Spatio-temporal chromatin dynamics during the DNA damage response — ASN Events
  1. Schedule
  2. Poster Session 2
  3. Spatio-temporal chromatin dynamics during the DNA damage response

Spatio-temporal chromatin dynamics during the DNA damage response (#264)

Belinda Wright 1 2 , Tara K Bartolec 3 , Jessie Zhang 3 , Tony J Cesare 3 , Elizabeth Hinde 1 2
  1. EMBL Australia Node in Single Molecule Science, University of New South Wales, Sydney, NSW, Australia
  2. ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, Sydney, NSW, Australia
  3. Genome Integrity Group, Children's Medical Research Institute, University of Sydney, WESTMEAD, NSW, Australia

The cell is constantly challenged by endogenous and exogenous agents which threaten the integrity of the genomic material. Faithful repair of any and all DNA damage is required to prevent the formation of highly deleterious mutations and chromosomal rearrangements. Compromised repair may lead to loss of cell viability or oncogenic transformation, particularly in the case of highly toxic double strand breaks. The DNA damage response (DDR) is a highly coordinated and specialised pathway whereby single- and double-stranded breaks are efficiently and specifically identified, and consequently repaired. Histone marks, protein factors and enzymatic steps involved in this response have been well studied; Mechanisms coordinating this complex and sequential set of interactions on a temporal and spatial scale have yet to be determined. One such mechanism is the regulation of chromatin architecture, both locally around a double strand break and globally throughout the nucleus to aid in the recruitment of DDR factors.

A novel technique developed by the Hinde lab facilitates real-time visualisation of chromatin compaction dynamics in live cells. U-2OS and HeLa cell lines, exogenously expressing H2B-GFP and H2B-mCherry, can be monitored using Förster resonance energy transfer (FRET) and fluorescence-lifetime imaging microscopy (FLIM), to provide a biophysical readout of chromatin compaction state across the three-dimensional space of the nucleus. Coupled with near infra-red laser micro-irradiation, which induces double strand breaks, live cell imaging of these cell lines using FLIM/FRET provides an ideal system to image the real-time changes in chromatin structure induced by the DDR.

We have determined the local and global chromatin dynamics that result from induction of double strand breaks. This includes the rapid decompaction of chromatin at the break site and compaction of chromatin in the boundary region surrounding the genomic lesions. The immediate response is then followed by global chromatin compaction changes throughout the nucleus, which return to the basal levels of chromatin compaction approximately six hours after break induction.

We are now probing, using enzymatic and/or CRISPR-mediated genetic inhibition, how Poly-ADP-ribosylation, phosphorylation, ubiquitiylation and SUMOylation in the DDR contribute to chromatin compaction changes during the cellular response to double strand breaks. We will report on our advances on this experimental front at the conference.

#LorneGenome