Deciphering the chromatin landscape induced around DNA double strand breaks
Deciphering the chromatin landscape induced around DNA double strand breaks
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DOI：
10.4161/cc.9.15.12412
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发表时间：
2010-08-01
期刊：
影响因子：
4.3
通讯作者：
Legube, Gaelle
中科院分区：
文献类型：
--
作者：
Massip, Laurent;Caron, Pierre;Legube, Gaelle
DNA双链断裂(DSB)是最有害的损伤形式之一，破译DSB周围诱导的染色质景观的细节对分子生物学家来说是一个巨大的挑战。染色质免疫沉淀，随后是微阵列杂交(CHIP-CHIP)或高通量测序(CHIP-SEQ)，是提供高分辨率蛋白质-基因组相互作用图的强大技术。然而，由于缺乏合适的DSB诱导技术，应用这些技术来研究DSB诱导的染色质变化受到了阻碍。我们最近开发了一种实验系统，利用融合到修饰的雌激素受体配体结合域(ASSI-ER)的限制性酶，在4-羟基他莫昔芬(4OHT)诱导下产生多个序列特异性和明确定位的DSB。(1)表达这种结构的细胞系是研究DSB修复过程中特定染色质变化的强大工具，能够高分辨率地分析DSB周围诱导的DNA修复复合体和染色质修饰。利用这个系统，我们最近在两条人类染色体上制作了第一张伽马H_2AX图谱，这是一种由DSB诱导的染色质修饰，并研究了它的扩散特性。(1)这里我们提供了描述细胞系的额外数据，给出了通过CHIP-SEQ获得的伽马H_2AX的全基因组图谱，并讨论了我们的系统在研究以前未描述的DSB修复方面的潜力。
DNA double strand breaks (DSBs) are among the most deleterious forms of lesions and deciphering the details of the chromatin landscape induced around DSBs represents a great challenge for molecular biologists.Chromatin Immunoprecipitation, followed by microarray hybridisation (ChIP-chip) or high-throughput sequencing (ChIP-seq), are powerful techniques that provide high-resolution maps of protein-genome interactions. However, applying these techniques to study chromatin changes induced around DSBs was previously hindered due to a lack of suitable DSB induction techniques.We have recently developed an experimental system utilizing a restriction enzyme fused to a modified oestrogen receptor ligand binding domain (AsiSI-ER), which generates multiple, sequence-specific and unambiguously positioned DSBs across the genome upon induction with 4-hydroxytamoxifen (4OHT).(1) Cell lines expressing this construct represent a powerful tool to study specific chromatin changes during DSB repair, enabling high-resolution profiling of DNA repair complexes and chromatin modifications induced around DSBs. Using this system, we have recently produced the first map of gamma H2AX, a DSB-induced chromatin modification, on two human chromosomes and have investigated its spreading properties.(1) Here we provide additional data characterizing the cell lines, present a genome-wide profile of gamma H2AX obtained by ChIP-seq, and discuss the potential of our system towards investigations of previously uncharacterized aspects of DSB repair.