NUTRITIONAL CONTROL OF ASPARAGINE SYNTHETASE

描述：营养控制对哺乳动物基因表达的影响很差 了解的过程对一般细胞营养和 许多疾病的发展。拟议的研究将继续我们的工作 人天冬酰胺合成酶基因转录调控的研究 在氨基酸缺乏之后。此外，我们还发现，AS 基因在转录水平上被葡萄糖剥夺激活，这是一个介导的过程 通过未折叠蛋白反应(UPR)信号转导途径。这 观察是新的，因为大多数UPR激活的基因都是ER结合的蛋白 与蛋白质加工有关。我们把人类定性为基因组 克隆并显示AS近端启动子有6个蛋白结合位点，位于 至少需要三个(I、II或III和V、VI)才能达到最大 营养控制。这些序列都不与已报道的哺乳动物的序列相符 UPR顺式元件。相反，AS启动子似乎包含两个独特的 介导对两种氨基酸的调节反应的顺式元件(V和VI位) 酸和葡萄糖限制。这些令人兴奋的结果表明，有两个 哺乳动物细胞中UPR转录调控的独立机制，一 其中一个与氨基酸信号重叠。全球假说是 在人类AS基因中有一组共同的独特的顺式元件 介导转录增加对多重营养感应的响应 小路。建议的实验既有标准的，也有创新的 进一步表征这些营养响应顺式作用的方法 并鉴定相应的转录因子。酵母菌 单杂交筛选、TJV交联和DNA亲和层析将 用于鉴定与AS启动子V和VI结合的蛋白质。 转导反式作用因子/FokI的Pin Point策略 核酸酶融合构建物将在体内记录特定的转录 与特定AS启动子结合的因子。诱变，EMSA， 转录因子诱骗和可能反式作用因子的过度表达 将进一步定义这些顺式元素，并探讨它们在 依赖营养的调节。拟议中的实验测试很重要 并将产生关于机制的有价值的新信息 哺乳动物细胞对氨基酸和葡萄糖变化的反应 可用性。

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.