Distinct modes of SMAD2 chromatin binding and remodeling shape the transcriptional response to NODAL/Activin signaling
- The Francis Crick Institute, United Kingdom
- Bioinformatics and Biostatistics, The Francis Crick Institute, United Kingdom
- Advanced Sequencing, The Francis Crick Institute, United Kingdom
Figures
Figure 1 with 4 supplements
The transcriptional response to acute, sustained and chronic NODAL/Activin signaling in P19 cells.
(A) Western blot showing SMAD2 phosphorylation timecourse upon Activin induction in P19 cells. Cells were treated as indicated and lysates were blotted using the antibodies shown. Arrows indicate …
Figure 1—figure supplement 1
Characterization of Activin/NODAL-induced transcription in P19 cells.
(A) Western blot for SMAD2/3 and TUBULIN (loading control) on lysates collected from P19 cells and two other mouse cell lines, C2C12 and EpH4 for comparison. Note that P19 cells express similar …
Figure 1—figure supplement 2
Characterization of the autocrine signal.
(A) Fragments per kilobase of exon per million fragments mapped (FPKM) values as obtained by RNA-seq (average of duplicates) for ligands and receptors involved in Activin/NODAL signaling. Shown are …
Figure 1—figure supplement 3
Activin/NODAL target genes are induced with different kinetics.
Left panel, a heatmap showing the log2FC values for the Activin-treated and untreated samples relative to the SB-431542-treated samples as determined by RNA-seq. Right panel, qPCR validation for …
Figure 1—figure supplement 4
Activin/NODAL target gene expression patterns require continuous Activin/NODAL signaling over a sustained time course.
(A) mRNA stability of Activin/Nodal target genes. Cells were treated with Actinomycin D (6 μM) for the indicated times to inhibit further mRNA synthesis. Levels of mRNA for a subset of target genes …
Figure 2 with 2 supplements
Combining RNA-seq with ChIP-seq for SMAD2 and Pol II leads to the definition of a high confidence dataset.
(A) IGV browser display of the Lefty1/Lefty2 genomic locus, showing tracks and MACS-called peaks for SMAD2 and tracks for Pol II Ser5P. Red lines below are regions for which ChIP-qPCR primers were …
Figure 2—figure supplement 1
Characterization of SMAD2 and Pol II binding in response to Activin signaling.
(A and B) IGV browser displays over the Lefty1/Lefty2 locus (A) or Pmepa1 locus (B) for the 2 replicates of the SMAD2 ChIP-seq experiment to show consistency between duplicates. The tracks and …
Figure 2—figure supplement 2
CRISPR/Cas9-induced deletion of the Lefty1 and Lefty2 upstream SBSs.
(A) Characterization of the P19 clones deleted for Lefty1 and Lefty2 upstream SBSs. Three individual clones are shown for each deletion. Fragments were amplified from genomic DNA by PCR using …
Figure 3
Activin-SMAD2 signaling regulates Pol II via de novo recruitment.
(A) Average metaprofiles for each of two replicates from the 1 hr Activin sample of the ChIP-seq for Ser5P or Ser2P isoforms of Pol II. Normalized read count across all genes is shown. The orange …
Figure 4 with 1 supplement
Localized SMAD2 chromatin binding does not directly correlate with transcription.
(A) Hierarchically-clustered heatmaps for each of the four different kinetic groups of target genes showing log2FC values relative to SB-431542 for gene expression as determined by RNA-seq (left), …
Figure 4—figure supplement 1
IGV browser displays of genes and associated peaks showing differential transcription and SMAD2 binding dynamics.
(A–C) IGV browser displays over the transiently induced gene Smad7 (A), Eomes (B) that is induced with delayed kinetics, and Tbx3 (C) which is a gene containing peaks which are more highly enriched …
Figure 5 with 3 supplements
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 …
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Figure 5—source data 1
H3K27Ac and H3K9Ac values detected in the 1 hr Activin sample.
- https://doi.org/10.7554/eLife.22474.015
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 …
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 …
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 …
Figure 6
Relationship between chromatin state and SMAD2 binding strength.
