Fig 1.
Rapid chromatin decompaction at the neural progenitor gene Pax6 in the caudal lateral epiblast following ERK1/2 dephosphorylation in the developing mouse embryo.
(A) E8.5 mouse embryo schematic, showing the caudal region and signalling pathways known to regulate neural differentiation. (B) Schematic of the hanging drop culture used to treat E8.5 mouse embryos with small molecule MEKi (PD184352) or vehicle control DMSO for 1 hour. (C and C’) Representative western blot of embryo lysates probed with antibodies against total (panERK1/2) and phosphorylated ERK1/2 (dpERK1/2) and LiCOR quantification data (n = 3 independent experiments, error bar = SEM, * p = <0.05) (S1 Data). (D) Position of fosmid probes flanking the Pax6 locus (interprobe probe distance ca. 45 kb). (D’) Representative examples of fosmid probes in individual NT and CLE nuclei (white dashed line) visualised with DAPI (blue) in each untreated condition, vehicle control (DMSO) and MEKi (in DMSO) treated. (D”) Interprobe distance measured in >50 nuclei in NT and CLE in each of three embryos in each condition (n = > 150 nuclei/condition/region, Mann–Whitney test/rank-sum test **** p ≤ 0.0001) (S1 Data). (E) Schematic showing caudal end explant (full tissue thickness taken) in E8.5 embryo. (E’) Chromatin from three biological replicates, each consisting of 30 pooled explants, were interrogated for H3K27me3 levels at TSSs of Pax6, known PRC target HoxD11 and at control regions (see text) compared to IgG background. Three biological replicates (circle, triangle, and diamond) and average (bar) shown, error bar = SEM (S1 Data). CLE, caudal lateral epiblast; FGF, fibroblast growth factor; IgG, immunoglobulin G; MEKi, MEK inhibitor; NT, neural tube; PRC, polycomb repressive complex; RA, retinoic acid; Raldh2, retinaldehyde dehydrogenase 2; RAR, retinoic acid receptor; TSS, transcription start site.
Fig 2.
In vitro differentiation of human ESC-derived NMP-L cells to NPs reveals locus decompaction correlates with dissociation of polycomb repressive complexes and transcriptional onset at PAX6.
(A) Schematic of differentiation regime used to generate first NMP-like cells (NMP-L (D3)) and then spinal cord progenitors (NPs (D8)) from human ESCs (D1 = Day 1. (B-B”‘) Transcription levels of BRA, SOX2, HOXD11, and PAX6 assessed by RT-qPCR in undifferentiated cells (hESCs), NMP-Ls (D3), and NPs (D8) for PAX6 additionally on day 5 (D5) and day 6 (D6) of differentiation (n = 3 independent experiments, indicated with circles, triangles, and diamonds, error bars = SEM) (S2 Data). (C-C”) FISH to assess chromatin compaction around the PAX6 locus in NMP-L (D3) and NP (D8). Two probes flanking the target locus (interprobe distance ca. 51 kb) were hybridised and labelling visualised in DAPI-stained nuclei (blue, outlined with white dashed line). Interprobe distance measurements in >50 nuclei in NMP-Ls and NPs in 3 individual experiments (n = > 150 nuclei/cell type, Mann–Whitney test/rank-sum test, *** p ≤ 0.001) (S2 Data). (D-D”‘) ChIP-qPCRs investigating polycomb repressive complex occupancy (Ring1B/PRC1 and Jarid2/PRC2) and the histone modifications H3K27me3 and H3K4me3 in NMP-L and NP cells (note, D8 Jarid2 is below IgG control) (n = 3 independent experiments, each indicated by circles), bar = average, error bar = SEM, * p ≤ 0.01, t test (S2 Data). ChIP-qPCR, chromatin immunoprecipitation quantitative PCR; ESC, embryonic stem cell; FISH, fluorescent in situ hybridisation; IgG, immunoglobulin G; NMP-L, NMP-like; NP, neural progenitor; RT-qPCR, reverse transcription quantitative PCR.
Fig 3.
Progressive increase in human neural gene accessibility during in vitro differentiation.
