NUTRITIONAL CONTROL OF ASPARAGINE SYNTHETASE

描述：哺乳动物基因表达的营养控制是一个很差的 了解过程是重要的一般细胞营养和 许多疾病的进展。拟议的研究将继续我们的工作 人天冬酰胺合成酶（AS）基因的转录调控 在氨基酸剥夺后。此外，我们还发现， 基因转录激活葡萄糖剥夺，一个过程介导 Unfolded Protein Response（UPR）信号转导通路。这 这一发现是新颖的，因为大多数UPR激活的基因是ER结合蛋白 与蛋白质加工有关。我们已经描述了人类AS基因组 克隆，并显示AS近端启动子有六个蛋白结合位点， 其中至少三个（I，II或III，以及V，VI）是最大需要的 营养控制这些序列都不对应于报道的哺乳动物 UPR顺式元件。相反，AS启动子似乎含有两个独特的 顺式元件（位点V和VI）介导对两种氨基酸的调节反应， 酸和葡萄糖限制。这些令人兴奋的结果表明， 哺乳动物细胞中UPR转录控制的独立机制， 与氨基酸信号传导重叠。总体假设是， 在人AS基因中有一组共同的独特顺式元件， 介导响应于多种营养物感应的转录增加 途径。建议的实验包括标准和创新 进一步表征这些营养响应顺式作用的方法 元件并鉴定相应的转录因子。酵母 单杂交筛选、TJV交联和DNA亲和层析将 用于鉴定与AS启动子位点V和VI结合的蛋白质。 涉及转染反式作用因子/FokI的Pin点策略 核酸酶融合构建体将在体内记录特异性转录 与特定AS启动子位点结合的因子。诱变，EMSA， 转录因子诱饵和可能的反式作用因子的过表达 将进一步定义这些顺式元件，并探讨它们在 营养依赖性调节拟议的实验测试很重要 假设，并将产生有关机制的宝贵的新信息 哺乳动物细胞对氨基酸和葡萄糖变化的反应 空房的

DESCRIPTION: Nutrient control of mammalian gene expression is a poorly understood process that is important to general cellular nutrition and the progression of numerous diseases. The proposed research will continue our work on transcriptional control of the human asparagine synthetase (AS) gene following amino acid deprivation. In addition, we have discovered that the AS gene is transcriptionally activated by glucose deprivation, a process mediated by the Unfolded Protein Response (UPR) signal transduction pathway. This observation is novel because most UPR activated genes are ER-bound proteins associated with protein processing. We have characterized a human AS genomic clone and shown that the AS proximal promoter has six protein binding sites, at least three of which (I, II, or III, and V, VI) are required for maximal nutrient control. None of these sequences corresponds to the reported mammalian UPR cis-element. Instead, the AS promoter appears to contain two unique cis-elements (sites V and VI) that mediate the regulated response to both amino acid and glucose limitation. These exciting results suggest that there are two independent mechanisms for UPR transcriptional control in mammalian cells, one of which overlaps with amino acid signaling. The global hypothesis is that there is a common set of unique cis-elements within the human AS gene that mediate increased transcription in response to multiple nutrient-sensing pathways. The proposed experiments contain both standard and innovative approaches to further characterize these nutrient-responsive cis-acting elements and to identify the corresponding transcription factors. Yeast one-hybrid screening, TJV cross-linking, and DNA affinity chromatography will be used to identify the proteins that bind to the AS promoter sites V and VI. The Pin point strategy involving transfection of trans-acting factor/FokI nuclease fusion constructs will document in vivo the specific transcription factors that bind to a particular AS promoter site. Mutagenesis, EMSA, Transcription Factor Decoy, and overexpression of likely trans-acting factors will further define these cis-elements and explore their role in nutrient-dependent regulation. The proposed experiments test important hypotheses and will generate valuable new information regarding the mechanisms by which mammalian cells respond to changes in amino acid and glucose availability.