scar-6 lncRNA transcript is ubiquitously expressed and nuclear enriched. (A) Image showing agarose gel electrophoresis of the PCR product derived from scar-6 lncRNA and ?-actin, amplified from cDNA synthesized using both oligo dT(dT) and random hexamer primers (RH). (B) Coding potentiality scores were calculated using CPC2 for scar-6 and other lncRNA and protein-coding genes (f10 and prozb). (C) Western blot of HSP90, ?-actin and H4 histone to confirm the purity of sub-cellular fractions of zebrafish cells. (D) The bar plot represents the relative abundance of the of scar-6, f10 and prozb in different subcellular fractions quantified using qRT-PCR. The scar-6 and prozb exhibit enrichment in the nucleus fraction and f10 shows equal enrichment in cytoplasm and nucleus. Data from 3 different experiments were plotted as relative abundance percentages ± SEM. (E) The expression profile of human SCAR-6 lncRNA across various tissues, as shown in the GTEx v8 database, is presented in transcripts per million (TPM). The box plot illustrates the data distribution, with the median indicated by a line inside each box, and the 25th and 75th percentiles represented by the lower and upper edges of the box, respectively. Outliers, defined as data points beyond 1.5 times the interquartile range, are also displayed.

CRISPR-Cas9 mediated mutant generation of scar-6 lncRNA gene. (A) DNA-PAGE gel illustrating the results of the heteroduplex mobility assay (HMA) conducted on the scar-6 targeted region in F1 scar-6 mutant adult zebrafish, with genotypes identified by in-dels. Extra bands in the PAGE gel represent heteroduplex templates formed due to heterozygosity in the target region. The red asterisk denotes heterozygous mutant animals. (B) Representative image showing blood vessels of 3 dpf zebrafish progeny derived from wild-type gib004Tg(fli1a:EGFP;gata1a:DsRed) and scar-6gib007?12/?12 zebrafish. 2.5× magnification; scale bar = 500 ?m. (C) Representative image showing secondary structure of scar-6 RNA under wildtype and 12-bp edited condition. The highlighted region represent change in the structure of lncRNA.

Knockdown of scar-6 shows no significant phenotype. (A) Schematic of antisense splice blocking morpholino oligo targeting the exon 1 splicing junction of scar-6 lncRNA. (B) Relative fold change expression of scar-6 upon of knockdown with splice blocking morpholino in zebrafish at 3 dpf. Data from 3 independent biological replicates plotted as mean fold change ± standard deviation; *P < 0.05 (two-tailed unpaired t-test). (C) Box and whisker plot representing the percentage of animals with haemorrhage phenotype in zebrafish embryos injected with 250 ?M of morpholino. Data from 4 independent experiments are represented by boxes indicating the interquartile range (25th to 75th percentiles), with the horizontal line within each box denoting the median. Whiskers extend to 1.5 times the interquartile range to define the minimum and maximum values, while individual points represent data from each experiment.: ns, not significant (two-tailed unpaired t-test). (D) Representative image of gib004Tg(fli1a:EGFP;gata1a:DsRed) zebrafish injected with different concentration of morpholino at 3 dpf. Red arrowhead denotes hemorrhage in the animals. 2.5× magnification, scale bar = 500 ?m. (E) Relative fold change expression of f10 and prozb upon knockdown of scar-6 lncRNA with splice blocking morpholino in zebrafish. Data from 3 independent biological replicates plotted as mean fold change ± standard deviation; ns, not significant (two-tailed unpaired t-test). (F) Coagulation assay plot calculating the time of occlusion (s) in different individual zebrafish in control and 500uM morpholino injected zebrafish. Each point represents the occlusion time of individual zebrafish segregated based on genotype; *P < 0.05 (Mann-Whitney U).

