Prostate Cancer Risk Locus at 8q24 as a Regulatory Hub by Physical Interactions with Multiple Genomic Loci across the Genome. M. Du1, T. Yuan1, K. Schilter1, R. Dittmar1, A. Mackinnon1, X. Huang1, M. Tschannen2, E. Worthey2, H. Jacob2, S. Xia1,3, J. Gao4, L. Tillmans5, Y. Lu6, P. Liu6, S. Thibodeau5, L. wang1 1) Pathology, Medical college of wisconsin, Milwaukee, WI; 2) Human Molecular Genetics Center, Medical College of Wisconsin, WI 53226; 3) Department of Oncology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China, 430030; 4) Beijing 3H Medical Technology Co. Ltd., Beijing, China, 100176; 5) Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 55905; 6) Department of Physiology, Medical College of Wisconsin, Milwaukee, WI 53226.

   Chromosome 8q24 locus contains regulatory variants that modulate genetic risk to various cancers including prostate cancer (PC). However, the biological mechanism underlying this regulation is not well understood. Due to lack of annotated genes, it is hypothesized that the 8q24 risk locus may affect other genes through long range chromatin interaction. To thoroughly survey the possible chromatin interactions, we developed a chromosome conformation capture (3C)-based multiple target sequencing (3C-MTS) technology to examine the 8q24 risk locus and its target loci in five prostate-derived cell lines and one lymphoblastoid cell line. By targeted enrichment of DNA fragments defined by 77 EcoRI sites covering all three PC risk regions (~302kb), we successfully identified multiple genomic regions that showed frequent intra- or inter-chromosomal interactions with the risk locus. We observed the most frequent inter-chromosomal interaction between CD96 intron 2 at 3q13 and 8q24 risk region 1. Subsequent 3C-qPCR assays and Fluorescence In Situ Hybridization (FISH) analysis confirmed the inter-chromosomal interaction. The second most common interaction occurred at MYC locus. We identified at least five interaction hot spots within the predicted functional regulatory elements at the 8q24 risk locus. We also found intra-chromosomal interaction genes PVT1, FAM84B, GSDMC and inter-chromosomal interaction gene CXorf36 in all six cell lines. Other gene regions appeared to be cell line-specific such as RRP12 in LNCaP, USP14 in DU-145 and SMIN3 in LCL. To predict potential functional consequences of these frequent contacts, we applied GREAT (Genomic Regions Enrichment of Annotations Tool) in the suggestive interaction regions and found that the 8q24 functional domains more likely interacted with genomic regions containing genes enriched in critical pathways such as Wnt signaling and promoter motifs such as E2F1 and TCF3. This result suggests that the risk locus may function as a regulatory hub by physical interactions with multiple genes important for prostate carcinogenesis. Using the 3C-MTS technology, for the first time, we are able to systematically examine the complete 8q24 risk locus and its potential target genes at genome-wide level. Further understanding genetic effect and biological mechanism of these chromatin interactions will shed light on the newly discovered regulatory role of the risk locus in PC etiology and progression.

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