FGF Signalling Regulates Chromatin Organisation during Neural Differentiation via Mechanisms that Can Be Uncoupled from Transcription

Changes in higher order chromatin organisation have been linked to transcriptional regulation; however, little is known about how such organisation alters during embryonic development or how it is regulated by extrinsic signals. Here we analyse changes in chromatin organisation as neural differentiation progresses, exploiting the clear spatial separation of the temporal events of differentiation along the elongating body axis of the mouse embryo. Combining fluorescence in situ hybridisation with super-resolution structured illumination microscopy, we show that chromatin around key differentiation gene loci Pax6 and Irx3 undergoes both decompaction and displacement towards the nuclear centre coincident with transcriptional onset. Conversely, down-regulation of Fgf8 as neural differentiation commences correlates with a more peripheral nuclear position of this locus. During normal neural differentiation, fibroblast growth factor (FGF) signalling is repressed by retinoic acid, and this vitamin A derivative is further required for transcription of neural genes. We show here that exposure to retinoic acid or inhibition of FGF signalling promotes precocious decompaction and central nuclear positioning of differentiation gene loci. Using the Raldh2 mutant as a model for retinoid deficiency, we further find that such changes in higher order chromatin organisation are dependent on retinoid signalling. In this retinoid deficient condition, FGF signalling persists ectopically in the elongating body, and importantly, we find that inhibiting FGF receptor (FGFR) signalling in Raldh2−/− embryos does not rescue differentiation gene transcription, but does elicit both chromatin decompaction and nuclear position change. These findings demonstrate that regulation of higher order chromatin organisation during differentiation in the embryo can be uncoupled from the machinery that promotes transcription and, for the first time, identify FGF as an extrinsic signal that can direct chromatin compaction and nuclear organisation of gene loci

Widespread reorganisation of pluripotent factor binding and gene regulatory interactions between human pluripotent states.

The transition from naive to primed pluripotency is accompanied by an extensive reorganisation of transcriptional and epigenetic programmes. However, the role of transcriptional enhancers and three-dimensional chromatin organisation in coordinating these developmental programmes remains incompletely understood. Here, we generate a high-resolution atlas of gene regulatory interactions, chromatin profiles and transcription factor occupancy in naive and primed human pluripotent stem cells, and develop a network-graph approach to examine the atlas at multiple spatial scales. We uncover highly connected promoter hubs that change substantially in interaction frequency and in transcriptional co-regulation between pluripotent states. Small hubs frequently merge to form larger networks in primed cells, often linked by newly-formed Polycomb-associated interactions. We identify widespread state-specific differences in enhancer activity and interactivity that correspond with an extensive reconfiguration of OCT4, SOX2 and NANOG binding and target gene expression. These findings provide multilayered insights into the chromatin-based gene regulatory control of human pluripotent states

Correction: FGF Signalling Regulates Chromatin Organisation during Neural Differentiation via Mechanisms that Can Be Uncoupled from Transcription.

[This corrects the article DOI: 10.1371/journal.pgen.1003614.]

ERK1/2 signalling dynamics promote neural differentiation by regulating chromatin accessibility and the polycomb repressive complex

Fibroblast growth factor (FGF) is a neural inducer in many vertebrate embryos, but how it regulates chromatin organization to coordinate the activation of neural genes is unclear. Moreover, for differentiation to progress, FGF signalling must decline. Why these signalling dynamics are required has not been determined. Here, we show that dephosphorylation of the FGF effector kinase ERK1/2 rapidly increases chromatin accessibility at neural genes in mouse embryos, and, using ATAC-seq in human embryonic stem cell derived spinal cord precursors, we demonstrate that this occurs genome-wide across neural genes. Importantly, ERK1/2 inhibition induces precocious neural gene transcription, and this involves dissociation of the polycomb repressive complex from key gene loci. This takes place independently of subsequent loss of the repressive histone mark H3K27me3 and transcriptional onset. Transient ERK1/2 inhibition is sufficient for the dissociation of the repressive complex, and this is not reversed on resumption of ERK1/2 signalling. Moreover, genomic footprinting of sites identified by ATAC-seq together with ChIP-seq for polycomb protein Ring1B revealed that ERK1/2 inhibition promotes the occupancy of neural transcription factors (TFs) at non-polycomb as well as polycomb associated sites. Together, these findings indicate that ERK1/2 signalling decline promotes global changes in chromatin accessibility and TF binding at neural genes by directing polycomb and other regulators and appears to serve as a gating mechanism that provides directionality to the process of differentiation

