Global Developmental Gene Programing Involves a Nuclear Form of Fibroblast Growth Factor Receptor-1 (FGFR1)

Abstract

Genetic studies have placed the Fgfr1 gene at the top of major ontogenic pathways that enable gastrulation, tissue development and organogenesis. Using genome-wide sequencing and loss and gain of function experiments the present investigation reveals a mechanism that underlies global and direct gene regulation by the nuclear form of FGFR1, ensuring that pluripotent Embryonic Stem Cells differentiate into Neuronal Cells in response to Retinoic Acid. Nuclear FGFR1, both alone and with its partner nuclear receptors RXR and Nur77, targets thousands of active genes and controls the expression of pluripotency, homeobox, neuronal and mesodermal genes. Nuclear FGFR1 targets genes in developmental pathways represented by Wnt/β-catenin, CREB, BMP, the cell cycle and cancer-related TP53 pathway, neuroectodermal and mesodermal programing networks, axonal growth and synaptic plasticity pathways. Nuclear FGFR1 targets the consensus sequences of transcription factors known to engage CREB-binding protein, a common coregulator of transcription and established binding partner of nuclear FGFR1. This investigation reveals the role of nuclear FGFR1 as a global genomic programmer of cell, neural and muscle development.

Fig 1. Genome-wide analyses of nFGFR1, RXR and Nur77 binding in pluripotent ESCs and RA-induced NCs.

(A) nFGFR1, (B) RXR and (C) Nur77 peaks are present on all chromosomes, in both ESCs and NCs. (D) Genomic distribution of nFGFR1, RXR and Nur77 peaks within proximal promoters (-1kb to +1 kb relative to TSS), distal promoters (-5 kb to -1 kb relative to TSS), genic and intergenic regions in ESCs and NCs. (E-G) Enrichment of FGFR1, RXR and Nur77 peaks within promoter and genic regions.

Fig 2. Genome-wide colocalization of nFGFR1, RXR and Nur77 peaks.

(A) Venn diagram illustrates the number of individual and overlapping nFGFR1, RXR and Nur77 binding sites. (B) nFGFR1, RXR and Nur77 peaks colocalize within all genomic regions. Specifically in the proximal promoter and NCs, the number of sites at which RXR or Nur77 were bound together with nFGFR1 was markedly higher than the number of sites at which RXR or Nur77 were bound without nFGFR1.

Fig 3. Binding of nFGFR1, RXR and Nur77 to expressed genes.

(A) Heatmap representation of genes that were differentially expressed in pluripotent ESCs and RA-induced NCs from three independent biological replicates. Out of 14,443 expressed genes, 1,834 were up-regulated and 1,477 were down-regulated in NCs [Fold Change (FC) ≥-/+2.0 and p-value <0.035 were considered significant]. Values are displayed as fragments per kb of transcript per million fragments mapped (FPKM). (B) Binding of nFGFR1, RXR and Nur77 within the proximal promoter of differentially regulated genes. In NCs, the population of regulated genes that are targeted by nFGFR1 (2,058 genes) was markedly higher than the population of regulated genes that are not (480 genes).

Fig 4. Ingenuity Pathway Analysis (IPA) of genes expressed differentially in ESCs and NCs and targeted by nFGFR1 only.

Top biological functions, networks and diseases identified. Networks illustrate the degree of gene upregulation (red) and downregulation (green) by color intensity. Genes that were bound by nFGFR1 and not differentially regulated according to our cut-off are displayed in gray. Solid lines represent direct, and dotted lines indirect, interactions between genes in the network. A complete interpretation of network shapes and interactions can be found in Material and Methods. P-values were calculated using the right-tailed Fisher’s exact test. (A-C) nFGFR1 binding to genes in ESCs: (A) biological functions and diseases; (B) top network controlling cell proliferation and survival; and (C) eight top canonical pathways. (D-F) Binding of nFGFR1 to genes in in RA-differentiated NCs: (D) biological functions and diseases; (E) networks; and (F) eight top canonical pathways.

Fig 5. nFGFR1 targets pluripotentcy core genes and motifs in pluripotent transcription-factors (TFs).

