LAUSR

BACKGROUND We found that cell-autonomous innate immune signaling causes global changes in the expression of epigenetic modifiers to facilitate nuclear reprogramming to pluripotency. A role of S-nitrosylation by inducible NO synthase (iNOS), an important effector of innate immunity, has been previously described in transdifferentiation of fibroblasts to endothelial cells. Accordingly, we hypothesized that S-nitrosylation might also have a role in nuclear reprogramming to pluripotency. METHODS We used murine embryonic fibroblasts containing a doxycycline-inducible cassette encoding the Yamanaka factors (Oct4, Sox2, Klf4 and c-Myc), and genetic or pharmacological inhibition of iNOS together with Tandem Mass Tag (TMT) approach, chromatin immunoprecipitation-qPCR, site directed mutagenesis, and micrococcal nuclease assay to determine the role of S-nitrosylation during nuclear reprogramming to pluripotency. RESULTS We show that an optimal zone of innate immune activation, as defined by maximal yield of induced pluripotent stem cells (iPSCs), is determined by the degree of NFkB activation; nitric oxide (NO) generation; S-nitrosylation of nuclear proteins; and DNA accessibility as reflected by histone markings and increased mononucleosome generation in a micrococcal nuclease assay. Genetic or pharmacological inhibition of iNOS reduces DNA accessibility and suppresses iPSC generation. The effect of NO on DNA accessibility is mediated in part by Snitrosylation of nuclear proteins, including MTA3, a subunit of Nucleosome Remodeling Deacetylase (NuRD) complex. S-nitrosylation of MTA3 is associated with decreased NuRD activity. Overexpression of mutant MTA3, in which the two cysteine residues are replaced by alanine residues, impairs the generation of iPSCs. CONCLUSIONS This is the first report showing that DNA accessibility and iPSC yield depends on the extent of cell-autonomous innate immune activation and NO generation. This "Goldilocks zone" for inflammatory signaling and epigenetic plasticity may have broader implications for cell fate and phenotypic fluidity.