CHD3 and CHD4 recruitment and chromatin remodeling activity at DNA breaks is promoted by early poly(ADP-ribose)-dependent chromatin relaxation

Abstract

One of the first events to occur upon DNA damage is the local opening of the compact chromatin architecture, facilitating access of repair proteins to DNA lesions. This early relaxation is triggered by poly(ADP-ribosyl)ation by PARP1 in addition to ATP-dependent chromatin remodeling. CHD4 recruits to DNA breaks in a PAR-dependent manner, although it lacks any recognizable PAR-binding domain, and has the ability to relax chromatin structure. However, its role in chromatin relaxation at the site of DNA damage has not been explored. Using a live cell fluorescence three-hybrid assay, we demonstrate that the recruitment of CHD4 to DNA damage, while being poly(ADP-ribosyl)ation-dependent, is not through binding poly(ADP-ribose). Additionally, we show that CHD3 is recruited to DNA breaks in the same manner as CHD4 and that both CHD3 and CHD4 play active roles in chromatin remodeling at DNA breaks. Together, our findings reveal a two-step mechanism for DNA damage induced chromatin relaxation in which PARP1 and the PAR-binding remodeler activities of Alc1/CHD1L induce an initial chromatin relaxation phase that promotes the subsequent recruitment of CHD3 and CHD4 via binding to DNA for further chromatin remodeling at DNA breaks.

Figure 1.

The PAR-dependent recruitment of CHD4 to sites of DNA damage is not triggered by PAR binding. (A) Recruitment of GFP-CHD4 to sites of microirradiation 120 seconds after DNA damage in the presence (PARPi) or absence (CTRL) of PARP inhibitor. Microirradiation is highlighted with green arrows. (B) Normalized kinetics of PAR levels as measured by mH2A1.1 macrodomain recruitment (red) and CHD4 recruitment (black) after microirradiation. Kinetics are normalized at a maximum of 1. (C) Schematic of PAR binding assay. The LacI-GFP trap is used to tether the GFP or YFP bait-protein to the LacO array and appears as a bright spot within the nucleus. In a non-damaged situation, there is no interaction of mCherry-PARP1 with the bait protein. Upon UV-irradiation induced DNA damage, both mCherry-PARP1 and the GFP/YFP-bait protein will recruit to the site of damage. If the bait protein is capable of binding PAR, PARylated mCherry-PARP1 that is released from the site of damage will interact with the bait protein at the LacO array, resulting in an increase in mCherry signal at the LacO array. If the bait protein cannot bind PAR, there will be no enrichment of mCherry-PARP1 at the LacO array. (D) YFP-macrodomain of mH2A1.1 recruits PARylated PARP1, which is abolished by PARP inhibition (PARPi). Inset shows the magnified LacO array. Post-irradiation images are shown at 120 seconds. (E) GFP-CHD4 does not recruit PARylated PARP1 after microirradiation. Post-irradiation images are shown at the indicated time points post-irradiation. Scale bars are 5 μm. The look-up-table shown on panel (A) is valid for all pseudocolored images.

Figure 2.

Chromatin relaxation is sufficient to promote the binding of CHD4 to chromatin. (A–C) Chromatin relaxation (A), CHD4 recruitment (B) and PAR levels (C) in cells over-expressing wild-type Alc1 (Alc1 WT, black) or the ATPase-dead (Alc1 E175Q, red) mutant after UV-microirradiation. (D) Pseudocolored confocal images of the same Hoechst-stained nucleus in isotonic and hypotonic media. Scale bar is 5 μm. (E) U2OS cells expressing GFP-CHD4 in isotonic or hypotonic solutions before (Pre) and after photobleaching (F, G) Normalized FRAP curves (F) and recovery times (G) of GFP-CHD4 in isotonic (iso) or hypotonic (hypo) solutions in wild-type (WT) or PARP1 knockout (P1KO) cells. PARP inhibition is denoted by PARPi. The look-up-table shown on panel (D) is valid for all pseudocolored images.

Figure 3.

CHD4 contributes to chromatin relaxation at sites of DNA damage. (A) Confocal image sequence of the photoconverted chromatin region upon micro-irradiation at 405 nm in U2OS nuclei expressing H2B-PAGFP presensitized by Hoechst labeling and that have been treated with scrambled or CHD4 siRNA. The enlargement of the photoconverted line at 120 s post-irradiation is used to assess chromatin relaxation at sites of DNA damage. Intensity profiles show the thickness of the line (μm) at 0 s (Black) and 120 s (Red). Scale bars are 2 μm. (B) Chromatin relaxation curves showing the kinetics of chromatin relaxation as measured by the width of the photoconverted line of scrambled (siScr, Black) or CHD4 knock-down (siCHD4 #1, Red, siCHD4 #2, Blue) cells. A ‘no damage’ control, nuclei without Hoechst sensitization, is shown in green. (C) Effect of two independent CHD4 (siCHD4) knockdowns on DNA damage-induced chromatin relaxation as compared to scrambled siRNA treatment (siScr). Boxplots show chromatin relaxation 120 seconds after microirradiation. (D) Western blot showing knockdown efficiency of CHD4 and expression levels of CHD3, PARP1 and Alc1. Tubulin is used as a loading control.

Figure 4.

CHD3 is recruited to sites of DNA damage and is required for DNA damage induced chromatin relaxation. (A) Recruitment kinetics of GFP-CHD3 (red) and GFP-CHD4 (black) to sites of DNA damage. (B) Recruitment of GFP-CHD3 to sites of microirradiation 120 seconds after DNA damage in the presence (PARPi) or absence (CTRL) of PARP inhibitor. Microirradiation is highlighted with green arrows. Scale bars are 5 μm. (C) GFP-CHD3 is tethered to the LacO array and co-expressed with mCherry-PARP1. Upon DNA damage, PARP1 recruits to microirradiation induced DNA breaks but does not accumulate at the tethered GFP-CHD3, indicating that CHD3 does not bind PAR. Scale bars are 5 μm. Inset shows the magnified LacO array. (D) Effect of two independent CHD3 (siCHD3) knockdowns on DNA damage-induced chromatin relaxation as compared to scrambled siRNA treatment (siScr). Boxplots show chromatin relaxation 120 seconds after microirradiation (E) Western blot showing knockdown efficiency of CHD3 and expression levels of CHD4, PARP1 and Alc1. Tubulin is used as a loading control.

Figure 5.

Model of two-phase chromatin remodeling at sites of DNA damage. DNA damage promotes the recruitment of PARP1 where it is activated. Activated PARP1 will PARylate proteins at the site of damage, including itself, and promote the recruitment of the chromatin remodeler, Alc1. Alc1 remodeling activity leads to chromatin relaxation in the vicinity of the DNA break. CHD3 and CHD4 bind to the relaxed chromatin and further remodel chromatin.