Quantifying site-specific chromatin mechanics and DNA damage response

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.

Figure 1

Bulk chromatin motion measurements are independent of bound probe. Nuclei in both cases are stained with Hoechst 33342 (a) U2OS cell transfected with GFP-Fibrillarin showing localization of Fibrillarin within nucleoli. (b) U2OS cell transfected with RFP-TRF1 showing TRF1 localization at telomeres. (c) MSDs of RFP-TRF1 (n = 17), in red, and GFP-Fibrillarin (n = 13), in green, tracked in untreated control U2OS cells. Lines lie atop each other indicating that measurements of MSDs taken from different probes yield equivalent results. Error bars are SEM.

Figure 2

Co-localization of TA and TetR loci with markers of euchromatin and heterochromatin markers, respectively. Co-localization of TA-mCherry with AcH3K9 indicating that the TA motif localizes to regions of euchromatin, and co-localization of TetR-mCherry with DiMeK9H3 indicating that the TetR motif localizes to regions of heterochromatin.

Figure 3

MSDs comparing mobility of the bulk chromatin motion to the four tracers. Bulk chromatin motion measured as cotransfected GFP-Fibrillarin. (a) TA-mCherry (TAmCh) denoted by red circles (n = 34), cotransfected GFP-Fibrillarin (Fib. (TAmCh)) denoted by green line (n = 30), (b) TetR-mCherry (TetRmCh) denoted by purple circles (n = 27), cotransfected GFP-Fibrillarin (Fib. (TetRmCh)) denoted by green line (n = 31), (c) TA-KillerRed (TAKR) denoted by red diamonds (n = 20), cotransfected GFP-Fibrillarin (Fib. (TAKR)) denoted by green line (n = 25), and (d) TetR-KillerRed (TetRKR) denoted by purple diamonds (n = 19), cotransfected GFP-Fibrillarin (Fib. (TetRKR)) denoted by green line (n = 21). Error bars are SEM.

Figure 4

Comparison MSDs showing temporal and global response of chromatin to DNA damage. (a) TA-KillerRed after 2 hours (2 hr_TAKR) denoted by orange diamonds (n = 22), cotransfected GFP-Fibrillarin (Fib. (2 hr TAKR)) denoted by green line (n = 26), and (b) TA-KillerRed after 2 hours (2 hr_TAKR) again denoted by orange diamonds (n = 22), TA-KillerRed (TAKR) denoted by red diamonds (n = 20), and GFP-53BP1, denoted by blue triangles (n = 12). Mobility is increased after 2 hours. 2 hr_TAKR displays dramatic skew at longer lag times. Error bars are SEM.

Figure 5

MSDs showing decoupled chromatin dynamics induced by DNA damage. (a) U2OS cell treated with bleocin for 2 hours prior to imaging showing the localization patterns exhibited by GFP-53BP1 and RFP-TRF1. (b) MSDs from RFP-TRF1 (n = 18) in U2OS cells treated with bleocin, shown in red, and GFP-Fibrillarin (n = 13) in untreated control U2OS cells, shown in green, showing similar mobility to each other despite global damage induction, but increased mobility in damage loci marked by GFP-53BP1 (n = 12), shown as blue triangles. Error bars are SEM. GFP-53BP1 previously used in Fig. 4b. GFP-Fibrillarin (Control) previously used in Fig. 1c.