Extended Data Fig. 2: ERK regulates RNAPII and TBP binding to control nascent transcription independently of PRC2 activity. | Nature

Extended Data Fig. 2: ERK regulates RNAPII and TBP binding to control nascent transcription independently of PRC2 activity.

From: Dynamic lineage priming is driven via direct enhancer regulation by ERK

Extended Data Fig. 2

a, Quantification of the changes in intronic reads of the RNA-seq time course shown in Extended Data Fig. 1n, showing rapid repression of nascent transcription of a panel of transcription factors. The curves represent a cubic spline interpolation of the discrete points (using the MATLAB function cscvn). The maximal fold changes are for the 4 h period tested and are taken from the fitted spline curves. n = 3 biologically independent samples. b, RT–qPCR analysis of nascent (intronic) RNA for the indicated genes in the presence or absence of the translation inhibitor cycloheximide. Cells were pre-treated with cycloheximide (20 µg ml−1) for 30 min before 4OHT addition. Data are mean ± s.e.m., n = 3 biologically independent samples. c, Box plots showing H3K27me3 deposition across gene bodies of ERK induced or repressed genes following 2 h ERK stimulation. No significant changes in response to ERK were observed (significance was tested using two-permutation tests with 2,000 simulations). n = 62,426 (global), n = 882 (induced) and n = 1,362 (repressed). Data are derived from two biologically independent samples. Boxes show median, first and third quartiles, whiskers show 1.5× IQR and outliers are shown as black dots. d, Immunofluorescence analysis showing H3K27me3 staining in cRAF-ERT2-expressing cells treated for 24 h with DMSO or the EZH2 inhibitor EPZ-6438 (EZHi), and subsequently stimulated for 2 h with 4OHT. Data are representative of three biologically independent samples. e, RT–qPCR analysis of a panel of induced and repressed genes showing little dependency on PRC2 enzymatic activity (based on EPZ-6438 treatment) for either induced or repressed genes other than Myc and cFos, both of which showed slightly increased induction in cells depleted of H3K27me3. Two-tailed Students t-test, *P = 0.02, **P = 0.001; Data are mean ± s.e.m., n = 3 biologically independent samples. f, A schematic depicting the strategy to define changes in RNAPII binding. g, A scatter plot showing the correlation of RNAPII binding in 227 differentially bound genes. The differentially bound genes were identified by counting the number of RNAPII ChIP reads that uniquely mapped to gene bodies in the 0 h, 8 h and 8 h + 2 h MEKi conditions. Pairwise comparisons using DESeq2 identified 180 genes with significant changes in RNAPII binding between 8 h and 8 h + 2 h MEKi and 126 between 8 h and 0 h. Concatenation of the two gene lists resulted in 227 genes bodies that show a positive correlation in RNAPII binding (Spearman correlation R = 0.79, P < 2.2 × 10−16) induced by ERK and reversed by inhibition of the pathway. n = 3 (0 h), n = 3 (8 h) and n = 2 (8 h + 2 h MEKi) biologically independent samples. h, Binding profiles of TBP to the TSSs of genes with changing RNAPII (left) and ChIP–seq tracks for the TSS of both Nanog and Spry4 (right). Data are derived from two biologically independent samples. i, A heat map showing the changes in nascent RNA expression of ERK-regulated genes associated with super enhancers (|log2(FC)| > 1, adj. P ≤ 0.01). n = 3 biologically independent samples. Super enhancer gene association was performed using GREAT (http://great.stanford.edu/).

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