CRISPRi of scar-6 locus exhibits haemorrhage phenotype. (A) Representation of CRISPR-dCas9-KRAB mediated inhibition of TF binding on scar-6 locus. (B) Representative image showing fluorescence for blood (gata1a:DsRed) in the cranial region of 3 dpf zebrafish and o-dianisidine stating of RBC blood cells in control and dCas-9-KRAB + sgscar-6 injected zebrafish. The black and white arrowhead denotes hemorrhage in the animals. 4× magnification; scale bar = 200 ?m. (C) Box and whisker plot representing the percentage of animals exhibiting haemorrhage phenotype in control and dCas-9-KRAB + sgscar-6 injected zebrafish. Data from 3 independent biological replicates are represented by boxes indicating the interquartile range (25th to 75th percentiles), with the horizontal line within each box denoting the median. Whiskers extend to 1.5 times the interquartile range to define the minimum and maximum values, while individual points represent data from each experiment; *p < 0.05 (two-tailed unpaired t-test).

Sub-TAD looping mediated enhancer-promoter interaction of scar-6/prozb locus. (A) Hi-C heatmap representation of Human SCAR-6 locus in HepG2 at 10 kb resolution from ENCODE database (ENCODE Project Consortium et al, 2020; Wang et al, 2018) and zebrafish scar-6 locus in brain tissue at 5 kb resolution from (Yang et al, 2020). (B) UCSC genome browser snapshot of data for CTCF binding peaks at 24 hpf of zebrafish for at scar-6 and prozb locus (Pérez-Rico et al, 2020). (C) Bar plot representing ChIP-qPCR quantifying fold enrichment using CTCF antibody for scar-6 locus in wild type and scar-6gib007?12/?12 mutant zebrafish. Data from 3 independent biological replicates plotted as mean fold enrichment ± standard deviation; *P < 0.05, **P < 0.01 (two-tailed unpaired t-test). (D) Schematic and western blot of DNA-pulldown assay was performed using streptavidin tagged scar-6 gene DNA in zebrafish, and immuno blotting was done using prdm14 antibody. N = 3.

Syntenic cardiovascular conserved associated region - 6 (scar-6) is a novel conserved lncRNA gene.

(A) Schematic of pipeline used for subset selection of syntenic lncRNAs with its neighboring protein-coding genes associated in the cardiovascular system. Overview of conserved syntenic block depiction of scar-6 locus between human and zebrafish. (B, C) UCSC genome browser snapshot of scar-6 locus in zebrafish and human. Strand-specific PCR and 3’RACE confirmed the transcript to be antisense to f10 in zebrafish. (D) Expression profile of scar-6 transcript (in TPM) across 8 different developmental stages of zebrafish using publically available RNA-seq data. (E) Expression profile of scar-6 transcript (in TPM) across 12 different tissues using publically available RNA-seq data. (F) Whole mount in situ hybridization expression analysis of scar-6, f10, and prozb transcripts of zebrafish in 3 dpf embryo. Sense probes were used as controls for f10 and prozb. No probe control was used for scar-6. Black arrow head denotes expression in liver and heart. 4× magnification scale bar = 500 μm. Source data are available online for this figure.

scar-6^{gib007Δ12/Δ12} mutant exhibits permeability defect with haemorrhage phenotype.

