UV-B MEDIATED CHANGES IN CHROMAIN REMODELING

描述（由申请人提供）：在这个FIRCA中，我们提议对UV-B照射后Mu转座子和细胞基因的染色质组织进行深入分析。重点是确定UV-B是否会改变染色质重塑蛋白和其他dna相关蛋白的丰度或翻译后修饰。接下来，我们将研究结合在Mu元件上的染色质蛋白和几种受UV-B处理上调或下调的宿主基因。在这项研究中，我们将询问是否有特定基因的变化，以及在治疗期间转录增加（或减少）的基因是否发生类似的变化。该项目最重要的是检查沉默Mu元素发生的变化，这可能解释UV-B如何重新激活这些转座子。在每个实验中都采用了明确的UV-B治疗方法和适当的基因型，这些基因型具有充分记录的UV-B反应，允许对每个假设进行强有力的检验。使用的方法将包括蛋白质组学来定义UV-B暴露后的核蛋白丰度和翻译后修饰，ChIP来定义在UV-B暴露前后与活性和沉默Mu元件不同相关的染色质因子，选择性微阵列杂交和RT-PCR检测来监测对UV-B非常敏感的RNAi敲除库存的转录物变化。这些研究旨在解决一个基本问题，即一个可变的环境因素是如何干扰细胞基因表达的，不仅在辐射部位，而且在远处的组织中。考虑到动物和植物细胞反应之间的广泛相似之处，这些数据应该有助于理解人类细胞对环境UV-B暴露的反应。由于南半球的臭氧变薄直接影响到阿根廷，人们对了解UV-B对人类、玉米等主要作物和整个生态系统的损害越来越感兴趣。即使是低剂量的UV-B暴露也会对生物体造成伤害：它是一种强致癌物，与人类皮肤癌有关。陆地植物尤其敏感，因为光合组织不可避免地暴露在UV-B下。因此，对植物损伤的研究可以帮助确定其他真核生物维持细胞功能所需的修复途径。

DESCRIPTION (provided by applicant): In this FIRCA we propose an in-depth analysis of chromatin organization at Mu transposons and cellular genes after UV-B irradiation. The focus is on establishing whether UV-B alters the abundance or post- translational modification of chromatin remodeling proteins and other DNA-associated proteins. Next we will examine the chromatin proteins bound at Mu elements and several host genes that are up- or down- regulated by UV-B treatments. In this study we will ask whether there are changes at particular genes and whether similar changes occur at genes whose transcript increase (or decrease) during treatment. Of paramount importance to the project is examining changes that occur at silenced Mu elements that might explain how UV-B can reactivate these transposons. Well-defined UV-B treatments and appropriate genotypes with well-documented UV-B responses are employed in each experiment permitting a robust test of each hypothesis. The methods used will include proteomics to define nuclear protein abundances and post-translational modifications following UV-B exposure, ChIP to define chromatin factors differentially associated with active and silent Mu elements before and after UV-B exposure, and selective microarray hybridization and RT-PCR assays to monitor transcript changes in RNAi knockdown stocks that are extremely UV-B sensitive. The studies are designed to address the fundamental question of how a variable environmental factor perturbs cellular gene expression, not only at the site of radiation but in distant tissues. Given the extensive parallels between cellular responses in animals and plants, these data should prove useful in understanding the responses of human cells to the challenge of environmental UV-B exposure. As ozone thinning in the Southern hemisphere impacts Argentina directly, there is heightened interest in understanding UV-B damage to people, major crops such as corn, and the entire ecosystem. Even low UV-B exposure harms organisms: it is a powerful carcinogen, implicated in skin cancer in humans. Land plants are particularly sensitive, because photosynthetic tissues are inevitably exposed to UV-B. Thus studies of plant damage can help define repair pathways required to maintain cellular functions in other eukaryotes.