Transdifferentiation Requires iNOS Activation: Role of RING1A S-Nitrosylation
Rationale: We have previously shown that innate immunity is necessary for transdifferentiation of fibroblasts to endothelial cells. A major signaling molecule involved in innate immunity is inducible nitric oxide synthase (iNOS). Accordingly, we hypothesized that iNOS-generated nitric oxide (NO) might enhance transdifferentiation.
Objective: To elucidate the role of NO in epigenetic plasticity during transdifferentiation.
Methods and Results: We exposed the BJ fibroblasts to transdifferentiation formulation that included endothelial growth factors and innate immune activator polyinosinic:polycytidylic acid (PIC) to induce endothelial cells (iECs). Generation of iECs was associated with iNOS expression and NO elaboration. In absence of PIC, or in presence of antagonists of NFκB or iNOS activity, NO synthesis and iEC generation was reduced. Furthermore, genetic knockout (in MEFs) or siRNA knockdown (in BJ fibroblasts) of iNOS nearly abolished transdifferentiation, an effect which could be reversed by iNOS overexpression. Notably, PIC induced nuclear localization of iNOS, and its binding to, and nitrosylation of, the epigenetic modifier RING1A as assessed by immunostaining, Co-IP and mass spectrometry. Nitrosylation of RING1A reduced its binding to chromatin, and reduced global levels of repressive histone marker H3K27trimethylation. Overexpression of a mutant form of RING1A (C398A) lacking the nitrosylation site almost abrogated transdifferentiation.
Conclusions: Overall, our data indicates that during transdifferentiation, innate immune activation increases iNOS generation of NO to S-nitrosylate RING1A, a key member of the polycomb repressive complex. Nitrosylation of RING1A reduces its binding to chromatin and decreases H3K27trimethylation level. The release of epigenetic repression by nitrosylation of RING1A is critical for effective transdifferentiation.
- Received August 16, 2016.
- Revision received August 26, 2016.
- Accepted September 13, 2016.