Figure 5. Changes in chromatin landscape around SBSs in response to signaling.

(A) IGV browser displays for the Lefty1 and Pou5f1 loci displaying ChIP-seq tracks for SMAD2, H3K9Ac, H3K27Ac and total histone H3 for the indicated treatments. For the SMAD2 ChIP-seq the MACS-called peaks are also shown. (B) Metaprofiles denoting coverage of total histone H3, H3K27Ac and H3K9Ac in a 5 kb window surrounding the average summit across SMAD2 consensus peaks. Each line within a plot traces the normalized read depth of H3 or acetylated H3 for each treatment. The left panels show metaprofiles for all 478 SMAD2 peaks, while the other panels only show those regions found near genes which are induced from an ‘on’ baseline or from an ‘off’ baseline. The right panels show metaprofiles for the subset of SMAD2 peaks which is associated with genes induced from an ‘off’ baseline and show changes in H3Ac enrichment from a low baseline as defined in Figure 6D. (C) Correlation plots showing normalized read depth (Log₂) (left graph) or log₂FC relative to SB-431542 (right graph) for H3K27Ac and H3K9Ac values, detected in the 1 hr Activin sample. (D) ChIP-PCR on the nucleosomes flanking the Lefty1 SBS (A) for H3K18Ac and H3K23Ac in P19 cells treated as indicated. Plotted are the means and SEM of three independent experiments. (E) ChIP-PCR showing enrichment of both SMAD2 and EP300 at the Lefty1 SBS in P19s treated as indicated. Plotted are the means and SEM of four independent experiments. (F) FAIRE-ChIP was performed on SB-431542, 1 hr Activin or 8 hr Activin-treated P19 cells. Soluble chromatin fractions were assayed for enrichment of the specific regions indicated which are highlighted in the IGV browser displays in panel (A) and Figure 2—figure supplement 1 and Figure 5—figure supplement 1. A representative experiment in shown (means ± SD). See Figure 5—figure supplement 1B for the averages of the three experiments and the statistical analyses. In D and E, n.s., not significant; ^**** corresponds to a p value of < 0.0001; ^*** corresponds to a p value of < 0.001 and ^** corresponds to a p value of < 0.01.

DOI: http://dx.doi.org/10.7554/eLife.22474.014

Figure 5—figure supplement 1. Validation of induced acetylation and FAIRE at and around SBSs.

(A) ChIP-PCR for either H3K27Ac, H3K9Ac or Histone H3 was performed on P19 cells treated as indicated. Regions corresponding to the SBS around Lefty1, Pmepa1, Trh (two peaks), Pou5f1 and Smad7 were amplified, as well as the regions directly flanking the peak where the acetylated nucleosomes are found. These are denoted as 5’ and 3’ to SBS. For Lefty1 and Smad7, enrichment of H3Ac was also determined at the TSS and in the region between the peak and TSS (intergenic). All amplified genomic regions are denoted in the IGV browser displays in Figure 5A and Figure 5—figure supplement 2. Enrichment of the modifications is also shown on the Gapdh TSS as a control (Bottom panels). A representative experiment is shown (means ± SD). (B) P19 cells were treated as indicated. The nucleosomes at the selected SBSs were assayed for FAIRE-PCR. Plotted are the means and SEM of three independent experiments. n.s., not significant; **** corresponds to a p value of < 0.0001; *** corresponds to a p value of < 0.001; ** corresponds to a p value of < 0.01.

DOI: http://dx.doi.org/10.7554/eLife.22474.016

Figure 5—figure supplement 2. IGV browser displays of genes and associated peaks showing changes in chromatin landscape.

IGV browser displays over the Pmepa1, Trh and Smad7 loci. Displayed are ChIP-seq tracks for SMAD2, H3K9Ac, H3K27Ac and total histone H3 in P19 cells which were treated as indicated. MACS-called peaks for SMAD2 are also shown, and the regions amplified in ChIP-PCR (Figure 5—figure supplement 1) are denoted below each panel.

DOI: http://dx.doi.org/10.7554/eLife.22474.017

Figure 5—figure supplement 3. Correlation between histone H3 acetylation changes and gene expression.

Hierarchically-clustered heatmaps for each of the four different kinetic groups of target genes. Color intensity denotes log2FC values relative to SB-431542 for either gene expression as determined by RNA-seq (left), or mean normalized read depth for H3K9Ac (middle) or H3K27Ac (right) over a 5 kb window surrounding the annotated TSS of each target gene. Untr, untreated.

DOI: http://dx.doi.org/10.7554/eLife.22474.018