An intronic region within FTO confers differentiation block in acute myeloid leukaemia through regulation of IRX3 and HOX [MeDIP-seq re-analyzed]

In acute myeloid leukaemia (AML), abnormal self-renewal and proliferation of a cell population defined as leukaemia stem cells (LSCs) results in the accumulation of undifferentiated blast cells in the bone marrow and blood. Gene expression analyses comparing LSCs to healthy haematopoietic stem cells (HSCs) revealed a significant upregulation of the transcription factor IRX3 in the LSC compartment, and functional experiments demonstrated that IRX3 contributes to a block in differentiation. Interestingly, IRX3 is minimally expressed in HSCs and not required for normal haematopoietic development. These discoveries highlight the necessity to investigate mechanisms underlying IRX3 misexpression in AML, as well as further investigate the mechanisms through which IRX3 expression contributes to the differentiation block. To investigate the mechanism of IRX3 misexpression in AML, we applied long-range chromatin interaction analyses, genome editing, enhancer-function studies and diverse bioinformatics tools in both AML cell lines and primary patient samples. We characterise for the first time a cluster of bona-fide enhancers and its associated transcript (eRNA), and describe a model through which these regulatory elements contribute to IRX3 expression. The cluster is composed of four modules with IRX3-regulatory activity in AML cells. Moreover, transcripts arise from the E2 module and aids in the function of the super-enhancer by falicitating a chromatin loop with the IRX3 promoter and maintaining high levels of H3K27ac at both the enhancer modules and IRX3 promoter. Overall design: Fujioka cells were a gift from Dr. Vaskar Saha (Children's Cancer Group, Manchester Cancer Research Centre). Cells were verified by STR analysis and confirmed to be mycoplasma-free. Primary human AML samples were obtained from the MCRC Tissue Biobank; their collection was approved by the South Manchester Research Ethics Committee. Biobank samples were screened for the expression level of IRX3. Please note that GSM5292613-GSM5292618 (in GSE174354) samples have been re-analyzed for the current study.