Nutritional Regulation of Ribosomal Protein Expression

哺乳动物细胞对氨基酸缺乏的反应是通过增加多种基因的转录，这一途径被称为氨基酸反应（AAR）。然而，这些事件在基因组水平上发生的分子机制尚不清楚。我们的总体假设是，人类S25核糖体蛋白基因代表了研究营养控制的模型，因为它包含一个氨基酸饥饿反应元件（AARE）来调节其转录。目标是表征S25基因的AAR顺式作用元件，然后使用该信息确定相应的转录因子。为了确定体内染色质结构的氨基酸依赖性变化，表明可能与转录因子相互作用的位点，我们将使用在氨基酸完全MEM或缺乏组氨酸的MEM中维持的人HepG2肝癌细胞，检测S25基因附近和内部的DNase I超敏感位点。为了更精确地描述AARE位点，我们将使用包含S25基因缺失/替代片段的报告质粒集合进行功能分析。AARE将进一步以高分辨率检测蛋白质- dna相互作用中氨基酸依赖的变化为特征，使用硫酸二甲酯在体内足迹，随后是单个核苷酸的突变。转染过量的AARE序列（转录因子诱饵）将测试相应的反式作用蛋白的吸附和随后的耗尽是否会抑制S25转录的氨基酸依赖调节。电泳迁移率位移分析（EMSA）数据，包括野生型和突变寡核苷酸，将与功能研究相关联，并将评估从氨基酸供给或氨基酸剥夺的细胞中提取的核提取物形成复合物的数量。如果AARE序列是新的，则采用酵母单杂交cDNA文库筛选或纯化反式作用蛋白DNA亲和层析法对AARE结合蛋白进行鉴定和克隆。将转录因子/FokI核酸酶融合蛋白导向特定顺式元件的Pin*Point策略将在体内记录因子结合的S25基因组位点。体内Pin Point试验补充并扩展了体外EMSA数据。拟议的实验测试了重要的假设，并将产生关于哺乳动物细胞对氨基酸可用性变化的反应机制的有价值的新信息。

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.