Abstract
ABSTRACTChanges in chromatin structure are key determinants of genomic responses. Thus, methods that enable such measurements are instrumental for investigating genome regulation and function. Here, we report further developments and validation of a streamlined method of histone-based fluorescence lifetime imaging microscopy (FLIM) that robustly detects chromatin compaction states in fixed and live cells, in 2D and 3D. We present a quality-controlled and detailed method that is simpler and faster than previous methods, and uses FLIMfit open-source software. We demonstrate the versatility of this chromatin FLIM through its combination with immunofluorescence and implementation in immortalised and primary cells. We applied this method to investigate the regulation of chromatin organisation after genotoxic stress and provide new insights into the role of ATM in controlling chromatin structure independently of DNA damage. Collectively, we present an adaptable chromatin FLIM method for examining chromatin structure and establish its utility in mammalian cells.
Highlights
In the nucleus, DNA is packaged into chromatin structures (Richmond and Davey, 2003) that determine the activity of genomic DNA in space and time (Bickmore, 2013; Dekker et al, 2017), and may contribute to non-genetic functions of the genome (Bustin and Misteli, 2016)
We extended the utility of the chromatin fluorescence lifetime imaging microscopy (FLIM) approach to human retinal pigment epithelial-1 (RPE1) cells (Fig. S2A,B), demonstrating that this method is an adaptable experimental approach for performing chromatin FLIM in fixed cells as a readout for the extent of FRET between two fluorescently tagged histones
Here, we report further developments that enable streamlining of chromatin FLIM for visualising chromatin structure and compaction states in live and fixed mammalian cells
Summary
DNA is packaged into chromatin structures (Richmond and Davey, 2003) that determine the activity of genomic DNA in space and time (Bickmore, 2013; Dekker et al, 2017), and may contribute to non-genetic functions of the genome (Bustin and Misteli, 2016) Such chromatin organisation is underpinned by regulatory epigenetic mechanisms, including histone modifications (Kouzarides, 2007). Interphase chromatin appears to exist in two clearly distinct states: open euchromatin and condensed heterochromatin (Bickmore and van Steensel, 2013). These chromatin states seem to be stable at steady-state conditions, they undergo dynamic reorganisation during genome transduction processes such as transcription (Therizols et al, 2014; Wang et al, 2014), or DNA repair (Lukas et al, 2011; Polo and Almouzni, 2015).
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