Chiu WT et al. (2014), Genome-wide view of TGFβ/Foxh1 regulation of th...

XB-ART-49634

Development 2014 Dec 01;14123:4537-47. doi: 10.1242/dev.107227.

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Genome-wide view of TGFβ/Foxh1 regulation of the early mesendoderm program.

Chiu WT , Charney Le R , Blitz IL , Fish MB , Li Y , Biesinger J , Xie X , Cho KW .

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Nodal/TGFβ signaling regulates diverse biological responses. By combining RNA-seq on Foxh1 and Nodal signaling loss-of-function embryos with ChIP-seq of Foxh1 and Smad2/3, we report a comprehensive genome-wide interaction between Foxh1 and Smad2/3 in mediating Nodal signaling during vertebrate mesendoderm development. This study significantly increases the total number of Nodal target genes regulated by Foxh1 and Smad2/3, and reinforces the notion that Foxh1-Smad2/3-mediated Nodal signaling directly coordinates the expression of a cohort of genes involved in the control of gene transcription, signaling pathway modulation and tissue morphogenesis during gastrulation. We also show that Foxh1 may function independently of Nodal signaling, in addition to its role as a transcription factor mediating Nodal signaling via Smad2/3. Finally, we propose an evolutionarily conserved interaction between Foxh1 and PouV, a mechanism observed in Pou5f1-mediated regulation of pluripotency in human embryonic stem and epiblast cells.

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Species referenced: Xenopus tropicalis
Genes referenced: agap1 atp1b2 bix1.1 cer1 chrd.2 ctnnb1 cyp27c1 foxa4 foxh1.2 gata4 gata6 gsc hes7.2 hhex ins itpka lefty mcidas mespb mex3c mix1 mixer nodal nodal1 nodal2 nodal3.1 nodal3.2 nodal3.4 odc1 otx2 pcdh8.2 pias4 pitx1 pitx2 pkdcc.1 pkdcc.2 pnhd pou5f3 pou5f3.2 pou5f3.3 psmd6 rasef rpl11 sia1 smad1 smad2 smad3 snai1 sox17a sox3 ssh1 tbx2 tcf12 tfap2a uspl1 vegt ventx2 ventx2.2 wnt11b
???displayArticle.antibodies??? Ctnnb1 Ab9 FLAG Ab1 FLAG Ab3 Foxh1 Ab2 Smad1 Ab11 Smad1 Ab5 Smad2 Ab4 Smad2 Ab8
???displayArticle.morpholinos??? foxh1 MO2 pou5f3.1 MO4 pou5f3.2 MO6 pou5f3.3 MO6

