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. ? ? ?