(A) Hierarchical clustering of average read intensity (Log2) over a 5 kb window surrounding all SMAD2 consensus peak summits for each indicated treatment is shown for H3K27Ac (left panel) and H3K9Ac …
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Figure 6—source data 1
Average read intensity over a 5 kb window surrounding all SMAD2 consensus peak summits for H3K27Ac and H3K9Ac.
- https://doi.org/10.7554/eLife.22474.020
Figure 7 with 4 supplements
FOXH1 is required for SMAD2 recruitment and nucleosome remodeling at a subset of Activin target genes.
(A) P19 cells were transfected with siRNAs directed against either Pou5f1 or Foxh1, along with a non-targeting control (NT). Following signal inhibition or Activin induction, qPCR was performed for …
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Figure 7—source data 1
The position of FOXH1 or POU5F1 motifs relative to the summit of all the consensus peaks.
- https://doi.org/10.7554/eLife.22474.022
Figure 7—figure supplement 1
TF binding sites under SMAD2 peaks.
(A) The top most enriched known and de novo motifs as obtained from a MEME-ChIP analysis on all SMAD2 peaks in the high confidence dataset. From the 478 consensus peaks found surrounding regulated …
Figure 7—figure supplement 2
The role of FOXH1 in SMAD2-mediated transcription.
(A) For experiment shown in Figure 7A, where gene expression following siRNA-mediated knockdown of Foxh1 or Pou5f1 was assessed, qPCR was performed on the untreated samples to determine expression …
Figure 7—figure supplement 3
Characterization of the in-house anti FOXH1 antibody.
Lysates were collected from wild type P19 cells transfected with either non-targeting (NT) or Foxh1 siRNAs and from P19 cells stably expressing MYC-tagged FOXH1 (see Figure 7—figure supplement 2B–D).…
Figure 7—figure supplement 4
The role of FOXH1 in SMAD2-mediated chromatin remodeling.
(A and B) P19 cells were transfected with either non-targeting (NT) or Foxh1 siRNAs, and were then signal inhibited (SB-431542) or stimulated with Activin for 1 hr after SB-431542 washout. The …
Figure 8 with 1 supplement
SMARCA4 is required for SMAD2 binding, nucleosome eviction and histone acetylation at a subset of Activin target genes.
(A) P19 cells were transfected with either non-targeting (NT) or Smarca4 siRNAs. Cells were then signal inhibited (SB-431542) or stimulated with Activin for 1 hr after SB-431542 washout. They were …
Figure 8—figure supplement 1
The role of SMARCA4 in SMAD2-mediated chromatin remodeling.
(A and B) P19 cells were transfected with either non-targeting (NT) or Smarca4 siRNAs, and were then signal inhibited (SB-431542), or stimulated with Activin for 1 hr after SB-431542 washout. The …
Figure 9
A model of dynamic SMAD2-dependent transcription.
The two modes of SMAD2 binding to either acetylated (green diamonds) nucleosome-depleted chromatin or closed, non-acetylated chromatin marked by H3K4Me1 (blue diamonds) upon Activin stimulation from …
Author response image 1
24 hr and 48 hr of Activin treatment partially recapitulate the untreated condition for pathway activation and induction of target genes.
(A) Western blot for pSMAD2, SMAD2/3 and TUBULIN (loading control) on lysates collected from cells treated overnight with SB-431542, followed by washout and stimulation for the indicated times with …
Author response image 2
Author response image 3
A) P19 cells express low level of SMAD3.Western blot for SMAD2/3 and TUBULIN (loading control) on lysates collected from three different mouse cell lines, the myoblast line, C2C12, the mammary epithelial cell line, EpH4 and P19 cells.
Note that P19 cells express similar level of SMAD2 compared to the others cell lines, but undetectable levels of SMAD3. B) Characterization of the in house FOXH1 antibody. Lysates were collected …
Additional files
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Supplementary file 1
RNA-seq and ChIP-seq datasets.
- https://doi.org/10.7554/eLife.22474.030
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Supplementary file 2
Gene set enrichment analysis
- https://doi.org/10.7554/eLife.22474.031
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Supplementary file 3
List of reagents used in the study.
- https://doi.org/10.7554/eLife.22474.032