(A) CRISPR-Cas9 mediated editing of scar-6 locus at exon 2 resulting in a stable 12-bp deletion zebrafish line named scar-6^gib007Δ12. (B) Chromatogram representing the sequence of wild type and homozygous mutant of scar-6^gib007Δ12. (C) Schematic representing in-cross of scar-6^gib007Δ12/+ zebrafish. (D) Pie chart representing mutation segregation of the progeny from in-cross of scar-6^gib007Δ12/+ zebrafish. The genotype of the progeny followed the Mendelian ratio. (E) Box and whisker plot representing the percentage of animals from the in-cross of scar-6^gib007Δ12/+ zebrafish exhibiting haemorrhage phenotype compared to wild-type control animals. Data from 3 independent in-cross of scar-6^gib007Δ12/+ zebrafish are represented by boxes indicating the interquartile range (25th to 75th percentiles), with the horizontal line within each box denoting the median. Whiskers extend to 1.5 times the interquartile range to define the minimum and maximum values, while individual points represent data from each experiment; **p = 0.0023 (two-tailed unpaired t-test). (F) Representative image showing the cranial region of 3 dpf zebrafish wild-type gib004Tg(fli1a:EGFP;gata1a:DsRed) and scar-6^{gib007Δ12/Δ12} zebrafish. The white arrowhead denotes haemorrhage in the animals. 40× magnification ; scale bar = 100 μm. (G) Bar plot representation of hemorrhage phenotype associated with genotyped scar-6^gib007Δ12/+ in-crossed zebrafish animals. Data from 3 independent in-cross of scar-6^gib007Δ12/+ zebrafish experiment plotted as mean percentage ± standard deviation. ****p-value ≤ 0.0001 (unpaired two-tail t-test). (H) Representative image showing intersegmental vessels (ISV) of 3 dpf wild-type and scar-6^{gib007Δ12/Δ12} zebrafish injected with Evans blue dye. 20× magnification; scale bar = 100 μm. (I) Quantitative analysis of vascular permeability in wild-type and scar-6^{gib007Δ12/Δ12} zebrafish injected with Evans blue dye. Data of 10 different ROI from 4 individual zebrafish from each wild-type and scar-6^{gib007Δ12/Δ12} zebrafish plotted as mean ± standard deviation; ****p-value ≤ 0.0001 (Mann–Whitney U). (J) Bar plot representing relative fold change in expression levels of f10, scar-6 and prozb between wild-type and scar-6^{gib007Δ12/Δ12} zebrafish. The prozb expression levels were 8-fold increase in the scar-6^{gib007Δ12/Δ12} zebrafish compared to the wild-type. Data from 3 independent biological replicates plotted as mean fold change ± standard deviation; ***p-value ≤ 0.001 (unpaired two-tail t-test). Source data are available online for this figure.

The scar-6 locus exhibits a functional role in the hemostatic process.

(A) Coagulation assay plot calculating the time of occlusion (s) in different individual zebrafish in control and progeny of in-cross scar-6^gib007Δ12/+ zebrafish performed blinded of the genotype of the animals. Combined data from 3 independent experiments with each point representing the occlusion time of individual zebrafish segregated based on genotype: ****p < 0.0001 (Mann-Whitney U). (B) Coagulation assay plot calculating the time of occlusion (s) in different individual zebrafish in control and different concentration of scar-6 IVT (100, 200, 500 ng/μl) injected embryos performed blinded of the injected concentration. Combined data from 3 independent experiments with each point representing the occlusion time of individual zebrafish segregated based on injected concentration; ****p < 0.0001 (Mann–Whitney U). (C) Survival curve representing survival percentage of closely monitored control (n = 52) and scar-6^{gib007Δ12/Δ12} (n = 24) zebrafish. p < 0.0001 (log-rank test). (D, E) Representative image at 30 dpf zebrafish of control and scar-6^{gib007Δ12/Δ12} group closely monitored for survival. The red arrowhead indicates haemorrhage and cranial edema in scar-6^{gib007Δ12/Δ12} mutant animals. 1.5× magnification scale bar = 200 μm. (F) Coagulation assay plot calculating the time of occlusion (s) in different individual zebrafish in control, in-cross progeny of scar-6^gib007Δ12/+, in-cross progeny of scar-6^gib007Δ12/+ injected with WT scar-6 IVT RNA(100 ng/μl) and in-cross progeny of scar-6^gib007Δ12/+ injected with scar-6^gib007Δ12 IVT RNA(100 ng/μl) performed blinded. Each point representing the occlusion time of individual zebrafish segregated based on injected concentration; ns, not significant, **p < 0.01, ****p < 0.0001 (Mann-Whitney U). (G) Representative image of the cranial region of zebrafish at 3 dpf of control wild-type, in-cross progeny of scar-6^gib007Δ12/+ and in-cross progeny of scar-6^gib007Δ12/+ injected with WT scar-6 IVT RNA(100 ng/μl). The white arrowhead denotes haemorrhage in the animals. 2.5× magnification scale bar = 200 μm. (H) Box and whisker plot representing the percentage of animals from control wild-type, in-cross of scar-6^gib007Δ12/+ zebrafish and, in-cross of scar-6^gib007Δ12/+ + scar-6 IVT RNA (100 ng/μl) exhibiting haemorrhage phenotype. Data from 3 independent experiments are represented by boxes indicating the interquartile range (25th to 75th percentiles), with the horizontal line within each box denoting the median. Whiskers extend to 1.5 times the interquartile range to define the minimum and maximum values, while individual points represent data from each experiment; ns, not significant (two-tailed unpaired t-test). (I) Relative fold change expression of scar-6, f10, and prozb when compared between control wild-type, scar-6^{gib007Δ12/Δ12} animals and scar-6^{gib007Δ12/Δ12} + scar-6 IVT RNA (100 ng/μl) animals. Data from 3 independent biological replicates plotted as mean fold change ± standard deviation; ns, not significant; **p < 0.01, ***p-value ≤ 0.001 (two-tailed unpaired t-test). Source data are available online for this figure.

