CDX2 cis-deregulation is driven by hijacking of an enhancer located in the PAN3 gene. (A) ChIP-seq signals of histone modifications (H3K4me3 and H3K27ac) of 1 representative CDX2/UBTF case (B_SL160) and ChIP-seq and ATAC-seq signals of NALM16, NB4, and K562 cell lines at the CDX2-PAN3 locus. CDX2 expression levels of cell lines are provided in supplemental Figure 5. The gray zone on CDX2/UBTF ALL and NALM16 traces indicate respectively the minimally deleted region and the duplicated region. The red box corresponds to the position of the enhancer identified in PAN3. (B) 4C-seq normalized signals of NALM16, NB4, K562, and Caco-2/TC7 cell lines with CDX2 promoter as view point. The red zone highlights the NALM16-specific called interaction (C) CRISPRi experimental scheme. In order to inactivate the enhancer in PAN3, we used the catalytically inactive CRISPR-associated protein 9 (dCas9) fused to the repressor KRAB with specific single-guide RNA. Representative example of a CRISPRi experiment showing CDX2 expression decrease in NALM16 after CRISPRi targeting the PAN3 enhancer, measured by qRT-PCR (D), and western blot (E).