Trait-associated noncoding variant regions affect TBX3 regulation and cardiac conduction
- Department of Medical Biology, Amsterdam Cardiovascular Sciences, Amsterdam University Medical Centers, University of Amsterdam, Netherlands
- Department of Clinical and Experimental Cardiology, Amsterdam Cardiovascular Sciences, Amsterdam University Medical Centers, University of Amsterdam, Netherlands
Figures
Figure 1
Identification of AVCS-specific accessible chromatin within the Tbx3 locus harboring GWAS-associated variant regions.
(A) UCSC genome browser view depicts the genomic location of human TBX3 and neighboring genes. GWAS SNPs associated with PR interval (van Setten et al., 2018) and QRS duration (van der Harst et al., …
Figure 2 with 2 supplements
Validation and characterization of STARR-seq datasets.
(A) Overview of the STARR-seq procedure on the murine/human TBX3/TBX5 locus. BACs spanning the entire murine or human TBX3/TBX5 locus are sheared to fragments of approximately 500–1000 bp. Fragments …
Figure 2—figure supplement 1
Overview of STARR-seq results in the human TBX3/TBX5 locus.
16 BACs spanning the entire TBX3/TBX5 locus were used to capture genomic fragments to generate STARR-seq libraries. Sequencing of the input library (DNA input) reveals an even coverage throughout …
Figure 2—figure supplement 2
Discrepancy between STARR-seq activity and luciferase reporter activity of selected regions.
(A) Overview of the murine Tbx3 locus with ATAC_AVJ, EMERGE and STARR_SG4 data. Fragments selected for luciferase assay, selected either on STARR-seq activity or EMERGE/ATAC peaks are depicted …
Figure 3 with 1 supplement
Approach for the identification of candidate REs potentially affected by GWAS variation.
(A) Active STARR regions within the human TBX3 locus, responding to either SG4- or Tcf-factors. Log2 ratios of the fold change of SG4- or Tcf-stimulated respons to pcDNA is depicted. Blue bars …
Figure 3—figure supplement 1
STARR-seq of the murine Tbx3/Tbx5 locus.
(A) Overview of the murine Tbx3/Tbx5 locus with STARR-seq data tracks for mSTARR_SG4 (SG4-vs-ctrl) and mSTARR_Wnt (Wnt-vs-ctrl). The displayed tracks represent log2 fold changes of enrichment of …
Figure 4 with 1 supplement
Identification of functional variants affecting RE candidate activity within human VR1 and VR2.
(A) Basal luciferase activity of the reference and alternative alleles for each RE candidate in HL-1 and COS-7 cells. Luciferase activities were normalized to the activity of the reference allele …
Figure 4—figure supplement 1
Regulatory activity of RE candidates.
(A) Basal activity of reference alleles of RE candidates in HL1 (light grey bars) and COS7 cells (dark grey bars). Luciferase activity for each RE candidate is normalized to the activity of the …
Figure 5 with 1 supplement
VR1 and VR2 are located within regulatory domains that redundantly drive AVCS expression.
(A) Zoom-in of the human TBX3 locus depicting distal variant region VR1, harboring SNPs for both PR interval and QRS duration, and proximal variant region VR2, harboring SNPs for PR interval. Mouse …
Figure 5—figure supplement 1
BAC 459M16-GFP recapitulates AVCS expression.
(A) Fluorescence of GFP in whole E14.5 459M16-GFP embryo (left) and isolated heart (right). Expression is seen in among others the whisker placodes, limb buds, mammary glands and developing retina. …
Figure 6 with 2 supplements
Genomic deletion of VR1 and VR2 in vivo.
(A) Mouse genomic regions targeted by TALEN- (VR1) or CRISPR-Cas9- (VR2) mediated genome editing. (B) Genotype ratios of ∆VR1 mutant and wildtype born pups, depicted as percentage of total number of …
Figure 6—figure supplement 1
Validation of genomic deletion of VR1 and VR2.
(A) UCSC browser view of the murine Tbx3 locus with the location of VR1 and VR2 depicted. Numbered arrows depict primers used for genotyping. Grey lettered arrows depict the location of regions …
Figure 6—figure supplement 2
Characterization of Tbx3ΔVR1/ΔVR1 and Tbx3ΔVR2/ΔVR2 mice.