(A) ATAC-seq peak tracks in NMP-L (D3), D5, D6, and NP (D8) cells for the genomic regions of PAX6, grey boxes are peaks within the gene body, black boxes, peaks outside of the gene body and red box genomic region of a known PAX6 enhancer. (B-B”) ChIP-qPCR for Jarid2, Ring1B, and H3K27me3 on D5, D6, and D7, note by D7 Jarid2 signal falls below detection (n = 3 independent experiments, each indicated by circles), bar = average, error bars = SEM, although there is a declining trend for Jarid2, there are no significant differences between samples, t test (S3 Data). (C-C’) Metaprofiles comparing chromatin accessibility in NMP-L (D3) and NP (D8) cells along the gene body genome-wide (C) and focussed on genes associated with increased accessibility in NPs (D8) (neural sites, C’) compared to NMP-L (S3 Data). (D) Venn diagram representing distribution of genomic regions more accessible in NPs (D8) (neural sites) compared to NMP-L (D3) and their appearance over time (D5 and D6). Comparison of accessible regions and associated genes on D5 and after D5 revealed approximately 19% of genes had additional accessible regions, while the majority were found in newly accessible genes, similarly 40% of D6 genes with additional regions became more accessible in NP (D8). (E) GO term analysis of genes associated with neural sites compared to NMP-L (D3) and genes associated with more accessible chromatin regions on D5 and D6 out of the neural sites (S3 Data). (F-F’) ATAC-seq peak tracks in NMP-L (D3), D5, D6, and NP (D8) cells for the genomic regions of RARB (E) and GLI3 (E’), grey boxes are peaks within the gene body. ChIP-qPCR, chromatin immunoprecipitation quantitative PCR; GO, gene ontology; NMP-L, NMP-like; NP, neural progenitor.
Fig 4.
ERK1/2 dephosphorylation induces precocious PAX6 expression, reduced polycomb protein occupancy and H3K27me3 at this locus and increases accessibility of hundreds of neural differentiation genes.
(A) Differentiation protocol generating NMP-L (D3) cells and spinal cord NPs by D8 and additional treatment with vehicle control DMSO or MEKi (PD184352) every 24 hours from D3 for the duration of the differentiation protocol. (B) Western blot confirming that exposure to MEKi lead to reduced ERK1/2 phosphorylation after 24 hours in differentiation conditions (D3-D4). (C) PAX6 transcript levels in NMP-L (D3), in control conditions (WT and DMSO) or in MEKi-treated cells on D5 and D6 and NP(D8) determined by RT-qPCR (n = 3 independent experiments indicated by circles, triangles, and diamonds, bar = average, error bars = SEM) (S5 Data). (D-D’) ChIP-qPCR for Ring1B and H3K27me3 on D6 note H3K27me3 below detection in MEKi conditions (n = 3 independent experiments indicated with circles, bar = average, error bars = SEM, * = p ≤ 0.05, t test, S5 Data). (E-E’) Venn diagrams representing distribution of genomic regions that become more accessible on D5 (E) and D6 (E’) in control condition and MEKi-treated cells compared to NMP-L (not restricted to regions accessible in NPs (D8)) as well as more accessible regions on D8. (F-F’) GO term analysis of genes associated with more accessible chromatin in D5 (F) and D6 (F’) in control condition and MEKi-treated cells compared to NMP-L (S5 Data). (G-G”) ATAC-seq peak tracks in NMP-L, D5 DMSO and MEKi, D6 DMSO and MEKi, and NP (D8) cells for the genomic regions of DCX (G), GABRB2 (G’), and PAX6 (G”), grey boxes outline peaks within the gene body, black boxes peaks outside of the gene body and red box genomic region of a known PAX6 enhancer, example comparisons between peaks in MEKi and DMSO conditions indicated with black arrowhead pairs. Note MEKi exposure does not alter levels of Ring1B protein while it promotes neural differentiation (S4 Fig). ChIP-qPCR, chromatin immunoprecipitation quantitative PCR; GO, gene ontology; MEKi, MEK inhibitor; NMP-L, NMP-like; NP, neural progenitor; RT-qPCR, reverse transcription quantitative PCR; WT, wild type.
Fig 5.
Bioinformatic analyses predict engagement of SOX factors following MEKi exposure and reveal distinct gene pathways occupied by RING1B in NMPs.
Top ten TFs identified by genomic footprinting of regions of increased accessibility detected by ATAC-seq following exposure to MEKi or control (WT) conditions on (A) D5 and (B) D6 (S8 Data); GO biological processes detected by GREAT; (C) that are associated with genomic intervals that change in Ring1B occupancy as detected by ChIP-seq between day 0 hESCs and day 3 NMPs-L cells (see Method); and associated with neural sites occupied by (D) Ring1B (purple—enriched for neural and patterning genes) and (D’) non-Ring1B in NMP-L cells. GO, gene ontology; hESC, human ESC; MEKi, MEK inhibitor; NMP, neuromesodermal progenitor; NMP-L, NMP-like; NP, neural progenitor; TF, transcription factor; WT, wild type.
