Fig. 3: Functional evaluation of the MNLP and correlated SNP variants by CRISPRi reveals the L allele regulatory effect on IRX4 level and L allele knock-in in LNCaP cells generates an active enhancer for IRX4. | Nature Communications

Fig. 3: Functional evaluation of the MNLP and correlated SNP variants by CRISPRi reveals the L allele regulatory effect on IRX4 level and L allele knock-in in LNCaP cells generates an active enhancer for IRX4.

From: A biallelic multiple nucleotide length polymorphism explains functional causality at 5p15.33 prostate cancer risk locus

Fig. 3: Functional evaluation of the MNLP and correlated SNP variants by CRISPRi reveals the L allele regulatory effect on IRX4 level and L allele knock-in in LNCaP cells generates an active enhancer for IRX4.The alternative text for this image may have been generated using AI.

a Annotated 5p15.33 PCa risk region and IRX4 genomic locus (hg19, chr5:1870000-1905000). Bottom track (red) shows IRX4 genomic region, middle track (orange) marks the correlated SNPs and MNLP positions. Upper track (gray) shows the mRNA expression levels of IRX4 after CRISPRi, mediated inhibition of indicated variants; each dot represents a unique gRNA with black dots indicating IRX4 expression in LNCaP (homozygous for S allele) and red dots indicate expression in VCaP (homozygous for L allele). To control CRISPRi inhibition effect on the IRX4 expression, three IRX4 promoter targeting gRNA were used as a positive control. As a negative control, three guide RNA were used ~3 kb upstream at the promoter region. Guide RNA locations and individual measurements of all three replicates are listed in Supplementary Fig. S3. b CRISPRi experiment in PC-3/AR PCa cell line demonstrates the L allele regulatory role in IRX4 expression. Suppressing the L allele by specific gRNAs showing ~ two-fold decrease on the IRX4 level. The suppression experiments were independently repeated three times (n = 3), and the average values are shown on the bars, while individual values are represented by dots. Error bars indicate the standard deviation of the three biological replicates. A two-sided t-test was used to calculate statistical significance. c Genome editing of LNCaP cells using HDR leading to knock-in of the L allele variant. H3K27Ac CHIP-seq at IRX4 genomic locus (hg19, chr5:1872000-1902000) in two individual knock-in clones and parental LNCaP cells. Light blue highlighted are shows the enhancer position. Knock-in 1 and Knock-in 2 clones both carrying a single copy of the L allele confer high epigenetic activity both measured by ATAC-seq and H2K27ac ChIP-seq in this region compared to the parental cell line (lack of L allele). d IRX4 mRNA expression levels in LNCaP parental and two knock-in clones. Knock-in 1 and Knock-in 2 clones both carrying a single copy of the L allele showing 2.4- and 3-fold IRX4 level increase compared to the parental cell line (lack of L allele). Bars represent the IRX4 expression levels using three technical replicates (individual dots) from each clone and control samples. A two-sided t-test was used to calculate statistical significance, error bars represent standard deviation.

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