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. 2019 Nov 20;8:e49921. doi: 10.7554/eLife.49921

Figure 3. Foxf is required for cardiopharyngeal-specific chromatin accessibility.

(A) Motif accessibility between libraries from chromVAR (Schep et al., 2017). Motifs were obtained and associated with Ciona transcription factors (TFs) as described in the Materials and methods. Deviations were computed for FGF signaling-dependent regions at 10 hpf and B7.5 replicates at 6 and 10 hpf. We calculated the differential accessibility of all motifs between conditions and time points. Only the most significant motif is shown for each TF. (B) Expression of transcription factors over time compared to enrichment of corresponding TF motifs in condition specific peak sets. log2(odds ratio) values (log2(OR), see Materials and methods) are shown for motifs that are significantly enriched in a peak set (one-tailed hypergeometric test, FDR < 0.05). Only TFs expressed in the B7.5 lineage are shown. (C) Differential expression of Foxf target genes (DE) vs. differential chromatin accessibility (DA) in FoxfCRISPR. ρ is the Spearman correlation of expression and accessibility for DA regions associated with DE genes. (D) Association between expression of Foxf target genes and accessibility of proximal regions which were both TVC-specific and closed in FoxfCRISPR as in Figure 2C. (E) A 3.6 kb region on chromosome 14 displaying expression profiles of RNA-seq and chromatin accessibility profiles of ATAC-seq normalized tag count. Foxf core binding site (GTAAACA) is displayed as blue line. The boxed region indicates a newly identified TVC-specific enhancer in Hand locus. Red arrow indicates a TVC-specific enhancer showing closed chromatin in FoxfCRISPR ATAC-seq. (F) Enhancer-driven in vivo reporter expression (green) of tested ATAC-seq peaks. TVCs marked with Mesp>H2B::mCherry (red). Numbers indicate observed/total of half-embryos scored. Experiment performed in biological replicates. Scale bar, 30 μm. Gene expression data for 6 hpf and ‘FGF-MAPK perturbation 10 hpf’ (Christiaen et al., 2008), and from 8 to 20 hpf (Razy-Krajka et al., 2014) were previously published.

Figure 3.

Figure 3—figure supplement 1. Conservation of DNA-recognition motifs of Foxf proteins.

Figure 3—figure supplement 1.

(A) Conservation of DNA-recognition motifs among Homo sapiens Hnf-3/forkhead and tunicates and amphioxus Foxf proteins. The protein sequences were retrieved from NCBI (www.ncbi.nlm.nih.gov), Ensembl (www.ensembl.org) and Aniseed (www.aniseed.cnrs.fr/aniseed) databases using Foxf of Ciona robusta as query sequence (see Supplementary file 1). Alignment of conserved amino-acid residues in Foxf orthologues was obtained using Clustal Omega (MView algorithm) (McWilliam et al., 2013). (B) Amino acid sequence alignment of DNA-recognition motif between Homo sapiens Hnf-3/forkhead (AAT74628.1) and Ciona robusta Foxf (NP_001071710.1). Asterisks indicate the structurally equivalenced residues between HFN3γ and GH5 as in Clark et al. (1993). (C) Amino acid sequence alignment in HFN3γ (107-223) and GH5 based on structural alignment and secondary structural elements indicated as rectangle for ɑ-helices and arrows for β-strand (structurally equivalenced residues are indicated in upper case) adapted from Clark et al. (1993).
Figure 3—figure supplement 2. General characterization of FoxfCRISPR.

Figure 3—figure supplement 2.

(A) ATAC-seq peaks displaying Foxf and Gata4/5/6 loci with expression profiles from RNA-seq and chromatin accessibility profiles from ATAC-seq (normalized by total sequencing depth). Known TVC-specific enhancers of Foxf (Beh et al., 2007) and Gata4/5/6 (Woznica et al., 2012) are in the boxed regions. (B) Differential gene expression determined by RNA-seq analysis of FoxfCRISPR vs. ControlCRISPR at 10 hpf. Colored dots represent genes that are differentially expressed (FDR < 0.05) in FoxfCRISPR vs. ControlCRISPR at 10 hpf. Genes not differentially expressed are shown in gray. (C) GSEA showing normalized enrichment score (NES) of gene sets in peaks ranked by difference in accessibility between FoxfCRISPR vs. ControlCRISPR at 10 hpf. Dark gray bars indicate significant enrichment. Light gray bars are not significant (FDR < 0.05). (D) Change in accessibility between ATAC-seq samples. Colored dots represent peaks associated with genes that are differentially accessible (FDR < 0.05 and |log2(FC)| > 0.5). (E) Association between expression and accessibility of Foxf up-regulated genes and peaks which were both closed in FoxfCRISPR and associated with TVC-specific peaks as in Figure 2C. Gene expression data for 6 hpf and ‘FGF-MAPK perturbation 10 hpf’ (Christiaen et al., 2008), and from 8 to 20 hpf (Razy-Krajka et al., 2014) were previously published.
Figure 3—figure supplement 3. Conserved binding motifs in TVC-specific Nk4/Nkx2-5 enhancer.