Signals regulating differentiation and expression patterns of <i>Pax6</i> and <i>Fgf8</i> along the elongating neural axis.

<p>(A) Summary of cell populations, signal localisation and interactions at the caudal end of the E8-8.5 mouse embryo, RA retinoic acid, RAR, retinoic acid receptor, FGF, fibroblast growth factor, PS, primitive streak, S, somite, <i>Raldh2, Retinaldehyde dehydrogenase 2</i>; (B) <i>Pax6</i> is expressed in the neural tube (B′) in transverse section (TS), but not in (B″) preneural tube or (B‴) stem zone; (C) <i>Fgf8</i> is expressed in the stem zone, but not in the neural tube (C′) in TS or in (C″) preneural tube, stem zone expression in TS (C‴). Grey dashed lines in B′, B″, B‴, C′,C″, C‴ outline cell populations in which nuclei were assessed in FISH experiments. Scale bar = 50 microns, asterisk indicates position of the node in all embryo images.</p

Inhibition of FGFR signalling in <i>Raldh2</i> mutants rescues higher order chromatin organisation around the <i>Pax6</i> locus, but not <i>Pax6</i> transcription.

<p>(A) <i>Pax6</i> transcripts are lacking in the neural tube of <i>Raldh2</i> mutants treated with DMSO or (B) with FGFR inhibitor PD173074; (C) Box-plot of inter-probe distances (µm²) for <i>Pax6</i> flanking probes in each tissue assessed in <i>Raldh2</i> mutant embryos or <i>Raldh2</i> mutants treated with PD173074, showing blocking FGFR signalling decompacts the <i>Pax6</i> locus in both stem zone, neural tube and somites; and examples of hybridised nuclei in <i>Raldh2</i> mutant embryos, D) stem zone, neural tube and somites, or treated with PD173074, (E) stem zone, neural tube and somites. (F) Graph of data distribution for fractional radius measurements in <i>Raldh2 −/−</i> and <i>Raldh2−/−</i> + PD173074 tissues, showing that the <i>Pax6</i> locus now shifts towards the nuclear centre in stem zone as well as in the neural tube when FGFR signalling is blocked.</p

Changes in chromatin compaction and nuclear position of <i>Pax6</i> and <i>Fgf8</i> loci during neural differentiation and following manipulation of retinoid and/or FGF signalling.

<p>(A) Schematic summarising changes in local chromatin organisation of <i>Pax6, Irx3</i> and <i>Fgf8</i> loci as neural differentiation commences in the elongating body axis of wildtype and FGFR signalling deficient (+PD173074) and for <i>Pax6</i> and <i>Fgf8</i> in retinoid deficient (<i>Raldh2−/−</i>) mouse embryos and when both these signalling pathways are attenuated. These data indicate that FGF signalling promotes chromatin compaction around <i>Pax6</i> and <i>Irx3</i> loci and regulates nuclear position of <i>Pax6, Irx3</i> and <i>Fgf8</i> gene loci during neural differentiation. Green and red dots represent flanking fosmid pairs and blue circle the nuclear edge, grey cross indicates likely loss of active <i>Fgf8</i>. (B) Summary of chronological steps towards neural differentiation deduced in this study, from the high FGF signalling context in the stem zone to the onset of neural gene expression in the high retinoid signalling environment of the neural tube.</p