Retinoic Acid Inducible Gene 1 Protein (RIG1)-Like Receptor Pathway Is Required for Efficient Nuclear Reprogramming

Abstract

We have revealed a critical role for innate immune signaling in nuclear reprogramming to pluripotency, and in the nuclear reprogramming required for somatic cell transdifferentiation. Activation of innate immune signaling causes global changes in the expression and activity of epigenetic modifiers to promote epigenetic plasticity. In our previous papers, we focused on the role of toll-like receptor 3 (TLR3) in this signaling pathway. Here we define the role of another innate immunity pathway known to participate in the response to viral RNA, the retinoic acid-inducible gene 1 receptor (RIG-1)-like receptor (RLR) pathway. This pathway is represented by the sensors of viral RNA, RIG-1, LGP2 and MDA5. We first found that TLR3 deficiency only causes a partial inhibition of nuclear reprogramming to pluripotency in mouse tail-tip fibroblasts, which motivated us to determine the contribution of RLR. We found that knockdown of iPS-1, the common adaptor protein for the RLR family, substantially reduced nuclear reprogramming induced by retroviral or by mmRNA expression of Oct 4, Sox2, KLF4 and cMYC (OSKM). Importantly a double knockdown of both RLR and TLR3 pathway led to a further decrease in iPSC colonies suggesting an additive effect of both these pathways on nuclear reprogramming. Furthermore, in murine embryonic fibroblasts expressing a dox-inducible cassette of the genes encoding OSKM, an RLR agonist increased the yield of iPSCs. Similarly, the RLR agonist enhanced nuclear reprogramming by cell permeant peptides of the Yamanaka factors. Finally, in the dox-inducible system, RLR activation promotes activating histone marks in the promoter region of pluripotency genes. To conclude, innate immune signaling mediated by RLR plays a critical role in nuclear reprogramming. Manipulation of innate immune signaling may facilitate nuclear reprogramming to achieve pluripotency.

Transflammation: Innate immune signaling in nuclear reprogramming

“…IPS-1 is an intermediary protein that is required for RLR-dependent IFN production in response to virus infection [93]. Knockdown of iPS-1, the common adaptor protein for the RLR family, substantially reduced nuclear reprogramming [2]. In addition, we found that LPS, an agonist of TLR4, also promotes the reprogramming mechanism (unpublished data).…”

“…These fibroblast-derived induced endothelial cells (iECs) expressed all of the expected immunohistochemical markers; manifested endothelial functions not present in fibroblasts (eg. generation of nitric oxide, formation of tubular networks in matrigel; uptake of acetylated LDL); and had a transcriptional profile (by RNA seq studies) that was highly similar to genuine endothelial cells [2]. Expected histone modifications were observed in the iECs (such as increased H3K4me3 and decreased H3K27me3 in endothelial specific gene promoters such as CD31).…”

“…More notably, iECs derived from these KD fibroblasts had markedly defective endothelial cell function. The RNA sequencing of these KD iECs revealed that they were incompletely reprogrammed [2]. …”

“…Specifically, the stimulation of pattern recognition receptors (PRRs) causes global alterations in the expression and activity of epigenetic modifiers to favor an open chromatin configuration. Activation of toll-like receptors (TLR) or RIG-1-like receptors (RLR) [2] trigger signaling cascades that result in NFκB or IRF-3 mediated changes in epigenetic plasticity that facilitate reprogramming. Another form of nuclear reprogramming is so-called direct reprogramming or transdifferentiation of one somatic cell to another lineage.…”

Paying the Toll in Nuclear Reprogramming

“…Based on this, we hypothesized that these cytoplasmic proteins, which regulate inflammatory and apoptotic responses could also enhance nuclear reprogramming. Our hypothesis was further strengthened when our attempt to reprogram fibroblasts from TLR3-knockout mice failed to completely abolish nuclear reprogramming, thereby suggesting that other pathways might also be involved (Sayed et al, 2017 ). Knockdown of the RLR pathway partially inhibited reprogramming, while addition of the RLR ligand enhanced the process, suggesting that RLR pathways are yet another PRR that might be involved in viral-based nuclear reprogramming (Sayed et al, 2017 ).…”