Transdifferentiation Requires iNOS Activation

Abstract

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.

Transflammation: Innate immune signaling in nuclear reprogramming

“…Our chromatin immunoprecipitation studies revealed that S-nitrosylation of RING1A caused it to dissociate from chromatin. This effect was associated with a reduction in H3K27 trimethylation, and an increase in H3K4 trimethylation [5]. These studies indicated that S-nitrosylation of RING1A impaired the repressive effects of PRC1, thereby facilitating transdifferentiation (Figure 1).…”

“…We found that iNOS-derived NO is a major effector of transdifferentiation [5]. Stimulation of innate immune signaling using the TLR 3 agonist poly I:C induces iNOS at the earliest stage of transdifferentiation, weeks before iECs are generated.…”

“…The critical importance of iNOS in transdifferentiation was revealed when we repeated our transdifferentiation protocol using pharmacological antagonists or genetic interventions to abrogate iNOS activity or expression. The iNOS antagonists significantly inhibited transdifferentation, and the generation of iECs from iNOS−/− fibroblasts was nearly abolished [5]. Thus iNOS activity is required for transdifferentiation of fibroblasts to endothelial cells.…”

“…We identified that the innate immune activation was required for the changes in the expression and activity of epigenetic modifiers that make the chromatin more accessible to transcriptional factors [1–5]. We have coined the term “transflammation” to describe the process by which innate immune signaling promotes epigenetic plasticity.…”

“…We have shown that transdifferentiation of human fibroblasts to endothelial cells also involves transflammation [3]. Recently, we also identified reactive oxygen species (ROS) [4] and reactive nitrogen species (RNS) [5] as mediators of innate immune signaling in nuclear reprogramming. Innate immune signaling plays a key role in nuclear reprogramming by regulating DNA accessibility (Figure 1).…”