Figure 5: The region encompassing rs55705857 acts as a MYC enhancer and rs55705857-G allele increases this activity.

(a) Chromatin Topological Domain structure and epigenetic and transcriptional features of the region around rs55705857 and MYC. Normalized Hi–C interaction frequency displayed as a two-dimensional heat map showing the topologic domain that includes the glioma risk allele rs55705857 as well as the MYC oncogene (Adapted from²¹). The frequency of interaction between two 40-kb genomic regions is indicated by the color intensity at their diagonal intersection. (b) Epigenetic and transcriptional features, obtained from UCSC Genome Browser, are as follows 1) The genomic region encompassing rs55705857 shows evidence of a high sequence conservation between evolutionarily-divergent species, 2) rs55705857 resides in DNaseI hypersensitivity site, detected in 3 cell-lines (one of which being hippocampal astrocyte cell line HA-h), 3) This genomic region displays H3K27ac and H3K4me1 signals, both of which distinguish active enhancers from inactive/poised enhancer elements, detected in H1-derived neuronal progenitor cells, 4) An RNA polymerase II binding site, as indicated by Pol2 ChIA-PET signal in HeLaS3 cells, overlaps the genomic region encompassing rs55705857, suggesting active-transcription, 5) This genomic region also displays a balanced short RNA transcription, characteristic of enhancer-associated RNAs (eRNAs), which is a hallmark of active enhancers. (small RNA-seq data). (c) The left panel shows the organization of luciferase assay constructs. HEK293 cells were transfected with Renilla luciferase plasmid pRL-CMV and one of three firefly luciferase plasmids: MYC promoter alone, MYC promoter plus enhancer region with rs55705857-A allele or MYC promoter plus enhancer region with rs55705857-G allele. As a control, MYC promoter was replaced with the CDX2 promoter and assayed in parallel. After 48–72 hours, luciferase expression was measured via Dual-Glo Luciferase Assay System. Average of 3 experiments are shown (errors bars indicate S.E.M). y-axis shows normalized luciferase activity in logarithmic scale. (*) Indicates a statistical significance level of p < 0.05.