Phasor histone FLIM-FRET microscopy quantifies spatiotemporal rearrangement of chromatin architecture during the DNA damage response
Phasor histone FLIM-FRET microscopy quantifies spatiotemporal rearrangement of chromatin architecture during the DNA damage response
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DOI：
10.1073/pnas.1814965116
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发表时间：
2019-04-09
影响因子：
11.1
通讯作者：
Hinde, Elizabeth
中科院分区：
文献类型：
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作者：
Lou, Jieqiong;Scipioni, Lorenzo;Hinde, Elizabeth
为了研究染色质结构在双链断裂（DSB）修复位点的时空组织方式，我们建立了一种生物物理方法来量化DNA损伤反应（DDR）过程中核小体水平的染色质致密化。该方法是基于相量图像相关光谱的组蛋白荧光寿命成像显微镜（FLIM）-福斯特共振能量转移（FRET）显微镜数据中获得的活细胞共表达H2 B-eGFP和H2 B-mCherry。这种多路复用的方法产生的时空地图的核范围内的染色质压实，当再加上激光微辐射诱导的DSB，量化的大小，稳定性和整个DDR紧凑的染色质焦点之间的间距。使用这种技术，我们确定共济失调毛细血管扩张突变（ATM）和RNF 8调节DSB的快速染色质解压缩和修复位点周围致密染色质灶的形成。这种染色质结构用于将修复位点与周围的核环境区分开来，并调节53 BP 1的迁移率。
To investigate how chromatin architecture is spatiotemporally organized at a double-strand break (DSB) repair locus, we established a biophysical method to quantify chromatin compaction at the nucleo-some level during the DNA damage response (DDR). The method is based on phasor image-correlation spectroscopy of histone fluorescence lifetime imaging microscopy (FLIM)-Forster resonance energy transfer (FRET) microscopy data acquired in live cells coex-pressing H2B-eGFP and H2B-mCherry. This multiplexed approach generates spatiotemporal maps of nuclear-wide chromatin compaction that, when coupled with laser microirradiation-induced DSBs, quantify the size, stability, and spacing between compact chromatin foci throughout the DDR. Using this technology, we identify that ataxia-telangiectasia mutated (ATM) and RNF8 regulate rapid chromatin decompaction at DSBs and formation of compact chromatin foci surrounding the repair locus. This chromatin architecture serves to demarcate the repair locus from the surrounding nuclear environment and modulate 53BP1 mobility.