The scar-6 locus exhibits cis-regulatory enhancer signature.

(A) UCSC genome browser snapshot of zebrafish scar-6 locus with chromatin annotation marks from danio-code data across 5 developmental stages. (B) UCSC genome browser snapshot of human SCAR-6 locus with regulatory signature track from ENCODE data, chromatin annotation of 8 different cell lines from Epigenome roadmap project and enhancer data from GeneHancer database. (C) Representative image of enhancer assay for human and zebrafish scar-6 locus in zebrafish injected with empty E1b-GFP, scar-6-E1b-GFP and human SCAR-6-E1b-GFP. Transient expression was observed in the F₀ animals in the cardiac region and in case of human SCAR-6 some expression was also observed in the trunk region. The red arrowhead indicates eGFP signal in the animal. 2.5× magnification; scale bar = 500 μm. (D) Bar plot of luciferase enhancer assay for human and zebrafish scar-6 locus in HHL-17; HEK297T; HUVEC/hTERT; and HepG2 cell line transfected with zebrafish scar-6-psi-Chek2 and human SCAR-6-psi-Chek2 plasmid. Enhancer function was observed in HUVEC/hTERT; and HepG2 cell line. Data from 3 independent biological replicates plotted as mean fold change ± standard deviation; ns, not significant, *p < 0.05, **p < 0.01, ****p < 0.0001 (two-tailed unpaired t-test). (E) Representative image of 3C experimental for scar-6 and prozb locus and bar plot representing relative fold change between wild type and scar-6^{gib007Δ12/Δ12} mutant zebrafish normalized with actb. The scar-6^{gib007Δ12/Δ12} zebrafish showed an increase in the scar-6/prozb looping. Data from 3 independent biological replicates plotted as mean fold change ± SEM. *p < 0.05 (two-tailed unpaired t-test). Source data are available online for this figure.

The scar-6 elncRNA gene epigentically regulates expression of prozb via prdm14-PRC2 complex.

(A) UCSC genome browser screenshot representing JASPER 2022 track with 12 bp deletion of scar-6 locus. (B) Bar plot representing ChIP-qPCR quantification in percentage input using prdm14 antibody as a target and mock as a control for scar-6 locus in wild type and scar-6^{gib007Δ12/Δ12} mutant zebrafish. Data from 3 independent biological replicates plotted as mean fold change ± standard deviation; ns, not significant, *p < 0.05 (two-tailed unpaired t-test). (C) Box and whiskers plot representing percentage methylation profile of proximal CpG island of scar-6 using targeted bisulfite sequencing of wild type and scar-6^{gib007Δ12/Δ12} mutant zebrafish per cytosine. Data from 4 independent biological replicates plotted with line at mean. (D) Bar plot representing fold enrichment of scar-6 elncRNA upon RNA-immunoprecipitation of prdm14 and Suz12 followed by qPCR of scar-6 elncRNA. The scar-6 elncRNA shows enrichment in the binding with prdm14 and Suz12. Data from 3 independent biological replicates plotted as mean fold change ± standard deviation; ***p-value ≤ 0.001, ****p < 0.0001 (two-tailed unpaired t-test). (E) Bar plot representing ChIP-qPCR quantification in percentage input using prdm14 antibody as a target and mock as a control for scar-6 locus in zebrafish injected with scar-6 elncRNA morpholino (scar-6 MO) and scrambled morpholino (scr MO). Data from 3 independent biological replicates plotted as mean fold change ± standard deviation; **p < 0.01, ***p < 0.001, ****p < 0.0001 (two-tailed unpaired t-test). (F) Schematic representation of prdm14-PCR-2 complex binding at scar-6 locus and being stabalized by the scar-6 elncRNA produced from the scar-6 elncRNA gene. Source data are available online for this figure.

