The 8q24 cancer risk variant rs6983267 shows long-range interaction with MYC in colorectal cancer

Abstract

An inherited variant on chromosome 8q24, rs6983267, is significantly associated with cancer pathogenesis. We present evidence that the region harboring this variant is a transcriptional enhancer, that the alleles of rs6983267 differentially bind transcription factor 7-like 2 (TCF7L2) and that the risk region physically interacts with the MYC proto-oncogene. These data provide strong support for a biological mechanism underlying this non-protein-coding risk variant.

Figure 1

Enhancer activity at the colon cancer risk locus and allele specific differences in enhancer activity and TCF7L2 binding. a) ChIP assay on Colo205, demonstrating a pattern consistent with enhancer activity. b) luciferase reporter assay demonstrating enhancer activity in two CRC lines. c) a representative luciferase assay showing increased enhancer activity of G over T alleles; performed on a total of 18 clones (9 G and 9 T over 3 days) (P=0.024). d) mass spectrometry plots from Sequenom analysis, showing preferential binding of TCF7L2 to risk allele (G) in immunoprecipitated DNA, as evidenced by differential peak heights (right panel) compared to control input DNA (left panel) (P=1.1×10⁻⁵).

Figure 2

Long-range physical interaction of the CRC risk variant rs6983267 with MYC in colon cancer cell lines. a) Physical map of the region interrogated by 3C, spanning a 395-kb distance with rs6983267 at one end and MYC at the other. The position of the constant fragment containing rs6983267 is marked by a red bar; positions of target fragments are marked by black bars, with an expanded view of MYC target fragments 5–9. b) Graph showing 3C interaction frequency of the constant fragment containing rs6983267 with each target fragment. The results demonstrate decreased interaction at 12 kb, 95 kb (fragments 2 and 3) and 395 kb (fragment 10), and increased interaction frequency in CRC cell lines at the MYC promoter (fragment 4) and the first half of the MYC structural gene (fragments 5 and 6), located ~330 kb away. Distance from rs6983267 (x-axis) is not represented to scale. The y-axis refers to number of molecules in 3C libraries from each interrogated ligation product. Labels above each data point in the graph refer to the name of each target fragment 1–10.