Genome wide analsis of the chromatin accessibility and gene expression in control and NUDT5 depleted U2OS cells [RNA-seq]

NUDT5 was reported to generate ATP to support local chromatin remodeling and transcription of hormone-responsive genes. Our research has found that NUDT5 is also required for DNA damage repair by homologous recombination. To investigate how NUDT5 regulates HR, we depleted NUDT5 in U2OS cells by siRNA and compared the transcriptome profiling (RNA-seq) of NUDT5 depleted cells with control cells to investigate whether NUDT5 regulate HR by altering the expression of essential components of the HR machinery. The results suggest that NUDT5 does not regulate HR by altering the expression of essential components of the HR machinery. To investigate whether NUDT5 contributes to chromatin remodeling after DNA damage, we compared the chromatin accessibility profiling (ATAC-seq) in NUDT5 depleted U2OS cells and control cells, either before or after ionizing radiation. The results suggest that NUDT5 silencing blocked most DNA damage-induced changes in chromatin accessibility following radiation exposure and implies that NUDT5 may support the activity of remodeling ATPases during damage repair. Overall design: RNA-seq analysis of U2OS cells transfected with siCtrl or siNUDT5 (samples in triplicate). ATAC-seq analysis of U2OS cells transfected with siCtrl or siNUDT5, treated with or without ionizing radiation at 5Gy (samples in duplicate).

已有研究报道，NUDT5可合成三磷酸腺苷（ATP），以介导激素响应基因的局部染色质重塑与转录过程。本研究证实，NUDT5同样参与同源重组（homologous recombination, HR）介导的DNA损伤修复。为探究NUDT5调控HR的具体机制，我们通过小干扰RNA（siRNA）在U2OS细胞中敲低NUDT5的表达，并将NUDT5敲低细胞与对照细胞的转录组测序（RNA-seq）结果进行对比分析，以明确NUDT5是否通过改变HR核心组分的表达来调控HR。实验结果显示，NUDT5并未通过改变HR核心组分的表达来调控HR。为探究NUDT5是否在DNA损伤后参与染色质重塑过程，我们分别在电离辐射处理前后，对NUDT5敲低的U2OS细胞与对照细胞的染色质可及性分析（ATAC-seq）结果进行对比。结果表明，沉默NUDT5会阻断辐射暴露后大多数DNA损伤诱导的染色质可及性变化，这提示NUDT5可能在损伤修复过程中维持重塑型ATP酶的活性。整体实验设计：对转染对照小干扰RNA（siCtrl）或靶向NUDT5的小干扰RNA（siNUDT5）的U2OS细胞进行RNA-seq分析，每组设置3个生物学重复。对转染siCtrl或siNUDT5的U2OS细胞施以5Gy电离辐射或不作处理，开展ATAC-seq分析，每组设置2个生物学重复。