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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-6gib007Δ12/Δ12 mutant zebrafish normalized with actb. The scar-6gib007Δ12/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.
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