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. Author manuscript; available in PMC: 2022 Jun 1.
Published in final edited form as: Nature. 2021 Nov 24;600(7890):731–736. doi: 10.1038/s41586-021-04116-8

Extended Data Figure 6. Endogenous enhancer connectome of COLO320-DM MYC ecDNA amplicon and effect of promoter sequence, cis enhancers, and BET inhibition on episomal reporter activation.

Extended Data Figure 6.

(a) Top to bottom: COLO320-DM H3K27ac HiChIP contact map (KR-normalized read counts, 10 kb resolution), reconstructed COLO320-DM amplicon, H3K27ac ChIP-seq signal, BRD4 ChIP-seq signal, WGS coverage, interaction profile of PVT1 and MYC promoters at 10kb resolution with FitHiChIP loops shown below, colored by adjusted p-value. Active elements identified by scATAC and overlapping H3K27ac HiChIP contacts named by genomic distance to MYC start site: −1132E, −1087E, −679E, −655E, −401E, −328E, −85E. (b) Comparison of HiChIP matrix normalization for COLO320-DM H3K27ac HiChIP at 10kb resolution. HiChIP signal is robust to different normalization methods. (c) Quantification of NanoLuc luciferase signal for plasmids with PVT1p-, minp-, or MYCp-driven NanoLuc reporter expression. Luciferase signal was calculated by normalizing NanoLuc readings to Firefly readings. Bar plot shows mean ± SEM. P values were calculated using a two-sided student’s t-test (n=3 biological replicates). (d) Violin plots showing mean fluorescence intensities and signal sizes of the NanoLuc reporter RNA in PVT1p-reporter and minp-reporter transfected cells. P-values were calculated a two-sided Wilcoxon test. (e) Schematic of PVT1 promoter-driven luciferase reporter plasmid with a cis-enhancer. Details of cis-enhancer are in Methods. (f) Bar plot showing luciferase signal driven by PVT1p, MYCp or the constitutive TKp with or without a cis-enhancer (mean ± SEM). All values are normalized to the corresponding promoter-only construct without a cis-enhancer. P values were calculated using a two-sided student’s t-test (n=3 biological replicates). (g) Dot plots showing fold change in luciferase signal (Firefly-normalized NanoLuc signal) in JQ1-treated over DMSO-treated COLO320-DM and COLO320-HSR cells after transfection with the PVT1p or the MYCp plasmid with or without a cis-enhancer. P values were calculated using a two-sided student’s t-test (n=3 biological replicates).