An intronic region within FTO confers differentiation block in acute myeloid leukaemia through regulation of IRX3 and HOX [4C-seq]

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. 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.

在急性髓系白血病（acute myeloid leukaemia, AML）中，被定义为白血病干细胞（leukaemia stem cells, LSCs）的细胞群出现异常自我更新与增殖，会导致骨髓与血液中未分化母细胞的累积。通过对比LSCs与健康造血干细胞（haematopoietic stem cells, HSCs）的基因表达分析发现，转录因子IRX3在LSC群体中显著上调；功能实验证实，IRX3可介导分化阻滞过程。值得注意的是，IRX3在HSCs中仅微量表达，且对正常造血发育非必需。上述发现凸显了探究AML中IRX3异常表达机制的必要性，同时也需进一步解析IRX3表达引发分化阻滞的具体分子机制。

为探究AML中IRX3异常表达的调控机制，本研究在AML细胞系与原代患者样本中开展了长程染色质相互作用分析、基因组编辑、增强子功能研究以及多类生物信息学工具分析。我们首次鉴定出一类真正的增强子簇及其关联转录本（增强子RNA, enhancer RNA, eRNA），并构建了此类调控元件参与IRX3表达调控的模型。该增强子簇包含四个具备AML细胞中IRX3调控活性的功能模块。此外，E2模块转录产生的转录本，可通过与IRX3启动子形成染色质环，并在增强子模块与IRX3启动子区域维持高水平的组蛋白H3赖氨酸27乙酰化（H3K27ac），从而促进该超级增强子发挥功能。

Fujioka细胞由Vaskar Saha博士（曼彻斯特癌症研究中心儿童癌症研究组）惠赠。本研究通过短串联重复序列（short tandem repeat, STR）分析验证了细胞身份，并确认其无支原体污染。原代人AML样本获取自曼彻斯特癌症研究中心组织生物样本库，其采集流程已获得南曼彻斯特研究伦理委员会的批准。我们对该生物样本库中的样本进行了IRX3表达水平的筛选。