PROJECT SUMMARY We discovered that innate immune activation facilitates nuclear reprogramming to pluripotency or to a different cell lineage. Our characterization of this process (Lee et al, Cell 2012) 1 has produced new insights into the mechanisms of nuclear reprogramming. We discovered that innate immune activation causes global changes in the expression of epigenetic modifiers, associated with histone markings that favor nuclear reprogramming. We intend to further elucidate the mechanisms by which innate immune activation facilitates direct reprogramming (transdifferentiation). A major factor involved in innate immune response is the inducible enzyme nitric oxide synthase (iNOS). Our data indicates that iNOS translocates to the nucleus during transdifferentiation and may directly bind and S-nitrosylate epigenetic modifiers. Accordingly, we intend to: 1. Assess the direct effects of S-nitrosylation on epigenetic regulators during transdifferentiation. We will assess the effect of S-nitrosylation on the activity of selected epigenetic factors, and/or their binding to co- factors or chromatin, using immunoblot analyses, IP and ChIP, and the effect of genetic or pharmacologic manipulation of iNOS. We will also elucidate the importance of iNOS translocation and its physical association with epigenetic modifiers in transdifferentiation using genetic and pharmacological methods to disrupt iNOS translocation and binding to selected epigenetic modifiers. 2. Characterize the effects of S-nitrosylation on DNA accessibility and fidelity of transdifferentiation. We will characterize open chromatin regions using DNAse-Seq and Mnase-Seq; bivalent chromatin markings by ChIP-seq, integrated with a global assessment of the transcriptional regulation (RNA-Seq) and determine the importance of iNOS in the fidelity of epigenetic changes required for activation of cell identity genes.