(A) Heatmap illustrating the expression patterns of core pluripotentcy genes (left) and associated proximal promoter binding of nFGFR1, RXR and Nur77 (right). (B) The pluripotent gene network is based on Chen et al.[25, 26]. The present investigation reveals core genes that have promoters targeted by nFGFR1 and were differentially regulated during the RA-induced transition from ESC to NC. (C) Expression of core pluripotentcy genes in the presence of dominant-negative nuclear FGFR1(SP-/NLS)(TK-). mRNA expression was measured in extracts from ESCs that had been transfected with either β-gal (control) or FGFR1(SP-/NLS)(TK-) and were then maintained in the presence of +LIF or +RA for 48 hours. In the presence of LIF, the block in nFGFR1 increased the basal expression of all genes examined. In RA-treated ESCs, the dominant-negative FGFR1 protein completely abolished the RA-induced downregulation of all members of the pluripotency core. p-value <0.05 * relative to β-gal+LIF; + relative to β-gal+RA. (D) Expression of core pluripotency genes in the presence of FGFR1(SP-/NLS). nFGFR1 in the presence of LIF repressed core pluripotency genes to an extent similar to RA alone. In the presence of LIF, transfection of FGFR1(SP-/NLS) induced the downregulation of all pluripotent genes. P-value <0.05 * relative to β-gal+LIF.

Fig 6. nFGFR1 regulates the expression of Homeobox genes in RA-treated NCs.

(A) Heatmap illustrating the expression patterns of genes within the Hoxa-Hoxd clusters (left), and the associated binding of nFGFR1, RXR and Nur77 to the proximal promoter (right). (B) Expression of Hoxa genes in the presence of dominant-negative nuclear FGFR1(SP-/NLS)(TK-). mRNA expression was measured by RT-qPCR, in extracts from ESCs that had been transfected with either β-gal (control) or FGFR1(SP-/NLS)(TK-) and were then maintained in the presence of +LIF or +RA for 48 hours. In the presence of LIF, blocking nFGFR1 increased the levels of expression of the Hoxa1, 4 and 7 mRNAs. In RA-treated ESC, blocking nFGFR1 significantly reduced the RA-induced expression of all genes of the Hoxa cluster other than Hoxa5. * p-value <0.05; * relative to Bgal+LIF; + relative to β-gal+RA. (C) Expression of Hoxa genes in the presence of nFGFR1. nFGFR1 activates Hoxa gene expression to an extent similar to RA treatment. In the presence of LIF, FGFR1(SP-/NLS) induced significant upregulation of Hoxa1 and Hoxa2. * p value <0.05; * relative to Bgal+LIF; (D) RA-induced expression of Hoxa cluster genes in the presence of nuclear FGFR1(SP-/NLS). In the presence of RA, transfection of FGFR1(SP-/NLS) led to a marked increase in the expression of all Hoxa cluster genes. P-value <0.05; * relative to β-gal+LIF; + relative to β-gal +RA.

Fig 7. A paradigm for ontogenic global genome programming by nFGFR1.

Genetic experiments position the Fgfr1 gene at the top of gene hierarchy that directs the development of multicellular animals. Fgfr1 governs gastrulation, as well as development of the major body axes, neural plate, central and peripheral nervous systems, and mesoderm by affecting the genes that control the cell cycle, pluripotency and differentiation [–6], and microRNAs. This regulation is executed by a single nuclear protein, nFGFR1, which integrates signals from RA and other development-initiating factors, cooperates with RXR, Nurs and multiple TFs, and targets thousands of genes, including ones that encode miRNAs and some within top ontogenic gene networks. nFGFR1 binding to promoters of genes that encode TFs, and the genomic sequences targeted by these TFs suggest a feed forward mechanism for fine-tuning complex developmental networks. Legend: (A) nFGFR1 binding within the proximal promoter of exemplary target genes in which color denotes up- (red) or down-regulation (green) during transition from ESCs to NCs (RNA-seq). Black indicates genes that are not differentially regulated according to our cutoff. Gray box denotes direct regulation by nFGFR1, as determined by loss- or gain-of-function experiments. * denotes gene directly regulated by nFGFR1 from previous studies. (B) In the cases of KLF4 and other TFs (TP53, SMAD, CTCF, MYC, OCT4, SOX2 and STAT3), nFGFR1 interacts both with the TF-encoding genes and the consensus sequences to which they bind. This implies a feed-forward mechanism, in which nFGFR1 controls both the generation of TFs and their downstream function to fine tune ontogenic gene networks.