Fig. 3: The enhancer variant rs73613962 modulates PRMT7 expression by regulating enhancer activity. | Nature Communications

Fig. 3: The enhancer variant rs73613962 modulates PRMT7 expression by regulating enhancer activity.

From: An enhancer variant at 16q22.1 predisposes to hepatocellular carcinoma via regulating PRMT7 expression

Fig. 3

a, b The association between the genotypes of rs73613962 and PRMT7 expression through linear regression analysis in GTEx liver tissues (a, the center white line and the black box in the plot indicate the median and the first to third quartile, respectively) and TCGA LIHC (b, the center white dot, the black limit, and the whisker represent the median, the first to third quartile, and the 95% confidence interval, respectively). c Gene editing of the region surrounding rs73613962 by CRISPR-Cas9. The above panel shows the basic principle of the Cas9 assay. The Cas9 protein binds to the neighboring sequence of the rs73613962 site guided by small guide RNAs (sgRNAs) targeting the upstream and downstream of this locus to induce the double-stranded breaks of these regions in the PRMT7 gene. qPCR detection of relative PRMT7 expression upon the controls without any mutation in these regions (Control_1, 2), and the mutations being edited in these regions (Mutation_1, 2, 3) are shown in the bottom panel (P = 0.0001, P = 0.0003, and P = 0.0231 in Mutation_1, 2, or 3 compared to Control_1, respectively). d, g Illustration of the dCas9 assay. The above diagram shows the CRISPR-interference (CRISPRi) assay. DCas9-KRAB binds to the upstream and downstream of rs73613962 guided by sgRNAs to inhibit PRMT7 expression (d). The bottom diagram shows the CRISPR-activation (CRISPRa) assay to activate the expression of PRMT7 guided by sgRNAs (g). e, f RNA level of PRMT7 detected by qPCR in the CRISPRi assay in the QGY-7703 (e, P = 0.0038 in dCas9-KRAB_gRNA (K_gRNA) compared to dCas9-KRAB_Control (K_Ctrl)) and HepG2 (f, P = 0.0011 in K_gRNA compared to K_Ctrl) cell lines. h, i RNA level of PRMT7 detected by qPCR in the CRISPRa assay in the QGY-7703 (h, P = 0.0007 in dCAS9-VP48_gRNA (V_gRNA) compared to dCAS9-VP48_Control (V_Ctrl)) and HepG2 (i, P = 0.0300 in V_gRNA compared to V_Ctrl) cell lines. Values are expressed as the mean ± SD, n = 3 in c, e, f, h, and i. *, **, and *** mean P-values less than 0.05, 0.01, and 0.001, respectively (two-sided student’ t-test).

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