Extended Data Fig. 3: ERK regulates ES cell enhancer activity.

a, RT–qPCR analysing the expression of eRNA at pluripotency super enhancers. Fgf4^−/− ES cells were stimulated for either 30 or 90 min with 40 ng ml⁻¹ recombinant FGF4 before analysis. *P < 0.05 and **P < 0.01, two-tailed Student’s t-test; data are mean ± s.e.m., n = 6 independent experiments. b, A scatter plot showing the changes in RNAPII binding and eRNA expression at regulated super enhancers (adj. P ≤ 0.01), showing a high level of correlation between RNAPII binding and eRNA expression in response to ERK (two-sided Spearman's correlation coefficient). n = 3 biologically independent samples. c, A scatter plot showing the changes in H3K27ac and EP300 enrichment at both putative traditional and super enhancers in response to ERK (see materials and methods for further details). ERK activation results in the correlated changes in EP300 binding and H3K27ac deposition at both super enhancers and traditional enhancers (two-sided Spearman's correlation coefficient). n = 636 (activated traditional enhancers), n = 1,726 (repressed traditional enhancers), n = 30 (repressed super enhancers) and n = 27 (activated super enhancers). Data are derived from two biologically independent samples. d, A volcano plot showing the expression pattern of ERK-regulated genes (taken from Fig. 1d, nascent RNA) associated with ERK-regulated enhancers. n = 3 biologically independent samples. e, A stacked bar chart showing the percentage of genes repressed or induced by ERK and associated with repressed (acDOWN) or activated (acUP) enhancers. Eighty-five per cent of genes associated with repressed enhancers are repressed themselves and, similarly, 83% of genes associated with activated enhancers are themselves induced by ERK. Gene association was performed using GREAT (http://great.stanford.edu/) with single-nearest-gene cut-off. f–i, ATAC profiles at ERK regulated traditional enhancers (f, g) and super enhancers (h, i) following 2 h stimulation, showing that changes in chromatin accessibility at traditional enhancers, but not at super enhancers, is reflective of their activity. n = 3 biologically independent samples. j, A scatter plot showing the changes in mRNA and protein levels for a panel of ES cell transcription factors in response to 6 h ERK signalling. All but NANOG show a degree of persistence into early differentiation. n = 3 biologically independent samples. k, mRNA and protein half-lives of a panel of ES cell transcription factors measured by actinomycin D and cycloheximide treatment, respectively. In brief, cells were treated with actinomycin D (1 μM), or cycloheximide (20 μg ml⁻¹) for time periods ranging from 3–6 h. Samples were collected and processed whereupon the respective half-lives were calculated as in extended data Fig. 1g. Data are mean ± s.e.m., n = 3 biologically independent samples. l, Bar chart showing the overlap of SOX2 and ESRRB binding with the list of enhancers defined in c. Overlap was determined using the UpSet module from Intervene. Data are derived from two biologically independent samples. m, Chow–Ruskey diagrams showing overlap of ERK-regulated tranditional enhancers with changing transcription factor binding, showing that early changes are largely maintained at later time points. Graphs are scaled to maximal changes at 8 h for each transcription factor. Data are derived from two biologically independent samples.

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