Phasor Histone FLIM-FRET Microscopy Maps Nuclear-Wide Nanoscale Chromatin Architecture With Respect to Genetically Induced DNA Double-Strand Breaks

Frontiers in Genetics

Jieqiong Lou²  Elizabeth Hinde²  Ashleigh Solano²  Zhen Liang³ 

[1] Cancer and RNA Laboratory, St. Vincent’s Institute of Medical Research, Fitzroy, VIC, Australia;Department of Biochemistry and Pharmacology, University of Melbourne, Melbourne, VIC, Australia;Department of Medicine, Melbourne Medical School, St Vincent’s Hospital, University of Melbourne, Fitzroy, VIC, Australia;School of Physics, University of Melbourne, Melbourne, VIC, Australia;

关键词: DNA repair;    chromatin;    histones;    fluorescence lifetime imaging microscopy (FLIM);    Förster resonance energy transfer (FRET);   

DOI  :  10.3389/fgene.2021.770081

来源: DOAJ

【 摘 要 】

A DNA double-strand break (DSB) takes place in the context of chromatin, and there is increasing evidence for chromatin structure to play a functional role in DSB signaling and repair. Thus, there is an emerging need for quantitative microscopy methods that can directly measure chromatin network architecture and detect changes in this structural framework upon DSB induction within an intact nucleus. To address this demand, here we present the phasor approach to fluorescence lifetime imaging microscopy (FLIM) of Förster resonance energy transfer (FRET) between fluorescently labeled histones in the DSB inducible via AsiSI cell system (DIvA), which has sufficient spatial resolution to map nuclear-wide chromatin compaction at the level of nucleosome proximity with respect to multiple site-specific DSBs. We also demonstrate that when phasor histone FLIM-FRET is coupled with immunofluorescence, this technology has the unique advantage of enabling exploration of any heterogeneity that exists in chromatin structure at the spatially distinct and genetically induced DSBs.

【 授权许可】

Unknown