Nutritional Regulation of Ribosomal Protein Expression

哺乳动物细胞对氨基酸缺乏的反应是通过一种称为氨基酸反应(AAR)的途径增加多种基因的转录。然而，这些事件在基因组水平上发生的分子机制还不是很清楚。我们的全球假设是，人类S25核糖体蛋白基因代表了一个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。目标是确定S25基因的AAR顺式作用元件的特征，然后利用该信息识别相应的转录因子(S)。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置，我们将使用在氨基酸完全的MEM培养液或缺乏组氨酸的MEM中维持的人HepG2肝癌细胞，检测S25基因附近和内部的DNase I超敏位点。为了更准确地描述AARE位点，我们将使用一组包含S25基因缺失/替换片段的报告质粒进行功能分析。AARE的进一步特征将是使用活体足迹法高分辨率检测蛋白质-DNA相互作用中依赖氨基酸的变化，随后是单个核苷酸的突变。用过量的AARE序列(转录因子诱骗)转染将测试相应反式作用蛋白的吸附和随后的耗尽是否抑制S25转录的氨基酸依赖的调节。凝胶迁移率漂移分析(EMSA)数据，包括野生型和突变的寡核苷酸，将与功能研究相关联，并将使用氨基酸供应或缺乏氨基酸的细胞的核提取物评估复合体的形成数量。如果AARE序列是新的，将采用酵母单杂交文库筛选或反式作用蛋白DNA亲和层析的方法来鉴定和克隆AARE结合蛋白。将转录因子/FokI核酸酶融合蛋白定向到特定顺式元件的Pin*Point策略将在体内记录用于因子结合的S25基因组位置。体内Pin Point试验补充和扩展了体外EMSA数据。拟议中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获得性变化做出反应的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.