Figure 3—figure supplement 3.

(A) Conservation of TVC-accessible elements of Nk4/Nk2-5 locus between Ciona robusta and Ciona savignyi. The transcript model is shown in black. Conservation score was calculated using mVISTA (http://genome.lbl.gov/vista/mvista/submit.shtml). Pink peaks indicate conserved non-coding sequences (> 65% identity per 80 bp). (B) Alignment of DNA region corresponding to ‘KhC8.2201’ peak between Ciona savignyi and Ciona robusta (bottom sequence). Only the core nucleotides are shown for putative Forkhead and Gata binding sites. (C) Match scores for TF binding motifs present in the boxed region (~1.2 Kb) including the I-intronic element of both Ciona robusta and Ciona savignyi Nk4/Nk2-5 transcripts. Only the highest scoring match for each TF is shown.
Figure 3—figure supplement 4. Foxf loss-of-function (FoxfCRISPR) caused closing of TVC-specific enhancers.

Figure 3—figure supplement 4.

(A–E) Foxf target gene loci chromatin accessibility profiles showing ATAC-seq (normalized by total sequencing depth) in the indicated conditions. Foxf core binding sites (GTAAACA) are displayed as blue lines. The transcript model is shown in black.
Figure 3—figure supplement 5. Conserved binding motifs in TVC-specific Hand enhancer.

Figure 3—figure supplement 5.

(A) Conservation of TVC-accessible elements of Hand locus between Ciona robusta and Ciona savignyi. The transcript model is shown in black. Conservation score was calculated using mVISTA (http://genome.lbl.gov/vista/mvista/submit.shtml). Violet peaks indicate conserved sequences (> 65% identity per 80 bp). (B) Alignment of DNA region corresponding to ‘KhC14.806’ peak between Ciona robusta and Ciona savignyi (bottom sequence). Putative Forkhead binding sites are evidenced in green box (only the core nucleotides are shown). (C) Match scores for TF binding motifs present in the boxed region (~0.8 Kb) upstream of the coding ATG in both Ciona robusta and Ciona savignyi Hand transcripts. Only the highest scoring match is shown for each TF.
Figure 3—figure supplement 6. Conserved binding motifs in Smurf enhancer.

Figure 3—figure supplement 6.

(A) Conservation of TVC-accessible elements of Smurf locus between Ciona robusta and Ciona savignyi. The transcript model is shown in black. Conservation score was calculated using mVISTA (http://genome.lbl.gov/vista/mvista/submit.shtml). Pink peaks indicate conserved non-coding sequences (> 65% identity per 80 bp). (B) Alignment of DNA region corresponding to ‘KhC4.606’ peak between Ciona robusta and Ciona savignyi (bottom sequence). Only the core nucleotides are shown for putative Forkhead and GATA binding sites. (C) Match scores for TF binding motifs present in the boxed region including the II-intronic element and exon III-IV of both Ciona robusta and Ciona savignyi Smurf transcripts. Only the highest scoring match is shown for each TF.
Figure 3—figure supplement 7. Accessible elements annotated to Foxf target genes.

Figure 3—figure supplement 7.

(A) Intersection of elements associated with Foxf target genes at 10 hpf (see Material and methods) and differentially accessible elements. Peaks closed in FoxfCRISPR are peaks more accessible in ControlCRISPR vs. FoxfCRISPR at 10 hpf. A two-tailed binomial test was performed on each intersection against the null hypothesis that the intersection’s constituent sets are independent (see Statistics under Materials and methods). (B) Subset of (A) showing only elements associated with Foxf target genes. Peaks closed at 6 hpf are peaks more accessible in Mesp> LacZ 10 hpf vs. Mesp > LacZ 6 hpf. (C) ATAC-seq peaks (113 regions) associated with Foxf target genes showing differential accessibility (FDR < 0.05, see Material and methods) over time. The log2(FC)for each time point is versus the average (avg) of all control samples.