Journal article

Phasor histone FLIM-FRET microscopy quantifies spatiotemporal rearrangement of chromatin architecture during the DNA damage response

Jieqiong Lou, Lorenzo Scipioni, Belinda K Wright, Tara K Bartolec, Jessie Zhang, V Pragathi Masamsetti, Katharina Gaus, Enrico Gratton, Anthony J Cesare, Elizabeth Hinde

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA | NATL ACAD SCIENCES | Published : 2019

Abstract

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 nucleosome level during the DNA damage response (DDR). The method is based on phasor image-correlation spectroscopy of histone fluorescence lifetime imaging microscopy (FLIM)-Förster resonance energy transfer (FRET) microscopy data acquired in live cells coexpressing 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 c..

View full abstract

Grants

Awarded by Australian NHMRC


Awarded by Australian Research Council (ARC)


Awarded by National Institutes of Health


Awarded by Cancer Council NSW


Awarded by Cancer Institute NSW


Awarded by ARC


Awarded by NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES


Funding Acknowledgements

We thank Elvis Pandzic, Alex Macmillan, Renee Whan, and the Biomedical Imaging Facility and the Mark Wainwright Analytical Centre at the University of New South Wales for enabling access to the Zeiss LSM 880; Scott Page and the Children's Medical Research Institute (CMRI) Australian Cancer Research Foundation (ACRF) Telomere Analysis Centre (supported by the ACRF) for assistance with imaging infrastructure; Leszek Lisowski and the CMRI Vector and Genome Engineering Facility for making Lentivectors; the Westmead Institute for Medical Research Flow Cytometry Centre [supported by the Cancer Council NSW and the Australian National Health and Medical Research Council (NHMRC)] for cell sorting; and David Priest and Georgia Kafer for their helpful discussion and feedback on the manuscript. K. G. is supported by Australian NHMRC Grant APP1059278 and Australian Research Council (ARC) Grants CE140100011 and LP140100967. E. G. is supported by National Institutes of Health Grants P41-GM103540 and P50-GM076516. A.J.C. is supported by Australian NHMRC Grants 1053195, 1106241, and 1104461; Cancer Council NSW Grant RG 15-12; and Cancer Institute NSW Grant 11/FRL/5-02. E.H. is supported by Australian NHMRC Grants APP1104461 and APP1124762 and ARC Grant DP180101387.