A novel nuclear FGF Receptor‐1 partnership with retinoid and Nur receptors during developmental gene programming of embryonic stem cells

Abstract

FGF Receptor-1 (FGFR1), a membrane-targeted protein, is also involved in independent direct nuclear signaling. We show that nuclear accumulation of FGFR1 is a common response to retinoic acid (RA) in pluripotent embryonic stem cells (ESC) and neural progenitors and is both necessary and sufficient for neuronal-like differentiation and accompanying neuritic outgrowth. Dominant negative nuclear FGFR1, which lacks the tyrosine kinase domain, prevents RA-induced differentiation while full-length nuclear FGFR1 elicits differentiation in the absence of RA. Immunoprecipitation and GST assays demonstrate that FGFR1 interacts with RXR, RAR and their Nur77 and Nurr1 partners. Conditions that promote these interactions decrease the mobility of nuclear FGFR1 and RXR in live cells. RXR and FGFR1 co-associate with 5'-Fluorouridine-labeled transcription sites and with RA Responsive Elements (RARE). RA activation of neuronal (tyrosine hydroxylase) and neurogenic (fgf-2 and fgfr1) genes is accompanied by increased FGFR1, Nur, and histone H3.3 binding to their regulatory sequences. Reporter-gene assays show synergistic activations of RARE, NBRE, and NurRE by FGFR1, RAR/RXR, and Nurs. As shown for mESC differentiation, FGFR1 mediates gene activation by RA and augments transcription in the absence of RA. Cooperation of FGFR1 with RXR/RAR and Nurs at targeted genomic sequences offers a new mechanism in developmental gene regulation.

Evidence‐Based Theory for Integrated Genome Regulation of Ontogeny—An Unprecedented Role of Nuclear FGFR1 Signaling

“…MEME‐ChiP and other methods (Terranova et al, 2015) verified earlier findings (Lee et al, 2012) that nFGFR1 associates not only with the core AGGTCA sequences targeted by the classical nuclear receptors Nur77 and/or RXR, but also to sequences that are not targeted by Nur77 and/or RXT. nFGFR1‐targeted sites encompass the consensus sites for CREB, STAT, Sp1, and Smad as well other diverse TF that interact with CBP, and thus may engage in the nFGFR1‐CBP mediated transcriptional regulation.…”

“…Biophotonic assays, including FLIP and FRAP, have demonstrated that cytoplasmic FGFR1 exists in three separate populations: (1) an immobile, newly synthesized Endoplasmic Reticulum (ER) population; (2) a highly mobile, non‐glycosylated, cytosolic population; and (3) a slowly diffusing, membrane receptor population (Dunham‐Ems et al, 2006). Nuclear accumulation of FGFR1 in live cells is promoted by an accelerated cytoplasmic to nuclear import, as well by as a reduced nuclear to cytoplasmic export (Lee et al, 2012, 2013; Stachowiak et al, 2012b). In addition, activation of cell surface FGFR1 by FGF‐2 induces FGFR1 internalization, which is dependent upon the ARF6, Dynamin2 and Rab5 endocytic machinery, and is inhibited by cell surface E‐cadherin adhesion complexes (Bryant and Stow, 2005).…”

“…Within a few hours, nFGFR1 accumulates in the nuclei of ESC,s as the cells exit from the cell cycle, and neurogenic and neuronal genes are upregulated. By 24 h, the cells display a neuronal morphology (including long neurites and growth‐cone endings), and express neuron‐specific β‐III tubulin (B), MAP2, neurofilament L, TH (Lee et al, 2012), as well as glutamate and acetylcholine receptors (Guan et al, 2001; Okada et al, 2004; Akanuma et al, 2012; Lee et al, 2012). After 96 h of at‐RA treatment, only single cells displayed characteristic glial morphology and GFAP immunoreactivity (B), (Lee et al, 2012).…”

“…In the absence of at‐RA, the average neurite outgrowth induced by active nuclear FGFR1(SP‐/NLS) is similar to the at‐RA induced outgrowth. Bar size—20 µm (Lee et al, 2012). (D) RA signaling is mediated by both retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which can act as homo or heterodimers on RA‐responsive elements (RAREs) within RA‐regulated genes (Lefebvre et al, 2010).…”

“…nFGFR1 forms CBP‐containing low mobility chromatin complexes with RXR, RAR, and Nurs. These complexes bind to RARE, NBRE, and NurRE sequences within RA‐activated Fgfr1, Fgf‐2, and Th genes, and synergistically activate isolated RA‐ and Nur‐responsive elements (Baron et al, 2012; Lee et al, 2012, 2013). …”