Nutritional Control of Asparagine Synthetase

描述（由申请人提供）：蛋白质/氨基酸的可用性是哺乳动物一般营养的关键因素，但氨基酸波动对细胞功能（如转录）的影响知之甚少。我们利用氨基酸剥夺培养细胞来研究营养物质对转录的控制。限制任何单一氨基酸的数量会抑制一般蛋白质的合成，但实际上会促进mrna亚群的翻译增加，包括转录因子ATF4。我们的假设是ATF4是氨基酸应激反应途径的关键介质，而人天冬酰胺合成酶（ASNS）基因是研究这一途径的模型。我们之前的工作已经记录了atf4通过启动子顺式元件营养传感反应元件-1 （NSRE-1）介导的ASNS基因激活。在与增强子结合的过程中，真核生物启动子上组装了一个“起始前复合物”，该复合物由至少四种多蛋白复合物组成：染色质重塑复合物、中介复合物、一般转录因子复合物和RNA Pol II全酶。ATF4及其子结构域在ASNS启动子因子募集中的作用将通过染色质免疫沉淀（ChIP）在ATF4-/-成纤维细胞和ATF4- sirna处理的HepG2肝癌细胞中研究（Specific Aim I）。ATF4结合蛋白将通过酵母2杂交鉴定，并通过co-IP、GST pull-down和ChIP （Specific Aim II）进行研究。为了鉴定ASNS启动子上的其他蛋白质，将分离含有atf4的复合物，通过质谱鉴定单个蛋白质，并通过co-IP和ChIP研究atf4相互作用的蛋白质（Specific Aim III）。这些研究将提供有关营养信号调动转录机制的机制的有价值的信息，从而通过基因表达改变细胞代谢。鉴于ATF4在应对氨基酸限制和其他形式的营养应激中的核心作用，我们认为了解其在一般转录机制组装中的功能至关重要。

DESCRIPTION (provided by applicant): Protein/amino acid availability is a critical factor in general nutrition in mammals, yet there is relatively little known about the impact of amino acid fluctuation on cell functions, such as transcription. We use amino acid deprivation of cultured cells to investigate transcriptional control by nutrients. Limiting the amount of any single amino acid suppresses general protein synthesis, but actually promotes increased translation of a subset of mRNAs, including the transcription factor ATF4. Our hypothesis is that ATF4 is a critical mediator of the amino acid stress response pathway and that the human asparagine synthetase (ASNS) gene is a model for the study of this pathway. Our previous work has documented ATF4-mediated activation of the ASNS gene through the promoter cis-element Nutrient Sensing Response Element-1 (NSRE-1). In association with enhancer binding, a "pre-initiation complex" is assembled at eukaryotic promoters, which is comprised of at least four multi-protein complexes: a chromatin remodeling complex, a Mediator complex, a general transcription factor complex, and the RNA Pol II holoenzyme. The role of ATF4 and its sub-domains in factor recruitment to the ASNS promoter will be studied by chromatin immunoprecipitation (ChIP) in ATF4-/- fibroblasts and ATF4-siRNA treated HepG2 hepatoma cells (Specific Aim I). ATF4 binding proteins will be identified by yeast 2-hybrid and studied by co-IP, GST pull-down, and ChIP (Specific Aim II). To identify other proteins at the ASNS promoter, ATF4-containing complexes will be isolated, individual proteins identified by mass spectrometry, and the ATF4-interacting proteins studied by co-IP and ChIP (Specific Aim III). These studies will yield valuable information about the mechanisms by which nutritional signals mobilize the transcriptional machinery and thereby, alter cellular metabolism through gene expression. Given the central role of ATF4 in the response to amino acid limitation, and other forms of nutritional stress, we believe that it is critical to understand its function in the assembly of the general transcriptional machinery.