Streamlined histone-based fluorescence lifetime imaging microscopy (FLIM) for studying chromatin organisation

Alice Sherrard, Paul Bishop, Melanie Panagi, Maria Beatriz Villagomez Torres, Dominic Alibhai, Abderrahmane Kaidi

Research output: Contribution to journalArticle (Academic Journal)peer-review

5 Citations (Scopus)
257 Downloads (Pure)

Abstract

Changes 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 its 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 ATM’s role 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.
Original languageEnglish
JournalBiology Open
Volume7
Issue number3
Early online date23 Mar 2018
DOIs
Publication statusE-pub ahead of print - 23 Mar 2018

Keywords

  • ATM
  • DNA Damage
  • Chromatin
  • Genome
  • Histones

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