是第一次基于微阵列的输出，在整个动物生理系统的背景下对基因网络进行功能研究。这些数据无疑将有助于更好地理解在健康和疾病状态下涉及生理系统可塑性的基因网络。

The driving force behind this project has been the need to rapidly exploit genomic information in order to obtain physiological understanding. We now know that mammals have approximately 30,000 genes. These data prompt two questions, firstly, where and when are these genes expressed, and secondly, what do these genes do? We have addressed these questions in a robust model system, namely the physiologically challenged vasopressin (VP) neurones of the hypothalamus. When an animal is dehydrated, the peptide hormone VP is released and travels through the blood stream to specific receptor targets located in the kidney, where it reduces the excretion of water, thus promoting water conservation. This is accompanied by a plethora of changes in the morphology, electrophysiological properties and biosynthetic and secretory activity of VP neurones. We wish to understand this functional plasticity and its physiological consequences in terms of the differential expression of genes. We have used microarray techniques that allow us to look at the expression of tens of thousands of genes in a single assay. We have thus compiled catalogues that represent comprehensive descriptions of the RNA populations expressed in different regions of the hypothalamus. Further, we have identified transcripts that are either up- or down-regulated as a consequence of chronic dehydration. We have now selected 5 genes for further study on the basis that they code for transcription factors, proteins that work in the cell nucleus to control the initiation of gene expression, and hence collectively govern the composition of all of the messenger RNAs of a cell. These genes might be important key mediators, or regulators, of VP neuronal plasticity. In order to test this hypothesis, we will:1. check that the array data are correct using independent methods;2. find out which genes these transcription factors regulate using a method called chromatin immunopreciptitation;3. use gene transfer into the whole organism to determine the functional consequences of the increased or decreased activity of target gene products in the control of water balance.This will be the first time that, based on a microarray output, a gene network will be studied functionally in the context of a whole animal physiological system. The data will undoubtedly lead to a better understanding of gene networks involved in the plasticity of a physiological system in health and disease states.