Controlling NFAT1 in T cells using engineered ncRNA transcriptional regulators

描述（由申请人提供）：在免疫应答期间，许多基因的表达在转录水平上上调或下调，基因中的DNA被复制成mRNA的过程。在T细胞中，转录激活因子NFAT1控制许多对外来抗原应答重要的基因的转录，并在免疫疾病中发挥关键作用。该研究的主要目标是设计和制造三种类型的非编码RNA （ncRNA）分子，通过将其带入通常由NFAT1控制的基因中，可用于增强或抑制T细胞中的mRNA转录。除了创建一类新的基因特异性ncRNA转录调节因子（目前还没有类似的方法来控制基因表达），这些研究将揭示NFAT1对T细胞活化时发生的转录程序的贡献程度，并鉴定以前不知道由NFAT1直接调节的基因。这些研究结果将为T细胞中NFAT1的转录控制提供新的见解，这对于理解正常免疫反应以及自身免疫性疾病、哮喘和过敏期间发生的不适当免疫系统反应至关重要。提案的目标是：具体目标1。创建针对人类NFAT1的ncRNA转录调控因子。结合NFAT1的RNA分子将被分离，然后融合到先前显示的调节转录的ncRNA结构域。这些嵌合ncrna将被优化以激活或抑制人类T细胞模型基因上nfat1驱动的转录。具体目标2。研究nfat1特异性工程ncRNA如何控制T细胞全基因组转录。为了确定工程嵌合ncRNA调节T细胞全基因组转录的有效性和特异性，我们将使用表达和启动子微阵列。这些实验将揭示由工程ncRNA直接调控的基因的补充，并通过扩展NFAT1。这些研究的创新之处在于，当T细胞被激活时，利用工程化的ncrna改变由NFAT1设定的转录程序。这种方法与研究细胞中转录因子功能的传统技术相比是独特的，转录因子通常会降低或消除蛋白质。相反，这种方法使用NFAT1将调节性ncrna引导到特定基因的启动子上。这些研究将为创建针对其他哺乳动物转录因子的ncRNA转录调节因子提供基础。此外，我们开发的ncrna可以用作发现一类新的免疫疗法的先导化合物。项目简介：适当控制基因表达对维持生命和避免许多疾病和癌症至关重要。这里描述的研究旨在设计一类新的分子，这些分子将在免疫反应期间增强或抑制人类T细胞中特定基因的表达。这些研究将测试一种控制人类细胞中基因表达的新方法，并为设计额外的分子来控制对人类细胞生长、发育和疾病重要的基因表达提供必要的基础。

DESCRIPTION (provided by applicant): During an immune response the expression of many genes is either upregulated or downregulated at the level of transcription, the process by which the DNA in genes is copied into mRNA. In T cells, the transcriptional activator NFAT1 controls the transcription of many genes important in the response to foreign antigen and plays critical roles in immune diseases. The broad objective of the proposed research is to design and engineer three types of non-coding RNA (ncRNA) molecules that can be used to either enhance or inhibit mRNA transcription in T cells by being brought to genes normally controlled by NFAT1. In addition to creating a new class of gene-specific ncRNA transcriptional regulators (no similar approach to controlling gene expression currently exists), these studies will reveal the extent to which NFAT1 contributes to the transcriptional program that occurs when T cells are activated, and identify genes not previously known to be directly regulated by NFAT1. The results of these studies will provide new insight into transcriptional control by NFAT1 in T cells, which is vitally important in understanding the normal immune response, as well as the inappropriate immune system responses that occur during autoimmune disorders, asthma, and allergy. The aims of the proposal are: Specific Aim 1. Create ncRNA transcriptional regulators that target human NFAT1. RNA molecules that bind NFAT1 will be isolated, and then fused to ncRNA domains previously shown to regulate transcription. These chimeric ncRNAs will be optimized to activate or repress NFAT1-driven transcription at a model gene in human T cells. Specific Aim 2. Study how an NFAT1-specific engineered ncRNA controls genome-wide transcription in T cells. To determine the efficacy and specificity with which an engineered chimeric ncRNA modulates genome-wide transcription in T cells, we will use expression and promoter microarrays. These experiments will reveal the complement of genes directly regulated by the engineered ncRNA, and by extension NFAT1. The innovation in these studies arises from the use of engineered ncRNAs to change the transcriptional program set by NFAT1 when T cells are activated. This approach is unique from conventional techniques to study the function of a transcription factor in cells, which typically lower or eliminate a protein. Rather, this approach uses NFAT1 to direct regulatory ncRNAs to the promoters of specific genes. These studies will provide the basis for creating ncRNA transcriptional regulators that target other mammalian transcription factors. In addition, the ncRNAs we develop could be used as lead compounds for the discovery of a new class of immune-therapeutics. PROJECT NARRATIVE: Properly controlling gene expression is essential to sustaining life and avoiding many diseases and cancers. The studies described here are aimed at engineering a new class of molecules that will either enhance or repress expression of specific genes in human T cells during the immune response. These studies will test a new approach to controlling gene expression in human cells and provide the groundwork needed to engineer additional molecules to control expression of genes important to growth, development, and disease in human cells.