BCR-ABL-mediated recruitment of STAT5 revealed by ChIPseq

Even in the era of ABL tyrosine kinase inhibitors, eradication of chronic myeloid leukemia (CML) stem cells still remains to be a prerequisite for the complete cure of the disease. Interferon-a (IFNa), which has been used long for the treatment for CML-chronic phase, are now being re-evaluated. However, the molecular mechanism involved in the action of IFNa on CML stem cells have not been elucidated yet. In this study we have found that IFNa upregulates CCAAT/Enhancer Binding Protein ß (C/EBPß), a transcription factor required for demand-driven granulopoiesis, through activation of STAT1 and STAT5 in BCR-ABL-expressing cells. Activated STAT1 and STAT5 were recruited to the newly identified 3' distal enhancer region of Cebpb, which contains tandemly aligned interferon-g activated sequences (GAS). Genome editing-mediated repression or deletion of the GAS elements significantly abrogated the IFNa-dependent upregulation of C/EBPß. IFNa induces differentiation and exhaustion of CML stem cells both in vitro and in vivo in a C/EBPß-dependent manner. In addition, IFNa upregulates C/EBPß and induces exhaustion of CD34+ CML stem cells obtained from CML patients. Collectively, these data clearly show that C/EBPß is a critical factor in IFNa signaling that induces differentiation and exhaustion of CML stem cells. Overall design: Examination of STAT5 binding in 3 conditions in a cell line即使在ABL酪氨酸激酶抑制剂（ABL tyrosine kinase inhibitors）时代，根除慢性髓性白血病（chronic myeloid leukemia, CML）干细胞仍是实现该病完全治愈的先决条件。干扰素-α（interferon-a, IFNa）长期用于CML慢性期的治疗，目前正被重新评估。然而，IFNa作用于CML干细胞的分子机制尚未阐明。本研究发现，在表达BCR-ABL的细胞中，IFNa通过激活信号转导子与转录激活因子1（STAT1）和信号转导子与转录激活因子5（STAT5），上调需求驱动型粒细胞生成所需的转录因子CCAAT/增强子结合蛋白β（CCAAT/Enhancer Binding Protein β, C/EBPβ）。激活的STAT1和STAT5被招募至新鉴定的Cebpb基因3'远端增强子区域，该区域包含串联排列的干扰素γ激活序列（GAS）。通过基因组编辑介导的抑制或敲除GAS元件，可显著抵消IFNa依赖的C/EBPβ上调作用。IFNa以C/EBPβ依赖的方式，在体外和体内诱导CML干细胞的分化与耗竭。此外，IFNa可上调C/EBPβ的表达，并诱导从CML患者体内分离的CD34+ CML干细胞发生耗竭。综上，上述数据明确表明，C/EBPβ是IFNa信号通路中诱导CML干细胞分化与耗竭的关键因子。总体实验设计：在3种实验条件下检测细胞系中STAT5的结合情况。