???displayArticle.gses??? GSE53652: Xenbase, NCBI

GSE53653: Xenbase, NCBI
GSE53654: NCBI

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	Fig. 1. Foxh1 is crucial for mesendoderm formation. (A) Distribution of foxh1 transcripts in X. tropicalis early gastrula. Total RNAs from animal, marginal and vegetal fragments were subjected to RT-qPCR analyses. tfap2a, ectodermal marker; t/brachyury, mesodermal marker; sox17a, endodermal marker; rpl11, expressed throughout the embryo. (B) Embryonic lysates from control or foxh1-MO injected embryos were subjected to immunoprecipitation followed by western blot using anti-Foxh1 antibody. Ctnnb1 protein levels in crude embryo lysates are unaffected by the MO. (C) Both Foxh1 morphant and SB431542-treated embryos exhibit gastrulation delay (vegetal views). (D) Early tailbud stage Foxh1 morphants displaying anterior defects and incomplete blastopore closure; SB431542-treated embryos lack distinctive A-P or D-V features. (E) Examination of foxh1 MO effects on different germ-layer markers by RT-qPCR. gsc, chrd, nodal1, nodal3 and mix1 are mesoendodermal markers; sox17a is an endodermal marker; ventx2.1 is a BMP target gene; sia1 is a Wnt target gene. The ectodermally enriched markers sox3 and foxh1.2 are included as non-Foxh1 targets for comparison. (F) Early gastrula cleared lysates were immunoprecipitated using either pan anti-Smad2 or anti-Smad1 polyclonal antibodies covalently coupled to beads. Bound proteins were subjected to western immunoblotting using anti-P-Smad2 or anti-P-Smad1, respectively. After detection, membranes were re-probed with anti-Smad2 or anti-Smad1 to show efficiency of the immunoprecipitations. Arrowhead indicates the P-Smad1 band; the lower band in the P-Smad1 lanes is low level primary antibody release from the beads.
	Fig. 2. Genome-wide survey of Nodal and Foxh1 targets in early gastrulae. (A-D) IGV genome browser views of gsc (A), nodal1 (B), cer1 (C) and hhex (D) genes. Track contents: (1) Foxh1 ChIP-seq; (2) Smad2/3 ChIP-seq; (3) RNA-seq of uninjected control embryos; (4) RNA-seq of foxh1 MO-injected embryos; (5) RNA-seq of mock-treated control embryos; and (6) RNA-seq of SB431542-treated embryos. The numbers in the upper left of each track indicate track heights. (E) Genome-wide analyses of Foxh1 and Smad2/3 peaks. Pie chart distributions of Foxh1 and Smad2/3 peaks across seven defined genomic features are shown. Randomized regions were also analyzed for comparison (right). These were generated by randomly redistributing the intervals of Foxh1 peaks throughout the genome. In parallel, Smad2/3 peak intervals were similarly randomized and showed a nearly identical genomic distribution (data not shown). (F) Distribution of Foxh1 (left) and Smad2/3 (middle) peaks within the intervals of 10 kb upstream of gene 5′ ends, gene bodies and 10 kb downstream of gene 3′ ends. Supplementary material Fig. S7 contains a distribution of randomly selected regions. As individual gene bodies are highly variable in length, we normalized these segments on a 0-100% scale (Zhang et al., 2012).
	Fig. 3. Motif analyses of Foxh1 and Smad2/3 peaks. Foxh1-bound (A) and Smad2/3-bound (B) regions (151 bp centered on peak summits) were retrieved to perform de novo motif analysis. Binding motifs in the sixth column were matched manually (see citations in ‘Reference’ column). Base positions in red in the ‘Regular Expression’ column match these published motifs. References: 1, Zhou et al., 1998; 2, Mason et al., 2010; 3, Chen et al., 2008; 4, Yoon et al., 2011; 5, Wilson et al., 1993; 6, Dennler et al., 1998; 7, Shi et al., 1998; 8, Zawel et al., 1998. While the search by STAMP of TRANSFAC identifies motif 5 in A as HEB, the search by TOMTOM of JASPAR and UniPROBE assigns this to Zic-related proteins.
	Fig. 4. Functional relationships between Foxh1 and Smad2/3 binding, and differential gene expression. (A-C) Kolmogorov-Smirnov (KS) tests for comparing relatedness between TF binding (ChIP-seq data sets) and loss-of-function analyses (RNA-seq data sets). Comparisons between Foxh1 peaks and either Foxh1-regulated targets (A) or Nodal-regulated targets (B). The x-axis represents genes ranked by ascending fold change (bottom scale), depicted as log2 ratios (top scale) between either foxh1 MO or SB431542 and controls. Log2 of ±0.583 corresponds to ±1.5-fold change. The y-axis scale is the running enrichment score (RES). (C) Comparison between Smad2/3 peaks and Nodal-regulated targets. (D) A pie chart that depicts the distributions of Nodal regulated targets that are (1) co-bound by both Foxh1 and Smad2/3 (blue); (2) bound by Foxh1 alone (red); (3) bound by Smad2/3 alone (green); or (4) not bound by either Foxh1 or Smad2/3 (purple). (E) A pie chart that depicts the distributions of 37 genes that are both Foxh1 direct targets (Foxh1 bound and change expression in response to Foxh1 MO) and Nodal targets (SB sensitive): 36 are activated by Foxh1 (blue), whereas only 1 is repressed (red). (F) Among 72 Foxh1 direct targets that are independent of Nodal regulation (SB insensitive), 45 are activated by Foxh1 (blue) and 27 are repressed. (G-J) RT-qPCR validations of the Nodal-independent Foxh1 targets. (G,H) mex3c and Xetro.A02401, from the group of 45 downregulated targets, were further validated by showing downregulation in foxh1 MO-injected embryos, when compared with control, but are unaffected by SB431542. (I,J) ssh1 and rasef were validated by showing upregulation after foxh1 MO injection but are unaffected by SB431542.