Prozb mediated endothelial cells activation via the PAR2-NF-kb pathway in scar-6^{gib007Δ12/Δ12} mutants.

(A) Representative image showing fluorescence for blood (gata1a:DsRed) in the cranial region of 3 dpf zebrafish and o-dianisidine stating of RBC blood cells in control and prozb IVT RNA (80 ng/μl) injected zebrafish. Black and white arrowhead denotes hemmorage in the animals. 4× magnification scale bar = 200 μm. (B) Stacked bar plot representing the phenotypic percentage of animals with haemorrhage and trunk deformities observed in control and prozb IVT RNA (80 ng/μl) injected zebrafish. Data from 3 independent biological replicates plotted as mean percentage ± standard deviation. **p < 0.01, ***p < 0.001 (unpaired two-tail t-test). (C) Bar plot representing relative fold change in expression of different PAR receptors (PAR1, PAR2a/b and PAR3) when compared between wild type and scar-6^{gib007Δ12/Δ12} mutant zebrafish. The PAR2a/b receptors expression levels were ~2 fold upregulated in scar-6^{gib007Δ12/Δ12} mutant zebrafish. Data from 3 independent biological replicates plotted as mean fold change ± standard deviation; ns, not significant, **p < 0.01, ****p < 0.0001 (two-tailed unpaired t-test). (D) Schematic representation of in-vivo PAR2 protease assay performed using eGFP-PAR2 domain-luciferase construct upon injecting into wild type and scar-6^{gib007Δ12/Δ12} mutant zebrafish followed by western blotting with luciferase antibody. The bar plot represents the quantification of cleavage activity in the wild type and scar-6^{gib007Δ12/Δ12} mutant zebrafish using the immunoblot quantification data. Data from 3 independent biological replicates plotted as mean ± standard deviation; **p < 0.01 (two-tailed unpaired t-test). (E) Western blot of NF-kB in wild type and scar-6^{gib007Δ12/Δ12} mutant zebrafish. The bar plot represents the quantification of the western blot from 3 independent biological replicates plotted as mean fold change ± standard deviation. *p < 0.05 (two-tailed unpaired t-test). (F) Bar plot representing relative fold change expression of vcam1a, vcam1b, icam1, inos2a, and inos2b when compared between wild type and scar-6^{gib007Δ12/Δ12} mutant zebrafish. Data from 3 independent biological replicates plotted as mean fold change ± standard deviation; ns, not significant, *p < 0.05, **p < 0.01 (two-tailed unpaired t-test). (G) Hypothetical schematic of prozb mediated endothelial cell activation through activation of PAR2-NF-κB pathway leading to upregulation of surface adhesion molecules (Icam1 and Vcam1) and downregulation of iNOS2a causing vascular dysfunction and haemorrhage in zebrafish. Source data are available online for this figure.

Epigenetic regulation of prozb through scar-6 locus is important for vascular function and hemostatic process.

Hypothetical schematic of scar-6 locus in wild-type zebrafish, where scar-6 elncRNA and prdm14-PRC2 complex hypermethylated the proximal CpG island and inhibits CTCF binding. In scar-6^{gib007Δ12/Δ12} mutant zebrafish. The 12 bp deletion affects the binding of the scar-6 elncRNA and prdm14-PRC2 complex leading to a partial change in methylation. This change allows the CTCF occupancy at the locus further mediating sub-TAD looping of the enhancer-promoter of prozb. This causes upregulation of prozb leading to endothelial cell activation via the PAR2-NF-κB pathway and causing vascular dysfunction leading to hemorrhage.