Quantifying site-specific chromatin mechanics and DNA damage response

Daniel B Whitefield,Stephen T Spagnol,Travis J Armiger,Li Lan,Kris Noel Dahl

doi:10.1038/s41598-018-36343-x

Abstract

DNA double-strand breaks pose a direct threat to genomic stability. Studies of DNA damage and chromatin dynamics have yielded opposing results that support either increased or decreased chromatin motion after damage. In this study, we independently measure the dynamics of transcriptionally active or repressed chromatin regions using particle tracking microrheology. We find that the baseline motion of transcriptionally repressed regions of chromatin are significantly less mobile than transcriptionally active chromatin, which is statistically similar to the bulk motion of chromatin within the nucleus. Site specific DNA damage using KillerRed tags induced in loci within repressed chromatin causes an increased motion, while loci within transcriptionally active regions remains unchanged at similar time scales. We also observe a time-dependent response associated with a further increase in chromatin decondensation. Global induction of damage with bleocin displays similar trends of chromatin decondensation and increased mobility only at 53BP1-labeled damage sites but not at non-damaged sites, indicating that chromatin dynamics are tightly regulated locally after damage. These results shed light on the evolution of the local and global DNA damage response associated with chromatin remodeling and dynamics, with direct implications for their role in repair.

Highlights

The human genome is four gigabases of double stranded DNA wound onto histones to form chromatin with loose spatial organization inside the nucleus[1]
We further explore the impact of DNA damage at different loci by inducing DNA double strand breaks (DSBs) using the KillerRed (KR) fluorescent protein bound to Transcription Activator (TA) and Tetracycline Repressor (TetR), respectively
We have previously demonstrated that ensemble chromatin dynamics on long time scales are independent of the chromatin-associated probes

Summary

Introduction

The human genome is four gigabases of double stranded DNA wound onto histones to form chromatin with loose spatial organization inside the nucleus[1]. TA binding to the TRE leads to transcriptional activation and concomitant chromatin decondensation, and TetR binding to the TRE reinforces transcriptional repression and chromatin condensation[5,6] While this allows the spatial advantage of examining specific chromatin territories, this method introduces challenges of data analysis from the tracking of a single point within a living cell. We find that transcriptionally active regions exhibit chromatin dynamics equivalent to bulk chromatin (as measured by chromatin probes bound inside nucleoli and at telomeres) and transcriptionally repressed regions have reduced mobility consistent with their tight condensation state. We observe that DSBs in transcriptionally repressed regions of chromatin, which typically have reduced mobility relative to bulk or transcriptionally active chromatin, exhibit enhanced dynamics more akin to bulk chromatin following DNA damage induction. The resulting effect decreases the probability of large length scale chromatin motion at long timescales, thereby reducing the potential for improper repair and translocations

Methods

Results

Conclusion