Transcriptional activation of IRX3 in T-ALL by loss of a promoter tether and enhancer hijack from the CRNDE locus. (A, top panel) UMI-4C contact profile generated by baiting the FTO intron 8 CTCF site in PF-382 (FTO^wt) cells. A green bar highlights the contacts that form a promoter tether between IRX3 and FTO intron 8. (A, bottom panel) UMI-4C contact profile generated by baiting the IRX3 proximal promoter in the PF-382 (FTO^wt) and ALL-SIL (FTO^int8del) T-ALL cell line. CTCF/CTCFL-binding sites across the FTO, IRX3, and CRNDE/IRX5 locus were in silico predicted by HOCOMOCO v11. (B) UMI count of interactions between the IRX3 proximal promoter and the CRNDE locus (P = .0005; Fisher exact test; UMI4Cats analysis package). (C) Expression (FPKM) of CRNDE, IRX5, and IRX3 across T-cell subsets by RNA-seq from the Blueprint Epigenome Project, sample TH91. (D) ChIP-seq for H3K27 acetylation (NCBI GEO accession #GSM1816978) and E-P loops generated by HiChIP for H3K27 acetylation in the ALL-SIL (FTO^int8del) T-ALL cell line about the FTO, IRX3, and CRNDE/IRX5 locus. Loops shown passed a q-value threshold of 0.01 at a bin size of 40 kb and were called by FitHiChIP. (E) Rank ordering of super-enhancers in the PF-382 (FTO^wt) T-ALL cell line by using H3K27ac ChIP-seq (NCBI GEO accession #GSE76783). Red points indicate genomic regions classed as super-enhancers which includes the CRNDE locus. (F) ATAC-seq at the CRNDE/IRX5 locus in PF-382 (FTO^wt) and PF-382 (FTO intron 8 CTCF site deleted) T-ALL cell lines, in addition to ChIP-seq for MYB (NCBI GEO accession #GSM2466687) and H3K27ac (NCBI GEO accession #GSM2037796) in PF-382 (FTO^wt) T-ALL cells. Shaded box represents peak region targeted to disrupt the CRNDE super-enhancer by CRISPR/Cas9 excision. (G) Expression level of IRX3 mRNA as determined by qPCR from ALL-SIL (FTO^int8del) T-ALL cell line after CRISPR/Cas9-mediated deletion of the CRNDE super-enhancer locus. (H) Proposed mechanism of action whereby promoter tethering of IRX3 to the relatively inert FTO intron 8 locus by CTCF binding allows infrequent E-P interactions and low transcriptional output. Subsequent focal deletion of this intronic CTCF site leads to loss of the promoter tether and enhancer hijack of the CRNDE super-enhancer. mRNA, messenger RNA.