Extended Data Fig. 1: ERK induction by cRAF-ER^T2 mimics endogenous FGF activity and directly regulates ES cell transcription.

a, A schematic representation of the inducible cRAF-ER^T2 construct. Versions with and without the FKBP(L106P) were used in this study and were found to be identical with respect to leakiness and induction. Stable cell lines were generated by random integration—all clones were screened for homogenous induction and maintained in selection for routine culture to prevent transgene variegation. b, Immunofluorescence analysis of pERK following 30 min of 4OHT treatment in cRAF-ER^T2-expressing ES cells showing homogenous induction of ERK phosphorylation. Data are representative of more than three biologically independent samples. c, Western blot showing loss of ERK and p90RSK phosphorylation following a 2 h induction and a subsequent 30-min treatment with 1 μM MEKi. All cell treatments were performed without medium changes to avoid confounding effects of serum stimulation. Data are representative of more than three biologically independent samples. d, Immunofluorescence analysis of pERK in Fgf4^−/− ES cells expressing cRAF-ER^T2, stimulated with either recombinant FGF4 (40 ng ml⁻¹) or 4OHT (250 nM) for the indicated times. The arrow indicates a cell with persistent pERK staining in FGF4-stimulated cells. Data are representative of three biologically independent samples. e, Schematic of a single ERK pulse. f, Gene ontology (GO) analysis of ERK-regulated genes at 8 h. Data are derived from two biologically independent samples. g, Comparison of the ERK-dependent half-life of wild-type NANOG and NANOG–eGFP showing no significant differences resulting from the eGFP tag. ERK activity was induced and in wild-type or NANOG–eGFP cells expressing cRAF-ER^T2 and NANOG expression was monitored at 3 or 6 h. The half-life was calculated as t_x(log(0.5))/log(N(t_x)/N(t₀)), where t_x is the duration of treatment, N(t_x) is protein concentration at time t_x and N(t₀) is initial protein concentration, and significance was tested using a two-tailed Student’s t-test. Data are mean ± s.e.m., n = 6 derived from 3 biologically independent samples measured at 3 and 6 h. h, ChIP–qPCR analysis of changes in H3K27me3 deposition across the NANOG locus between 24 and 36 h of ERK induction, significant increases between 24 and 36 h are denoted by *P = 0.05, **P = 0.02 (one tailed student t-test; data are mean ± s.e.m., n = 3 biologically independent samples). i, A heat map showing effective suppression of a panel of ERK-induced genes on treatment in the presence of actinomycin D. Values are log₂-transformed and displayed between the range of −5 and 5. n = 3 biologically independent samples. j, A box plot comparing the mRNA degradation rates of ERK target genes following a 4 h treatment with actinomycin D, with (4OHT) or without (EtOH) ERK activation, showing no significant contribution from ERK (two-sided Wilcoxon test). n = 415; data are derived from n = 3 (4OHT) and n = 2 (EtOH) biologically independent samples. Boxes show median, first and third quartiles, whiskers show 1.5× IQR and outliers are shown as black dots. k, PCA of gene expression in cells stimulated with ERK (4OHT) for 6 h with or without the proteasome inhibitor MG132. PC1 includes the variance caused by proteasomal inhibition, whereas PC2 captures the effect of ERK stimulation. n = 3 biologically independent samples. l, Scaled gene expression values for either repressed genes (top) or induced genes (bottom) that contribute to the variance in PC2 overlaid with the respective eigenvectors from k. The correlation threshold was set at 0.7. PC analysis and correlation scores were derived using ExAtlas. Data are derived from three biologically independent samples. m, Expression changes in NANOG–eGFP levels in response to ERK stimulation in cells expressing wild-type KLF2 or KLF4, or their respective putative ERK-regulated phosphorylation-site mutants (serine to alanine). Stable cell lines were derived for each gene and an empty vector served as a negative control. Expression of each protein (wild type and S>A mutant) was confirmed by western blot analysis (data not shown). A modest, but significant reduction in the magnitude of NANOG–eGFP repression was observed only in cells expressing KLF4(S123A) at 4 h (one-tailed Students t-test P = 0.02). Data are mean ± s.e.m., n = 3 biologically independent samples. n, RNA-seq time-course analysis of the first 4 h of ERK induction with robust mRNA differences only being observed at 4 h (top; y axis represent the number of significantly changing genes) and heat map of row-scaled values (bottom). n = 3 biologically independent samples.

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