IRX3 is a homeobox transcription factor essential for normal embryonic development but with no detectable expression across the normal T-lineage. In contrast, aberrant expression of IRX3 is observed in ~50% of patients with T cell acute lymphoblastic leukaemia (T-ALL). Prior studies have confirmed that IRX3 acts as a potent oncogene through its ability to induce leukaemias in vivo (Somerville et al. 2018). To date, the mechanism of transcriptional activation for IRX3 in T-ALL is unknown. We investigated whether aberrations of the noncoding genome are a cause of dysregulated IRX3 expression.
Data was generated by the following methods; HiChIP to map enhancer-promoter interactions and identify candidate cis-regulatory elements (cCREs), WGS and other approaches to identify copy number aberrations, ddPCR to determine locus-specific copy number calls, ChIP-Seq to determine the binding of transcription factors in noncoding sequences, CRISPR/Cas9 genome editing followed by qPCR to ascertain the functional impact of noncoding variants, and UMI-4C to determine chromatin looping interactions from the IRX3 promoter.
We identified a cCRE 3’ to IRX3 situated within FTO intron 8, that harbours heterozygous deletions in T-ALL genomes and matched supraphysiological expression of IRX3. Notably these deletions impinge on a CTCF binding site. Mimicking this deletion in an IRX3-ve FTO (wt/wt) cell line leads to a ≥ 4-fold increase in IRX3 mRNA. Furthermore, chromatin looping data from the IRX3 proximal promoter between cell lines with, and without ΔFTO intron 8 identified a significant increase in promoter contacts with the CRNDE/IRX5 locus positioned 5’ to IRX3. Remarkably, this locus harbours a super-enhancer determined by rank ordering. These data reveal that activation of IRX3 in T-ALL is caused by FTO intron 8 deletions and consequential enhancer hijacking.
We postulate that similar deletions of the noncoding genome especially those that impinge on CTCF sites, may cause dysregulated oncogene expression.