Fig 6.
Transient ERK1/2 dephosphorylation induces dissociation of Jarid2 and Ring1B and chromatin decompaction in NMP-L cells at the PAX6 locus but does not reduce H3K27me3 nor elicit transcription.
(A) Differentiation protocol used to generate NMP-L (D3) cells and additional treatment with vehicle control DMSO or MEKi for 12 hours. (B-B’) Representative western blot of cell lysates probed with antibodies against total (panERK1/2) and dual-phosphorylated-ERK1/2 (dpERK1/2) and LiCOR quantification data (n = 3 independent experiments, error bar = SEM) (S10 Data). (C-C”) ChIP-qPCRs investigating the histone modification H3K27me3 and polycomb repressive complex occupancy in NMP-L (D3) cells treated with MEKi or DMSO for 12 hours (n = 3 individual experiments indicated with circles, bar = average, * = p ≤ 0.05, t test) (S10 Data). (D-D”) Transcription levels of PAX6, HOXD11, and JARID2 assessed by RT-qPCR in undifferentiated cell (hESCs), NMP-L cells untreated, vehicle control (DMSO) treated or MEKi treated (n = 3 individual experiments, no significant differences between samples, t test) (S10 Data). (E-E’) FISH to assess chromatin compaction around the PAX6 locus in NMP-Ls untreated and treated with DMSO or MEKi for 12 hours. Two probes flanking the target locus (interprobe distance ca. 51 kb) were hybridised and visualised by differential labelling nuclei (outlined with white dashed line) visualised with DAPI (blue). Interprobe distance measurements in >50 nuclei in the three conditions in three individual experiments (n = > 150 nuclei/cell type, Mann–Whitney test/rank-sum test, *** p ≤ 0.001) (S10 Data), this decompaction correlated with a 2.5-fold decrease in number of base pairs per nm compared to both controls (untreated NMP-L (D3): 205 bp/nm, DMSO NMP-L (D3): 218 bp/nm and MEKi NMPL (D3): 87 bp/nm). ChIP-qPCR, chromatin immunoprecipitation quantitative PCR; MEKi, MEK inhibitor; NMP-L, NMP-like; RT-qPCR, reverse transcription quantitative PCR.
Fig 7.
Model of steps leading to engagement of the neural differentiation programme at exemplar gene PAX6.
In chick and mouse embryos, decline in FGF and downstream effector kinase ERK1/2 signalling takes place as NMP cells commence neural differentiation, mediated by rising levels of retinoid (RA) signalling, which represses Fgf8 (reviewed in [25]). Mouse NMPs are characterised by phosphorylated ERK1/2 (dpERK1/2) and compact chromatin across the exemplar neural differentiation gene Pax6, which is decorated with the PRC2 gene silencing mark H3K27me3. In human NMP-L cells, PRC2 (Jarid2) and PRC1 (Ring1B) occupy the locus and H3K27me3 is accompanied by gene activation associated histone modification H3K4me3, identifying this as a bivalent locus poised for transcription. During differentiation, loss of ERK1/2 signalling leads to chromatin decompaction across PAX6 in mouse embryo NMPs and in human NMP-L cells, where it is accompanied by loss of PRCs (PRC2 and PRC1). There is also evidence that levels of PRC2 protein Jarid2 decline during neural differentiation. Loss of ERK1/2 activity results in genome-wide increase in chromatin accessibility across neural genes, involving regulation of PRC and other chromatin accessibility complexes, opening sites that become bound by neural TFs (SOX family), detected prior to PAX6 transcription. The gene silencing histone mark H3K27me3 persists after PRC2 is lost and is removed coincident with transcription onset: ERK1/2 regulation of chromatin accessibility and neural TF binding appears distinct from this later step, which may depend on retinoid signalling, known to be required for PAX6 transcription in mouse and chick embryos. As transient loss of ERK1/2 activity removes PRC2 and PRC1 and these complexes are not reinstated on resumption of ERK1/2 signalling, decline in ERK1/2 activity and so PRC occupancy (and other chromatin remodelling complexes) acts as a gating mechanism that confers differentiation directionality as well as synchronising accessibility for neural TF binding and so engagement of the neural differentiation programme. FGF, fibroblast growth factor; NMP, neuromesodermal progenitor; NMP-L, NMP-like; RA, retinoic acid; PRC, polycomb repressive complex; TF, transcription factor.