The roles of microRNAs in regulatory T-cell development and function

In the development of an organism, the differentiation of cells into diverse tissue types is determined by the expression of specific sets of genes. Understanding the regulation of this process is important for developing therapeutic approaches because many diseases involve disruptions in the regulation programme. Much has been learned about the mechanisms controlling the production of messenger RNAs from genes. Recently an additional pathway has been found to be important, and this occurs after the messenger RNA has been produced. It involves a separate class of RNA molecules called microRNAs (miRNAs), which are small RNAs of only about 22 nucleotides. miRNAs regulate gene expression by binding to homologous sequences in messenger RNAs and inhibiting their translation into protein. Regulation by miRNAs is essential for development because knocking out a critical gene (Dicer) required for miRNA synthesis results in death at an early embryonic stage. miRNAs are also important at later stages in the development of many tissue types. Furthermore, mis-regulation of miRNA expression has been found to be important in diseases such as cancer, and altered regulation may also be involved in ageing. Therefore, the role of miRNAs in the regulation of gene expression is important in the development of an organism and the maintenance of health during its lifetime. My interest has been in the regulation of gene expression during the development of the immune system, particularly in lymphocytes. In past work we examined the importance of miRNAs in the development of T cells, which are a specific class of lymphocytes. We knocked out the Dicer gene only in T cells and found a significant decrease in a class of T cells called regulatory T (Treg) cells. Treg cells are required for turning off the immune response and are important in diseases such as autoimmunity and cancer. Therefore, a large amount of work has been done to understand the mechanisms regulating the developmental choice between conventional T cells and Treg cells. Since miRNAs are critical, understanding how they function could lead to new therapeutic approaches in diseases involving the immune response. Therefore, in this application, I propose to identify the individual miRNAs important for Treg development and function then find and characterize the critical genes they regulate. To identify the important miRNAs in the development and function of Treg cells, we have begun by measuring the levels of miRNAs in Treg cells compared to conventional T cells. From these data we have identified 6 candidate miRNAs that are differentially and also abundantly expressed. We plan to overexpress and block these miRNAs in experiments utilizing both in vitro culture conditions we have developed and also in vivo mouse model systems that test the ability of the manipulated Treg cells to prevent autoimmunity. In these experiments only the manipulation of important miRNAs will affect Treg development and function To identify genes regulated by functional miRNAs, we will begin by using miRNA target prediction programs. The predicted targets will be verified using synthetic reporter genes containing the gene sequence of interest linked to a second gene encoding an easily measurable protein. These reporter genes will be expressed in cells and tested for regulation by a miRNA. Specificity of the regulation will be determined utilizing reporters containing a mutation in the miRNA target site. The relevance of this regulation on the endogenous gene will be tested by altered expression of the specific miRNA. To characterize target gene regulation in Treg development, we will overexpress and knockdown the expression of these genes utilizing the above in vitro and in vivo systems. The importance of signaling pathways in which these gene products function will also be analyzed. The experiments in this proposal will greatly add to our knowledge of how miRNAs function in development and disease.

MicroRNAs (miRNAs) are small RNAs (~22mers) that post-transcriptionally regulate gene expression by binding to target sequences in messages and modulating their translation, stability, and/or localization. Gene regulation by miRNAs is critical at many developmental stages in organisms ranging from worms to man. My interest lies in the roles miRNAs play in T cell development. We previously examined miRNA function in T cell development by creating and characterizing a conditional knockout of Dicer, which is the RNase required for miRNA synthesis. Loss of Dicer resulted in a depletion of regulatory T (Treg) cells, which are required for downregulation of the immune response. How miRNAs influence Treg development and function will be examined as follows. 1. Identify miRNAs required for development. As a first step we have profiled the expression levels of miRNAs in Treg cells compared to conventional T cells. About 6 miRNAs were shown to be abundant and differentially expressed. Their function will be tested by overexpression and knockdown experiments in in vitro Treg differentiation systems we have developed. 2. Identify miRNAs required for function. In addition to development, miRNAs are required for the function of Treg cells. Similar to above, we will examine the importance of individual miRNAs in Treg cell function using in vitro and in vivo suppression assays. 3. Identify and characterise genes regulated by miRNAs. Using bioinformatic approaches, we will identify candidate genes potentially regulated by critical miRNAs. These will be tested in reporter assays along with mutants containing disrupted miRNA target sites. Regulation of the endogenous gene will be confirmed by altered expression of the critical miRNA. The importance of these target genes in Treg development will be characterised using overexpression and knockdown experiments. Finally we will analyse how this regulation affects signaling pathways in Treg development and function.

The role of microRNAs in the regulation of gene expression is important in both the development of an organism and the maintenance of health during its lifetime. This proposal focuses on identification of miRNAs critical to the development and function of regulatory T cells, which regulate the immune system and are important in diseases such as cancer and autoimmunity. Who will benefit? The project examines the fundamental mechanisms in the development and function of the immune system. The results will contribute to the scientific community's body of knowledge about the immune system and will benefit scientists in academic, government and commercial environments. It could trigger other original and groundbreaking research in the UK and internationally, or lead to the application of this knowledge in the development of novel therapies targeted to autoimmunity, cancer, and other disorders associated with ageing. The research proposal will also support training of a postdoctoral scientist. This will include acquisition of techniques in molecular biology, bioinformatics and immunology, alongside general development of academic research skills in critical thinking and presentation of results to the scientific community and general public. Training young scientists is critical to ensuring strong leadership in the future and achieving UK policy aims of increasing research and development and growing the UK's reputation as a hub for original, innovative, and cutting edge scientific exploration and discovery. How will they benefit? The basic science learned from this proposal will be published in high impact journals and presented at international meetings to disseminate the results to other researchers across the globe and particularly in the UK. It will support the emergence of new ideas, collaborations and cross-fertilisation, and contribute to efforts in designing new therapeutics towards disease and maintenance of health. This will help increase the reputation of the UK as an international hub for original and groundbreaking science research. The results will also be disseminated to the general public through outreach programs of the RVC, which convey to potential students and lay audiences the experiences of scientists and the value of their research. This will help to further public understanding of biological functions and potential medical applications to common health concerns. It could also encourage more young people to consider careers in science. The postdoctoral scientist will benefit by gaining both technical expertise and career development. Skills for performing and analyzing the experimentation programmes will come from experiences in the laboratory and presenting their work to other scientists and the public. The RVC has launched a code of practice for researchers and undertakes a generic skills training needs analysis for researchers, offering a specific staff development program tailored to their needs. What will be done to ensure they benefit? To communicate this research to the scientific community, the Principal Investigator has a strong track record of publishing in high impact science journals and is regularly invited to deliver seminars and conference presentations in the UK and abroad. This will be a key area of focus as emerging results are collected. To share the research results with the general public, the RVC encourages its staff to participate in outreach activities and has engaged a PR agency (Mistral Group Ltd) to provide full press office support for communicating research findings. In addition, if potential therapeutic targets are identified in the course of this work, the College's technology transfer unit, RVC Enterprise, is well placed to explore opportunities for commercial development. The PI also has significant experience and a successful track record in training and mentoring young scientists and will personally oversee the postdoctoral proposed work.