1. The prostate cancer risk variant rs regulates multiple gene expression through extreme long-range chromatin interaction to control tumor progression
by Yuyang Qian, Lei Zhang, Mingyang Cai, Hongxia Li, Heming Xu, Hongzhen Yang, Zhongfang Zhao, Suhn Kyong Rhie, Peggy J. Farnham, Jiandang Shi, and Wange Lu
Science
Volume 5(7):eaaw6710
July 17, 2019
Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).

2. Fig. 1 The chromatin locus containing rs55958994 is a functional enhancer in PCa cells.
The chromatin locus containing rs 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 rs associated enhancer region with the nonrisk allele “(C)”; T, luciferase reporter with the rs associated enhancer region with the risk allele (T). Data represent means ± SEM of three independent experiments. ***P < (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 < (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 < (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.
Yuyang Qian et al. Sci Adv 2019;5:eaaw6710
Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).

3. Fig. 2 Transcriptomic changes following the deletion of the rs55958994-associated enhancer.
Transcriptomic changes following the deletion of the rs 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.
Yuyang Qian et al. Sci Adv 2019;5:eaaw6710
Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).

4. Fig. 3 The rs55958994 risk enhancer is a hub for intrachromosomal and interchromosomal interactions.
The rs 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.
Yuyang Qian et al. Sci Adv 2019;5:eaaw6710
Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).

5. Fig. 4 Rescue of tumorigenic phenotypes in enhancer-deleted 22Rv1 cells following the re-expression of candidate enhancer targets.
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 < (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 < (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.
Yuyang Qian et al. Sci Adv 2019;5:eaaw6710
Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).

6. Fig. 5 Risk allele of SNP rs in 22Rv1 cells affected the expression levels of CNTN1 and CDH23.
Risk allele of SNP rs in 22Rv1 cells affected the expression levels of CNTN1 and CDH23. (A) Site-directed mutation by CRSPR-Cas9 and sequencing of SNP rs nonrisk (C) and risk (T) alleles in 22Rv1cells. (B) CDH23 and CNTN1 expressions are increased in cells with risk (T) allele of rs compared with WT 22Rv1 cells with (C) allele of rs , while no significant alteration of ITGA5 expression was detected in cells with risk (T) allele of rs Data represent means ± SEM of three independent experiments. **P < n.s., not significant.
Yuyang Qian et al. Sci Adv 2019;5:eaaw6710
Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).

7. Fig. 6 SNP rs55958994 is a functional SNP in 22Rv1 cells.
SNP rs is a functional SNP in 22Rv1 cells. (A) Soft agar colony formation assays comparing WT 22Rv1 cells with (C) allele of rs and 22Rv1 cells with (T) allele of rs Quantification is at right. Data represent means ± SEM of three independent experiments. *P < (B) Transwell assays in WT 22Rv1 cells with (C) allele of rs and 22Rv1 cells with (T) allele of rs 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 < (C) FACS analysis of the cell surface markers CD44 and CD24 in WT 22Rv1 cells with (C) allele of rs and in 22Rv1 cells with (T) allele of rs
Yuyang Qian et al. Sci Adv 2019;5:eaaw6710
Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).