A cardiac-related, promoter-proximal, regulatory element shapes chromatin and JAG1 transcription

Figures

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  Figure 1.. Identification of R7 as a promoter-proximal regulatory element with enhancer-like activity at the JAG1 locus. (A) Schematic of the E1b minimal promoter luciferase reporter system. Candidate regions were cloned upstream of E1b. (B) Luciferase activity of candidate regions R1–R10 in AC16 cells. R5 and R7 showed significantly higher activity than the E1b control, with R7 showing the strongest activity. (C) Genomic location of R7 relative to the JAG1 transcription start site (TSS). R7 is located 253 bp upstream of the JAG1 TSS. (D) Schematic of R7 reporter constructs, including forward R7 upstream of E1b (R7), reverse-orientation R7 upstream of E1b (R7_Reverse), and R7 upstream of luciferase without the E1b minimal promoter (R7_ΔE1b). (E) Luciferase activity of the R7 constructs shown in (D). R7_Reverse retained reporter activity, whereas R7_ΔE1b showed activity comparable with the forward R7 construct. (F) Schematic of CRISPR/Cas9-mediated R7 deletion in AC16 cells. (G) RNA-seq read coverage across the JAG1 locus in WT and R7_KO cells. (H) RNA-seq quantification of JAG1 transcript abundance. (I) RT-qPCR analysis of JAG1 mRNA expression. (J) Western blot analysis and quantification…

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  Figure 2.. R7 deletion affects AC16 cell-cycle progression, cell viability, and apoptosis, with partial alleviation by JAG1 restoration or overexpression. (A) RT-qPCR analysis of JAG1 mRNA expression in WT, R7_KO, JAG1-RE, and JAG1-OE cells. JAG1-RE indicates restoration of JAG1 expression to near-WT levels, whereas JAG1-OE indicates higher JAG1 overexpression in R7_KO cells. (B) Representative PI-staining flow-cytometry profiles for cell-cycle analysis. (C) Quantification of cell-cycle phase distribution. (D) Quantification of G2/M-phase cells. R7 deletion increased the G2/M population, which was reduced by JAG1 restoration or overexpression. (E) CCK-8 assay showing relative cell viability over time. (F) Representative Annexin V-FITC/PI flow-cytometry plots for apoptosis analysis. (G) Quantification of apoptotic cells. Data are shown as mean ± SD. Statistical significance was determined by a two-tailed unpaired t test for the indicated comparisons. **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant.

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  Figure S1.. R7 deletion reduces EdU incorporation and cell migration in AC16 cells. (A) Representative EdU incorporation images of WT and R7_KO AC16 cells. Nuclei were stained with Hoechst, and EdU-positive cells were detected by azide 555 staining. Quantification of EdU-positive cells is shown on the right. Scale bars, 50 μm. (B) Representative wound-healing images of WT and R7_KO AC16 cells at 0 and 16 h after scratching. Quantification of wound closure/migration rate is shown on the right. Scale bars, 200 μm. Data are shown as mean ± SD. Statistical significance was determined by a two-tailed unpaired t test. **P < 0.01, ***P < 0.001.

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  Figure 3.. R7 deletion alters local chromatin contacts at the JAG1 locus. (A) In situ Hi-C interaction heatmaps from human right ventricular tissue across a ±500 kb region centered on the JAG1 locus, showing the local TAD containing JAG1. (B) 4C-seq profiles anchored at a viewpoint approximately 17 kb upstream of the JAG1 TSS in WT and R7_KO AC16 cells. Tracks show viewpoint-specific contact signals in WT and R7_KO cells, together with the difference track (Diff), across the JAG1 locus.

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  Figure 4.. R7 deletion reduces local chromatin accessibility and is associated with heart-related biological processes. (A) CTCF ChIP-seq and ATAC-seq profiles across the JAG1 locus in WT and R7_KO AC16 cells. In WT cells, overlapping CTCF-binding and ATAC-seq signals were mainly observed around R7 and within the second intron of JAG1. After R7 deletion, the broader ATAC-seq signal surrounding R7 was reduced, including the adjacent promoter-proximal region and the second intron. (B) Line plots and heatmaps showing ATAC-seq signal intensity within ±3 kb of peak centers in WT and R7_KO AC16 cells. Differentially accessible peaks were grouped as increased (Up), decreased (Down), or non-significant (NoSig). (C) Venn diagram showing the overlap between genes associated with differentially accessible ATAC-seq peaks and differentially expressed genes identified by RNA-seq in R7_KO versus WT cells. (D) Gene Ontology Biological Process (GO-BP) enrichment analysis of the overlapping genes shown in (C). The top 10 significantly enriched GO-BP terms ranked by adjusted P-value are shown, with cardiac-associated terms highlighted in red. Signal intensities are normalized as reads per kilobase per million…

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  Figure S2.. R7 deletion is associated with reduced genome-wide ATAC-seq and H3K27ac signal intensity. (A) Aggregate plot and heatmap of ATAC-seq signal showing a global reduction in chromatin accessibility in R7_KO cells compared with WT cells. (B) Aggregate plot and heatmap of H3K27ac ChIP-seq signal showing reduced H3K27ac signal intensity after R7 deletion.

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  Figure 5.. R7 deletion alters transcription-associated chromatin features at the JAG1 locus. ChIP-seq signal tracks for H3K27ac, H3K4me3, RNA polymerase II (Pol II), and SRF in WT and R7_KO AC16 cells across the JAG1 locus. In WT cells, these transcription-associated signals were enriched around the R7-associated promoter-proximal region and within the second intron of JAG1. After R7 deletion, H3K27ac, H3K4me3, Pol II occupancy, and SRF binding were reduced across this promoter–intron regulatory region. Signal intensities are normalized to RPKM.

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  Figure S3.. Public cardiac developmental ChIP-seq datasets show regulatory chromatin signals near the mouse Jag1 and human JAG1 loci. (A) Public p300 ChIP-seq profiles around the mouse Jag1 locus across the indicated cardiac developmental stages (GSE195901). E indicates embryonic day, and P indicates postnatal day. (B) Public H3K27ac and CTCF ChIP-seq profiles around the human JAG1 locus during iPSC-derived cardiomyocyte differentiation (GSE116862). D indicates differentiation day after initiation of cardiac differentiation. The shaded region indicates the R7-containing promoter-proximal region.