(A,B) PR interval and QRS duration in Tbx3+/+ (WT; n = 9), Tbx3ΔVR1/+ (het; n = 12) and Tbx3ΔVR1/ΔVR1 (hom; n = 10) mice measured by surface ECG. No significant difference between the different …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Chlorocebus aethiops) | COS-7 | ATCC | RRID:CVCL_0224 | Fibroblast cell line |
| Cell line (Mus musculus) | HL-1 | William C. Claycomb (Claycomb et al., 1998) | RRID:CVCL_0303 | Cardiomyocyte cell line |
| Genetic reagent (Mus musculus) | Tbx3∆VR1/+; Tbx3∆VR1/∆VR1 | This paper | See Materials and methods, section ‘TALEN/CRISPR/Cas9-mediated genome editing of VR1 and VR2’ | |
| Genetic reagent (Mus musculus) | Tbx3∆VR2/+; Tbx3∆VR2/∆VR2 | This paper | See Materials and methods, section ‘TALEN/CRISPR/Cas9-mediated genome editing of VR1 and VR2’ | |
| Genetic reagent (Mus musculus) | Tbx459M16-GFP/+ | This paper | See Materials and methods, section ‘BAC modification and immunohistochemistry’ | |
| Recombinant DNA reagent | pSTARR-seq_human | Arnold et al., 2013 | Addgene; Plasmid #71509 | |
| Commercial assay or kit | Nextera DNA library prep kit | Illumina | FC-121–1030 | |
| Commercial assay or kit | NEBNext DNA Library Preparation Kit | New England Biolabs | NEB E6000S | |
| Commercial assay or kit | NEBNext Multiplex Oligos for Illumina | New England Biolabs | NEB E7335S | |
| Software, algorithm | EMERGE | van Duijvenboden et al., 2016 |
Additional files
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Supplementary file 1
Supplementary Tables 1—11.
Supplementary Table 1. Transcription factor binding motif enrichment in genomic regions accessible in E14.5 atrioventricular junctions. Transcription factor binding motif enrichment in 67 genomic regions accessible in E14.5 atrioventricular junctions (AVJ) or E14.5 ventricles. Enrichment was compared to enrichment in a number of random control sequences. Enrichment is depicted as total number of regions with the motif and the percentage of the total number of tested regions. P-values depict statistical significance of enrichment. Supplementary Table 2. Transcription factor binding motif enrichment in active STARR-regions. Transcription factor binding motif enrichment in genomic regions within both mouse (A) and human (B) TBX3 locus that respond to Smad/Gata (SG4) or Tcf (Wnt) factors as demonstrated by STARR-seq. Enrichment of binding motifs for Smad/Gata- and Tcf-factors is depicted. Enrichment was compared to enrichment in a number of random control sequences. Enrichment is depicted as total number of regions with the motif and the percentage of the total number of tested regions. P-values depict statistical significance of enrichment. Supplementary Table 3. Murine RE candidates within VR1 and VR2. Murine RE candidates selected based on accessible chromatin in AV junction cardiomyocytes (ATAC_AVJ), EMERGE prediction, or mSTARR_SG4/Wnt activity. Overlap is depicted of candidate regions with ChIP-seq peaks from publicly available datasets for Gata4 (differentiated cardiomyocytes) (Luna-Zurita et al., 2016), Hand2 (E10.5 heart) (Laurent et al., 2017) and H3K27ac (E11.5 heart) (Nord et al., 2013). Supplementary Table 4. Human RE candidates within VR1 and VR2. Human RE candidates selected based on mouse RE homology (mm9 liftover), EMERGE prediction, or hSTARR_SG4/Wnt activity. Regions overlapping GWAS SNPs (SNP id, trait association and respective p-value are listed) were selected for further analysis. Supplementary Table 5. TALEN/CRISPR target sequences for deletion of VR1 and VR2 from the murine genome Supplementary Table 6. qPCR primer sequences Supplementary Table 7. Overview of BACs used for the generation of STARR-seq libraries Supplementary Table 8. Primer sequences for the amplification of active STARR-seq regions for validation by luciferase reporter assay Supplementary Table 9. Primer sequences for the amplification of RE candidate fragments Supplementary Table 10. Mouse functional genomic datasets included in merge as possible predictors Supplementary Table 11. Human functional genomic datasets included in merge as possible predictors.
- https://cdn.elifesciences.org/articles/56697/elife-56697-supp1-v1.docx
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Transparent reporting form
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