Epigenetic Gene Regulation by the Ocular Environment

点击翻译按钮获取中文摘要

DESCRIPTION (provided by applicant): Immune privilege is maintained in the eye by multiple cellular mechanisms that prevent damaging inflammatory immune responses against pathogens or self-antigens in the eye. These cellular mechanisms include production of immunosuppressive cytokines and neuropeptides in the ocular environment such as transforming growth factor-beta, and alpha-melanocyte stimulating hormone, low MHC expression, lack of lymphatic drainage, a blood-ocular barrier, Fas Ligand expression, and anterior chamber associated immune deviation. We have successfully identified a molecular mechanism that may play a role in maintaining ocular immune privilege and provides a gene-based explanation for the exceptionally high success rate of corneal transplants in low risk patients without donor-typing and matching. Based on strong evidence that DNA methylation occurs in foreign cells when they are introduced into the eye, the hypothesis of this research project is that the ocular environment induces epigenetic methylation that down regulates gene expression in foreign tissues within the eye and mitigates destructive inflammatory ocular immune responses. The following specific aims and methods address key aspects of this hypothesis: (i) Identify the methyltransferases up regulated by ocular environment responsible for gene methylation, using micro array analysis, real-time PCR, and Western blot analysis, ii) Test whether methylation initiated by the ocular environment regulates gene expression by specifically methylating individual genes or by inducing global methylation and chromatin remodeling, using promoter-reporter vector assays, methylation specific PCR and chromatin immunoprecipitation techniques, (iii) Isolate and identify the factor(s) in the ocular environment responsible for triggering epigenetic gene regulation, using protein fractionation, mass spectroscopy, and proteome analysis. A better understanding of how the eye utilizes the molecular mechanisms of epigenetic regulation to maintain ocular immune privilege and prevent destructive innate and adaptive immune responses that threaten the survival of transplanted corneal tissue will provide the basis for development of new gene regulation-based strategies to improve chances of successful corneal transplants in high-risk patients whose prognosis for accepting a transplant are extremely poor due to persistent corneal inflammation.