Nutritional Regulation of Ribosomal Protein Expression

哺乳动物细胞通过增加多种基因的转录来响应氨基酸剥夺，该途径将被称为氨基酸响应（AAR）。 然而，这些事件发生在基因组水平的分子机制还没有很好地理解。 我们的全球假设是，人类S25核糖体蛋白基因代表了一个模型，用于调查营养控制，因为它包含一个氨基酸饥饿反应元件（AARE），调节其转录。 目的是表征S25基因的AAR顺式作用元件，然后使用该信息来鉴定相应的转录因子。 为了确定体内染色质结构中的氨基酸依赖性变化，表明与转录因子相互作用的可能位点，我们将使用维持在氨基酸完全MEM培养基或缺乏组氨酸的MEM中的人HepG 2肝癌细胞测定S25基因附近和内部的DNase I超敏位点。 为了更精确地描绘AARE位点，我们将使用含有S25基因缺失/取代片段的报告质粒的集合进行功能分析。 AARE将进一步通过使用硫酸二甲酯体内足迹法高分辨率检测蛋白质-DNA相互作用中的氨基酸依赖性变化，然后进行单个核苷酸的突变来表征。 用过量的AARE序列（转录因子诱饵）转染将测试相应反式作用蛋白的吸附和随后的消耗是否抑制S25转录的氨基酸依赖性调节。 使用野生型和突变寡核苷酸的电泳迁移率变化分析（EMSA）数据将与功能研究相关，并将使用来自氨基酸喂养或氨基酸剥夺细胞的核提取物评估复合物形成的量。 如果AARE序列是新的，酵母单杂交cDNA文库筛选或纯化反式作用蛋白的DNA亲和层析将用于鉴定和克隆AARE结合蛋白。 用于将转录因子/FokI核酸酶融合蛋白导向特定顺式元件的Pin*Point策略将记录用于体内因子结合的S25基因组位点。 体内PinPoint测定补充并扩展了体外EMSA数据。 拟议的实验测试重要的假设，并将产生有价值的新信息的机制，哺乳动物细胞响应氨基酸的可用性的变化。

Mammalian cells respond to amino acid deprivation by increasing the transcription of a wide variety of genes by a pathway that will be referred to as the Amino Acid Response (AAR). However, the molecular mechanisms by which these events occur at the genomic level are not well understood. Our global hypothesis is that the human S25 ribosomal protein gene represents a model for investigating nutrient control, because it contains an amino acid starvation response element (AARE) that regulates its transcription. The goal is to characterize the AAR cis-acting element of the S25 gene and then use that information to identify the corresponding transcription factor(s). To identify amino acid-dependent changes in chromatin structure in vivo indicating possible sites of interaction with transcription factors, we will assay for DNase I hypersensitive sites near and within the S25 gene using human HepG2 hepatoma cells maintained in amino acid-complete MEM medium or MEM lacking histidine. To more precisely delineate the AARE site, we will perform functional analysis using a collection of reporter plasmids containing S25 gene deletion/substitution fragments. The AARE will be further characterized by high resolution detection of amino acid-dependent changes in protein-DNA interactions using dimethyl sulfate in vivo footprinting, followed by mutation of individual nucleotides. Transfection with an excess of the AARE sequence (Transcription Factor Decoy) will test whether adsorption and subsequent depletion of the corresponding trans- acting proteins suppresses amino acid-dependent regulation of S25 transcription. Electrophoresis Mobility Shift Analysis (EMSA) data, with wild-type and mutated oligonucleotides, will be correlated with the functional studies and will assess the amount of complex formation using nuclear extracts from amino acid-fed or amino acid-deprived cells. If the AARE sequence is novel, yeast one-hybrid cDNA library screening or purification of the trans-acting protein DNA affinity chromatography will be used to identify and clone the AARE binding protein. The Pin*Point strategy for directing a transcription factor/FokI nuclease fusion protein to specific cis-elements will document the S25 genomic sites for factor binding in vivo. The in vivo Pin Point assay complements and extends the in vitro EMSA data. 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 availability.