EBF1 nuclear repositioning instructs chromatin refolding to promote therapy resistance in T leukemic cells. [DND41_ATACseq]. EBF1 nuclear repositioning instructs chromatin refolding to promote therapy resistance in T leukemic cells. [DND41_ATACseq]

Purpose: To investigate the mechanisms of 3D genome organization in drug-resistant T-ALL Methods: We used multiple epigenomics, chromatin conformation, and transcriptomic assays to study the mechanisms of chromatin adaptation in GSI-sensitive and GSI-resistant T-ALL Results: We report here that T/B cell lineage determining transcription factors are differentially expressed in GSI-resistant T-ALL cells, driving enhancer switching and genome folding reorganization events to promote GSI-resistance. Conclusions: These observations suggest a general mechanism that diffenrential activity of pioneering factors can be exploited to evade addiction to oncogenic signals. Overall design: For comparison of chromatin accessibility in parental and GSI-resistant DND41 cells, parental DND41 was treated with DMSO or 125 nM GSI for 24 hours and GSI-resistant cells were refreshed with media containing 125 nM GSI for 24 hours. For assessing the impact of LEF1 or TCF1 loss on parental DND41 genome, DND41-Cas9 cells carrying LRG2.1-LEF1-g3 or LRmCherry2.1-TCF7-g5 were sorted 6 days post transduction. To assess the impact of LEF1 and TCF1 loss on parental DND41 genome, DND41-Cas9 carrying LRG2.1-LEF1-g3 and LRmCherry2.1-TCF7-g5 were sorted 3, 6 and 9 days post transduction. DND41-Cas9 carrying LRG2.1 and LRmCherry2.1 were sorted 3 days post transduction as control.To assess the impact of EBF1 loss on GSI-resistant DND41 genome, DND41-Res-Cas9 with LRmCherry2.1-EBF1-g7 were sorted 3, 6 and 9 days post transduction and control cells were sorted 3 days post transduction. ATAC-seq experiment was performed in triplicates.

研究目的：探究耐药性T细胞急性淋巴细胞白血病（T-ALL）的三维基因组组织机制。
研究方法：本研究采用多组表观基因组学、染色质构象及转录组学实验手段，解析γ分泌酶抑制剂（GSI）敏感型与GSI耐药型T-ALL细胞的染色质适应机制。
研究结果：本研究发现，在GSI耐药型T-ALL细胞中，T/B细胞谱系决定性转录因子呈现差异表达，该差异表达驱动增强子转换与基因组折叠重排事件，进而促进GSI耐药表型的产生。
研究结论：本研究结果提示一类通用机制：先驱因子的差异活性可被肿瘤细胞利用，以逃逸致癌信号通路的成瘾性依赖。
整体实验设计：
1. 为比较亲本与GSI耐药型DND41细胞的染色质开放状态，将亲本DND41细胞分别用二甲基亚砜（DMSO）或125 nM GSI处理24小时，同时将GSI耐药型细胞置于含125 nM GSI的培养基中复培养24小时。
2. 为评估LEF1或TCF1单敲除对亲本DND41基因组的影响，将携带LRG2.1-LEF1-g3或LRmCherry2.1-TCF7-g5的DND41-Cas9细胞在转导后第6天进行分选。
3. 为评估LEF1与TCF1联合敲除对亲本DND41基因组的影响，将同时携带LRG2.1-LEF1-g3与LRmCherry2.1-TCF7-g5的DND41-Cas9细胞分别在转导后第3、6、9天进行分选；并以转导后第3天分选的携带空载体LRG2.1与LRmCherry2.1的DND41-Cas9细胞作为对照。
4. 为评估EBF1敲除对GSI耐药型DND41基因组的影响，将携带LRmCherry2.1-EBF1-g7的DND41-Res-Cas9细胞分别在转导后第3、6、9天进行分选，同时以转导后第3天分选的对应对照细胞作为对照。
5. ATAC测序（ATAC-seq）实验设置三次生物学重复。