	Fig. 5. Functional analysis of PouV genes in regulating Nodal targets. (A) RT-qPCR analysis of pou5f3.1, pou5f3.2 and pou5f3.3 transcript levels at egg, 128-cell, blastula (stage 9), early (stage 10) and mid-gastrula (stage 10.5) stages. Transcript levels were normalized to the pou5f3.1 level in egg RNA. (B) RT-qPCR analysis of pou5f3.1, pou5f3.2 and pou5f3.3 in animal, marginal and vegetal fragments of the gastrula (stage 10-10.5) stage embryo. (C) RT-qPCR of mesendodermal targets in PouV-depleted embryos at early gastrula (stage 10.5) and mid-gastrula (stage 11). (D) ChIP-qPCR strategy to show FLAG-Pou5f3.2 binding to Pou motif-containing regions within Foxh1 peaks. (E) Sequential ChIP-qPCR analyses for Foxh1 and PouV co-binding on Nodal targets. Chromatin from embryos expressing FLAG-Pou5f3.2 was immunoprecipitated using anti-Foxh1 antibody, followed by a second immunoprecipitation using anti-FLAG antibody or anti-IgG antibody (negative control).
	Figure S1. Generation Of Anti-Foxh1 Antibody. Localization of conserved domains in Foxh1 orthologs from Xenopus tropicalis, human and mouse. Blue boxes represent the Forkhead (FH) DNA binding domains; red boxes represent Fast/Foxh1 motifs (FM); purple boxes represents Smad-Interacting Motifs (SIM) (Randall et al., 2004). Green line (amino acids 14-113) indicates N-terminal region of X. tropicalis Foxh1 used for polyclonal antibody generation.
	Figure S2. Phenotypic rescue of Foxh1 Morphant (left panel) by co-injecting Foxh1 specific morpholino together with Foxh1 RNA (right panel).
	Figure S3. Dosage-dependent effect of SB431542 on Nodal-regulated target genes. gsc and chrd showed stronger repression by SB from 10 μM to 100 μM. Whereas ventx2.1 (BMP target), mex3c (nodal independent Foxh1 direct target, see main text), and ef1a1 (house keeping gene), were not affected.
	Figure S4. foxh1 shows negative autoregulation. foxh1 transcript levels are significantly up-regulated (~14 fold) upon foxh1 MO injection, while other Foxh1 targets, chrd, gsc, and otx2, were consistently down-regulated.
	Figure S5. Differential responses of genes in SB431542 treatment and foxh1-MO injection. A. Scatter plot showing expression levels (FPKM) of all genes at early gastrula stage in SB431542-treated compared to control embryos. B. Scatter plot for foxh1-MO injected embryo versus controls. Gray and black lines demarcate expression differences greater than 1.5 fold or 2 fold, respectively. Blue; down-regulated, red; up-regulated genes.
	Figure S6. ChIP-qPCR assays of several genomic regions validating the efficiency of the ChIP protocol. A. ChIP using anti-Foxh1 antibody was able to enrich cis-regulatory regions of the genes gsc, mix1, cer1 and otx2, as opposed to negative control regions in the odc1 and ventx2.1 promoters. B. ChIP using anti-Smad2/3 antibody was able to enrich mix1 and cer1 promoters and pitx2 intron1 regions, as opposed to odc1 and ventx2.1 promoters. PE, proximal element; ARE, activin response element.
	Figure S7. Plot of the distribution of a randomized set of peaks along the lengths of gene bodies, and 10kb upstream and downstream of the genes shows no obvious peak enrichment along these window, as compared to Foxh1’s in Fig. 2F.
	Figure S8. RT-qPCR validation of 6 more genes that are Foxh1 direct targets and are independent of Nodal regulation. A-C. Foxh1-activated targets. D-F. Foxh1 repressed targets.
	Figure S9. Morpholino rescue of Nodal independent Foxh1 direct target genes.
	Figure S10. gsc, nodal2 and mespb expressions were perturbed by PouV MOs injection(also see Fig. 5C), and could be significantly rescued by co-injection of RNAs encoding PouV proteins at both st 10.5 and st 11.
	Figure S11. A list of top 10 motifs discovered under Smad2/3-bound regions by MEME.
	Figure S12. Yoon et al. (2011) published a Smad2/3 ChIP-seq analysis on Xenopus tropicalis early gastrula stage embryos. A comparison between our Smad2/3 ChIP-seq data with these results has been performed using our own analysis pipeline. A. Two hundred one peaks were identified from Yoon et al.’s dataset, whereas 939 peaks were identified from our current data. This disparity may be caused by a number of factors including differences in total reads recovered, read mapping efficiency, ChIP efficiency, etc. Using our analysis pipeline, from a total of ~70M reads, we obtained ~39M uniquely mapped reads. From the Yoon et al.’s dataset of ~15M total reads, ~7M reads mapped uniquely. We used stringent criteria to assign peaks, which included a q-value < 0.05 and the requirement that bound regions are independently identified by two different peak callers, MACS2 and SISSRS.Despite these differences, ~70% (137/201) of peaks identified from Yoon et al.’s data overlapped with our peaks, suggesting high reproduciblity and reliability of both datasets. B. Assigning peaks to genes and comparison of these to differential gene expression data from RNA-seq on control versus SB431542-treated embryos reveals only 4 additional direct Nodal target genes (pkdcc.2, pnhd, wnt11b, and Xetro.A00154 [an unannotated gene that by blast and synteny appears to be mcidas, which is related to geminin]) in the Yoon et al. dataset that are missing from our analysis.

References [+] :

Agius, Endodermal Nodal-related signals and mesoderm induction in Xenopus. 2000, Pubmed, Xenbase