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The prostate cancer risk variant rs55958994 regulates multiple gene expression through extreme long-range chromatin interaction to control tumor progression

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Science Advances  17 Jul 2019:
Vol. 5, no. 7, eaaw6710
DOI: 10.1126/sciadv.aaw6710
  • Fig. 1 The chromatin locus containing rs55958994 is a functional enhancer in PCa cells.

    (A) H3K27ac ChIP-seq data from PCa cell lines are shown. (B) Luciferase reporter activity in 22Rv1, C4-2B, and LNCaP cells transfected with luciferase reporters. Ctrl, luciferase reporter without the inserted enhancer; C, luciferase reporter with the rs55958994-associated enhancer region with the nonrisk allele “(C)”; T, luciferase reporter with the rs55958994-associated enhancer region with the risk allele (T). Data represent means ± SEM of three independent experiments. ***P < 0.001. (C) Soft agar colony formation assays comparing wild-type (WT) 22Rv1 cells and three enhancer-deleted cell lines [knockouts (KOs) 1 to 3]. Quantification is at right. Data represent means ± SEM of three independent experiments. ***P < 0.001. (D) Transwell assays in WT 22Rv1 cells and three enhancer-deleted cell lines (KOs 1 to 3). Cells that had migrated to lower chambers were stained with 0.1% crystal violet. Quantification is at right. Data represent means ± SEM of three independent experiments. ***P < 0.001. (E) Fluorescence-activated cell sorting (FACS) analysis of the cell surface markers CD44 and CD24 in WT 22Rv1 cells and three enhancer-deleted cell lines (KOs 1 to 3). FITC, fluorescein isothiocyanate; PE, phycoerythrin.

  • Fig. 2 Transcriptomic changes following the deletion of the rs55958994-associated enhancer.

    (A) Comparison of gene expression in enhancer KO and WT cell lines. The heat map and dendrogram show clustering of differentially expressed genes in three KO and four WT 22Rv1 cell lines. (B) Annotation enrichment analysis of genes significantly down-regulated in KO compared to WT cells (fold change < 1 or 1.5 and adjusted P < 0.1). (C) Annotation enrichment analysis of genes significantly up-regulated in KO compared to WT conditions (fold change > 1.5 and adjusted P < 0.1). GTPase, guanosine triphosphatase.

  • Fig. 3 The rs55958994 risk enhancer is a hub for intrachromosomal and interchromosomal interactions.

    (A) Circos plot showing genome-wide interactions indicated by curves extending from the bait locus; intrachromosomal and interchromosomal interactions are in red and blue, respectively. Interactions reproducible in at least two biological replicates are shown. (B) A comparison of gene expression fold changes based on RNA-seq and the corresponding Capture-C signal counts are shown. A total of 2936 genomic loci with differential expression (adjusted P < 0.1) are shown on the plot. Among them, 219 loci with both reproducible interaction (Capture-C support > 1) and significant expression change between KO and WT (adjusted P < 0.1 and log 2 fold change > 1 or <−1) are shown in blue. (C) qRT-PCR analysis of mRNA (independent mRNA library) levels of potential target genes of the risk enhancer based on analysis in enhancer-deleted (KO) cells. Data represent means ± SEM of three independent experiments. ***P < 0.001, **P < 0.01 compared with corresponding KO cells.

  • Fig. 4 Rescue of tumorigenic phenotypes in enhancer-deleted 22Rv1 cells following the re-expression of candidate enhancer targets.

    Quantification of soft agar colony formation assays of WT 22Rv1 cells, three enhancer-deleted cell lines (KOs 1 to 3), and KO cells re-expressing CNTN1 (A), ITGA5 (B), or CDH23 (C). Data represent means ± SEM of three independent experiments. ***P < 0.001. (D) Quantification of Transwell assays of WT 22RVv1 cells, three enhancer-deleted cell lines (KOs 1 to 3), and KO cells re-expressing CNTN1. Data represent means ± SEM of three independent experiments. ***P < 0.001. (E) The percentage of CD44+CD24 cells in WT 22Rv1 cells, three enhancer-deleted cell lines (KOs 1 to 3), and CNTN1 re-expressed KO cells.

