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Fig. 3 The variant rs1821848 regulates the transcriptional activity of ARID3B (A) Epigenetic annotation of regions surrounding rs1821848 and rs3826041 in craniofacial tissues. The H3K4me1, H3K4me3, and H3K27ac profiles were generated across four Carnegie stages (CS13, CS14, CS15, and CS17). Each dataset includes replicates from either primary ChIP-seq (upper panels) or imputed ChIP-seq (lower panels). (B and C) Luciferase assay using allele-specific plasmids with the putative promoter sequence flanking rs1821848 or the enhancer sequence flanking rs3826041. The pGL3-basic or pGL4-promoter luciferase reporter vector was used as a baseline control. Luciferase activity was normalized to the Renilla signal (n = 3). (D and E) H3K4me3and H3K27ac bind to the region flanking rs1821848, as determined by chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP‒qPCR). The enrichment was quantified relative to the amount of input DNA, and IgG was used as a negative control. (F) Schematic diagram showing the procedure of rs1821848 activation via the CRISPR activation system. (G and H) ARID3B expression was measured via real-time qPCR and western blotting. sgRNA #1, sgRNA #2, and sgRNA #3 were designed to target rs1821848, and nontargeting sgNC was used as a negative control. (I and J) ARID3B-overexpressing vectors (pcDNA3.1-ARID3B) with either the risk or non-risk allele of rs1821848 were synthesized and transfected into HEPM and HEK293 cell lines. The expression level of ARID3B was detected by RT‒qPCR (I) and western blotting (J). The empty vector (pcDNA3.1) was used as a baseline control. Data are presented as mean ± SEM (n = 3). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; ns, not significant.