Fig. 4: Hypermethylation replaces CTCF binding sites in PFA ependymoma. | Nature Communications

Fig. 4: Hypermethylation replaces CTCF binding sites in PFA ependymoma.

From: 3D genome mapping identifies subgroup-specific chromosome conformations and tumor-dependency genes in ependymoma

Fig. 4: Hypermethylation replaces CTCF binding sites in PFA ependymoma.The alternative text for this image may have been generated using AI.

a Proposed mechanism of epigenetic oncogene activation in PFA ependymoma tumors. Top: The CTCF insulator is replaced by DNA methylation allowing enhancer to activate oncogene. Below: The oncogene is separated from an enhancer by topological barrier. Created with BioRender.com. b The volcano plot of differential CTCF binding sites between PFA and ZFTA ependymoma tumors (min p-value: 0.1). CTCF binding sites significantly hypermethylated in PFA are marked in orange (min q-value: 0.05). c Comparison of CTCF binding strength (CTCF ChIP-seq, x-axis, min p-value 0.1, min fold change: 0.5) and DNA methylation (WGBS, y-axis, min q-value: 0.05, min difference: 0.1) at differential CTCF binding sites between PFA and ZFTA ependymoma tumors. d Heatmap of WGBS-derived DNA methylation at the 300 most significant differential CTCF binding sites in three PFA (left) vs. three ZFTA (right) ependymoma tumors. e Genome browser visualization of PFA ependymoma-specific DNA loops that associate two PFA enhancers (E1 and E2) with the ARL4C gene. Tracks for RNA-seq, H3K27ac, CTCF and Hi-C derived DNA loops are obtained by merging PFA (9EP1,9EP9, 7EP18) or ZFTA (11EP22, 4EP53, 7EP41), respectively. f WGBS-derived DNA methylation and CTCF ChIP-seq data from PFA and ZFTA tumors at PFA-specific hypermethylated CTCF loci. g ARL4C expression is positively correlated with activity of enhancer E1 (chr2:237763494 − 237764993) in ependymoma tumors (n = 24). h Genetic (CRISPR-Cas9) time-course inhibition of ARL4C in PFA cells (EPD210FH) using a control sgRNA and two individual sgRNA constructs. All constructs are GFP tagged and GFP positive cells are sorted by FACS. Normalized data represent mean from n = 2 independent experiments. i Expanded view of the CTCF motif targeted by CRISPR-Cas9: two sgRNAs and protospacer adjacent motif (PAM) direct Cas9 nuclease to the motif. Sequencing of target site demonstrates the formation of indels (insertion or deletions). j qPCR reveals increased ARL4C expression up on targeting CTCF by CRISPR-Cas9 in ZFTA cells. Results are normalized to control gRNA and data represent mean from n = 2 independent experiments. k Images depict ARL4C expression in ZFTA cells (EP1NS) after targeting the CTCF binding site by either gControl/Cas9 or gCTCF#1/Cas9 at 10 days post-infection.

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