Regulation of Gene Expression by Dietary Fat

描述(申请人提供)：至少三十年来，美国公众一直被鼓励减少饮食中总脂肪的摄入量，并增加饮食中多不饱和脂肪酸与饱和脂肪酸的比例。然而，脂肪酸在细胞内发挥作用的分子细节还没有被很好地理解。长期目标是了解饮食中添加多不饱和脂肪是如何调节肝脏细胞内代谢的。本应用的目的是以G6PD作为我们的模型基因，确定多不饱和脂肪酸在基因表达过程中抑制转录后步骤的步骤。该应用的中心假设是，G6PD转录本的有效剪接需要将特定的剪接共激活蛋白与Pre-mRNA外显子中的剪接增强子序列结合。在饮食中添加多不饱和脂肪会抑制剪接共激活子的活性，减少前mRNA的剪接，从而针对转录物进行降解。其基本原理是，这种机制并不是G6PD独有的，而是参与中间代谢的其他基因所共有的，包括那些也在转录步骤中受到调控的基因。这种额外的调节水平将为细胞提供冗余的机制，以确保这些蛋白质的调节表达，并对不断变化的环境条件做出更快的反应。G6PD为研究这种新的转录后调控形式提供了一个理想的模型，因为没有转录变化使数据的解释变得容易得多。鉴定外显子剪接增强子元件序列的实验在特定目标1中描述。特定目标2测试剪接共激活蛋白是否与RNA元件结合，以及元件突变是否阻止该蛋白质的结合。该项目的一个长期目标是确定饮食脂肪调节基因表达的机制。在特定目标3中，表达G6PD RNA报告基因的转基因小鼠将被用于测试外显子剪接增强子是否参与基因表达的饮食调节。确定饮食脂肪改变细胞内代谢的分子途径对于了解肥胖、糖尿病和动脉粥样硬化等与过度摄入脂肪有关的疾病的分子基础至关重要，并可以通过饮食或药物干预改进这些疾病的治疗策略。

DESCRIPTION (provided by applicant): For at least three decades, the American public has been encouraged to decrease their consumption of total dietary fat and to increase the ratio of polyunsaturated to saturated fatty acids in the diet. Yet the molecular details by which fatty acids act within cells is not well understood. The long-range goal is to understand how the addition of polyunsaturated fat in the diet regulates intracellular metabolism in the liver. The objective of this application is to define the steps by which polyunsaturated fatty acids inhibit posttranscriptional steps during gene expression using G6PD as our model gene. The central hypothesis of the application is that efficient splicing of the G6PD transcript requires the binding of a specific splicing co-activator protein to a splicing enhancer sequence in an exon of the pre-mRNA. The addition of polyunsaturated fat to the diet inhibits the activity of the splicing co-activator, decreasing pre-mRNA splicing and thereby targeting the transcript for degradation. The rationale is that this mechanism is not unique to G6PD but is shared by other genes involved in intermediary metabolism including those that are also regulated at transcriptional steps. This additional level of regulation would provide the cell with both redundant mechanisms to ensure regulated expression of these proteins and a more rapid response to changing environmental conditions. G6PD provides an ideal model to study this novel form of posttranscriptional regulation because the absence of transcriptional changes makes interpretation of the data much easier. Experiments to characterize the sequence of the exon splicing enhancer element are described in Specific Aims 1. Specific Aim 2 tests if a splicing co-activator protein binds the RNA element and if mutations in the element block binding of this protein. A long-term goal of this project has been to identify the mechanism by which dietary fat regulated gene expression. In Specific Aim 3, transgenic mice expressing G6PD RNA reporters will be used to test if the exon splicing enhancer is involved in dietary regulation of gene expression. Identification of the molecular pathway by which dietary fat alters intracellular metabolism is critical to understanding the molecular basis for diseases such as obesity, diabetes and atherosclerosis that are associated with over-consumption of fat, and can lead to improved strategies for treatment of these diseases via diet or drug intervention.