  • Fig. 5 Risk allele of SNP rs55958994 in 22Rv1 cells affected the expression levels of CNTN1 and CDH23.

    (A) Site-directed mutation by CRSPR-Cas9 and sequencing of SNP rs55958994 nonrisk (C) and risk (T) alleles in 22Rv1cells. (B) CDH23 and CNTN1 expressions are increased in cells with risk (T) allele of rs55958994 compared with WT 22Rv1 cells with (C) allele of rs55958994, while no significant alteration of ITGA5 expression was detected in cells with risk (T) allele of rs55958994. Data represent means ± SEM of three independent experiments. **P < 0.01. n.s., not significant.

  • Fig. 6 SNP rs55958994 is a functional SNP in 22Rv1 cells.

    (A) Soft agar colony formation assays comparing WT 22Rv1 cells with (C) allele of rs55958994 and 22Rv1 cells with (T) allele of rs55958994. Quantification is at right. Data represent means ± SEM of three independent experiments. *P < 0.05. (B) Transwell assays in WT 22Rv1 cells with (C) allele of rs55958994 and 22Rv1 cells with (T) allele of rs55958994. Cells that had migrated to lower chambers were stained with 0.1% crystal violet. Quantification is at right. Data represent means ± SEM of three independent experiments. *P < 0.05. (C) FACS analysis of the cell surface markers CD44 and CD24 in WT 22Rv1 cells with (C) allele of rs55958994 and in 22Rv1 cells with (T) allele of rs55958994.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/7/eaaw6710/DC1

    Fig. S1. Fine-mapped PCa risk SNPs in 12q13 region.

    Fig. S2. SNP rs55958994 is associated with enhancer activity in cervical, liver, and breast cancer cell lines.

    Fig. S3. CRISPR-Cas9–mediated deletion of the rs55958994-associated enhancer.

    Fig. S4. Re-expression of target genes in enhancer-deleted PCa cells promotes cancer initiation, growth, and progression.

    Fig. S5. Re-expression of KRT8 in enhancer-deleted PCa cells did not rescue the defect of soft agar colony formation and invasive migration ability and the decrease in CSC population.

    Fig. S6. Validation of target gene expression in enhancer KO cells.

    Fig. S7. 3C-PCR analysis confirms interaction of the rs55958994-associated enhancer and target gene loci.

    Fig. S8. Comparison of transcriptomic changes following deletion of the rs55958994-associated enhancer and mutation of rs55958994.

    Table S1. Guide RNAs for CRISPR-Cas9–mediated deletions.

    Table S2. Primers used in qRT-PCR experiment.

    Table S3. Primers for 3C-PCR assay.

    Table S4. Guide RNAs for CRISPR-Cas9–mediated SNP editing.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Fine-mapped PCa risk SNPs in 12q13 region.
    • Fig. S2. SNP rs55958994 is associated with enhancer activity in cervical, liver, and breast cancer cell lines.
    • Fig. S3. CRISPR-Cas9–mediated deletion of the rs55958994-associated enhancer.
    • Fig. S4. Re-expression of target genes in enhancer-deleted PCa cells promotes cancer initiation, growth, and progression.
    • Fig. S5. Re-expression of KRT8 in enhancer-deleted PCa cells did not rescue the defect of soft agar colony formation and invasive migration ability and the decrease in CSC population.
    • Fig. S6. Validation of target gene expression in enhancer KO cells.
    • Fig. S7. 3C-PCR analysis confirms interaction of the rs55958994-associated enhancer and target gene loci.
    • Fig. S8. Comparison of transcriptomic changes following deletion of the rs55958994-associated enhancer and mutation of rs55958994.
    • Table S1. Guide RNAs for CRISPR-Cas9–mediated deletions.
    • Table S2. Primers used in qRT-PCR experiment.
    • Table S3. Primers for 3C-PCR assay.
    • Table S4. Guide RNAs for CRISPR-Cas9–mediated SNP editing.

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