Genome-wide chromatin contacts of super-enhancer-associated lncRNA identify LINC01013 as a regulator of fibrosis in the aortic valve

Figures

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  Fig 1. Super-enhancer (SE) in valve interstitial cells (VICs). A) Gene ontology enrichment for SE in VICs (adjusted P-value). B) ATAC-seq signal relative to distance from SE. C) Pie chart showing that 30% of VICs SE intersect with lncRNA (lncRNA-SE); SEs for which no lncRNA is transcribed are defined as regular SE (R-SE). D) The ranking of super-enhancer (ROSE) algorithm indicates that lncRNA-SE rank higher than R-SE in VICs. E) Signal intensity for open chromatin (ATAC-seq) was higher at the core of lncRNA-SE compared with R-SE. F) Box plot revealing higher ATAC-seq signal for lncRNA-SE.

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  Fig 2. CAVD associated lncRNA-SE and 3D mapping. A) Chromosome 6 interaction matrices at the indicated resolutions from H3K27ac HiChIP in VICs. B) Heat map of lncRNA expression in 10 control and 19 calcified aortic valves. C) Venn diagram of genes mapped by chromatin contact with differentially expressed lncRNA and genes encompassed in the GO for extracellular matrix organization displaying that only CCN2 is common to both sets. D) Virtual 4C representations of chromatin interactions at 6q23.2 revealing cell line specific contact between lncRNA-SE, LINC01013 and CCN2 promoter region. Tracks for H3K27ac ChIP-seq and HiChIP (1D), H3K4me1 ChIP-seq, SE-lncRNA, ATAC-seq signal and H3k27ac HiChIP (3D).

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  Fig 3. LINC01013 locus characterization. A) Chromatin conformation capture (3C) assay demonstrating contact between LINC01013 and CCN2 promoter regions (n = 4). B) Quantitative PCR (qPCR) primers design represented by the arrows to amplify specifically the long or the short transcripts of LINC01013 (3 or 2 exons). C) qPCR assay confirming increased RNA expression of LINC01013 in mineralized (n = 27) compared to non-mineralized (n = 17) aortic valves. D) ChIP-qPCR indicating higher H3K27ac signal in calcified valves at the LINC01013 promoter (CTL: n = 4, CAVD: n = 7).

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  Fig 4. LINC01013 regulates the transcription at CCN2. A) Cell fractionation and qPCR showing LINC01013 expression in both the nuclear and cytosolic compartments (n = 3). B-D) LINC01013 (n = 9) (B) and CCN2 (n = 8) (C) RNA levels and CCN2 protein (n = 6) (D) decrease following treatment with LINC01013 ASO. E) RNA immunoprecipitation assay (RIP) indicating an interaction between the LINC01013 and NELF-E protein, western blot demonstrating NELF-E immunoprecipitation (n = 4). F) ChIP-qPCR revealing a rise in NELF-E protein at the CCN2 promoter after treatment with LINC01013 ASO (n = 7). G-H) ChIP-qPCR showing increased RNAPII-Ser5P (n = 8) (G) occupancy at the CCN2 promoter after treatment with the ASO against LINC01013, but unchanged total RNAPII (n = 4) (H).

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  Fig 5. LINC01013 is associated with the TGF-β pathway. A) Protein-protein interaction (PPI) network from calcified aortic valves and B) gene ontology enrichment of the proteins included in the network show enrichment for the TGF-β signaling pathway (adjusted P value).

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  Fig 6. TGF-β mediates LINC01013 activation. A-B) Western blot (n = 6) (A) and pro-collagen-Iα measurements (n = 7) (B) indicate decreased collagen following LINC01013 ASO treatment. C) TGF-β treatment induces LINC01013 expression (n = 8). D-E) TGF-β-mediated increase of CCN2 mRNA expression (n = 8) (D) and protein level (n = 6) (E) rely on LINC01013 expression. F-G) The rise in COL1A1 expression (n = 8) and pro-collagen-Iα secretion (n = 7) following TGF-β treatment relies on LINC01013 expression. H) TGF-β promotes CCN2 expression (n = 7). I) CCN2 ASO (H) negates TGF-β-induced COL1A1 expression level (n = 7). J) Cartoon illustrates the process whereby LINC01013 controls the expression of CCN2; dashed line is a hypothetical process as a direct interaction of LINC01013 with DNA at the CCN2 locus remains to be demonstrated.