Overexpression of the WWE domain of RNF146 modulates poly-(ADP)-ribose dynamics at sites of DNA damage

Abstract

Protein poly-ADP-ribosylation (PARylation) is a post-translational modification formed by transfer of successive units of ADP-ribose to target proteins to form poly-ADP-ribose (PAR) chains. PAR plays a critical role in the DNA damage response (DDR) by acting as a signaling platform to promote the recruitment of DNA repair factors to the sites of DNA damage that bind via their PAR-binding domains (PBDs). Several classes of PBD families have been recognized, which identify distinct parts of the PAR chain. Proteins encoding PBDs play an essential role in conveying the PAR-mediated signal through their interaction with PAR chains, which mediates many cellular functions, including the DDR. The WWE domain identifies the iso-ADP-ribose moiety of the PAR chain. We recently described the WWE domain of RNF146 as a robust genetically encoded probe, when fused to EGFP, for detection of PAR in live cells. Here, we evaluated other PBD candidates as molecular PAR probes in live cells, including several other WWE domains and an engineered macrodomain. In addition, we demonstrate unique PAR dynamics when tracked by different PAR binding domains, a finding that that can be exploited for modulation of the PAR-dependent DNA damage response.

Keywords: Macrodomain; PAR binding domains; RNF146; WWE domain; poly-ADP-ribose (PAR).

Figure 1.

WWE domain mediated detection of PARylation levels and temporal dynamics in live cells. (A) Alignment of the amino acid sequences of the WWE domains from RNF146(92–168), RNF146(100–182), TRIP12(749–836), Deltex2(8–97) and Deltex2(98–174); (B) Graphic depicting a PAR binding domain (PBD) fused to EGFP that is used to detect levels of and temporal dynamics of PAR chains in live cells; (C) Confocal micrograph images of cells expressing WWE-EGFP fusions in ES-2 cells following laser micro-irradiation at multiple time points, white scale bar denotes 20µm; (D) Plot depicting the recruitment dynamics of WWE domains RNF146(92–168), RNF146(100–182) and TRIP12(749–836) in ES-2 cells to sites of laser micro-irradiation (405nm laser), N ≥ 28 cells, recruitment intensity normalized to nucleus fluorescent intensity background; (E) Relative recruitment peak intensity of the RNF146(100–182), RNF146(92–168), and TRIP12(749–836) WWE domains expressed in ES-2 cells. Each point represents a single cell recruitment focus, graph shows mean ± SEM; (F) Peak recruitment time of the RNF146(100–182) and RNF146(92–168) WWE domains expressed in ES-2 cells. Each point represents a single cell recruitment focus, graph shows mean ± SEM; (G) Plot depicting the dissociation dynamics of the RNF146(100–182) and RNF146(92–168) WWE domains foci during 20 minutes time span following laser-induced DNA damage in ES-2 cells. N ≥ 36 cells. Exclusion percentages were 0% for RNF146(100–182) and 33.3% (12 foci) for RNF146(92–168). No exclusion for TRIP12(749–836) as it did not show any recruitment. NS: no significance, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; a Kruskal-Wallis test was used for panel E, a Mann-Whitney test for panel F and a Kaplan-Meier test for panel G.

Figure 2.

WWE domain of RNF146(100–182) and the engineered Af1521(K35E/Y145R) macrodomain as molecular probes to track PARylation dynamics in live cells. (A) Schematic representation of the RNF146(100–182) WWE domain (fused to EGFP) or of the Af1521 macrodomain (fused to EGFP) bound to PAR chains at sites of DNA damage; (B) Confocal micrograph images of EGFP-fused RNF146(100–182), Af1521(WT) or Af1521(K35E/Y145R) expressed in U2OS cells, white scale bar denotes 20µm; (C) Recruitment of Af1521(K35E/Y145R)-EGFP in U2OS cells following pre-treatment with vehicle (DMSO, 0.1%, 60 minutes), the PARPi ABT-888 (10μM, 60 minutes) or the PARGi PDD00017273(10μM, 60 minutes), to sites of laser micro-irradiation (405nm laser), following BrdU sensitization (10μM, 24 hours), N≥20 cells, recruitment intensity normalized to nucleus fluorescent intensity background; (D) Recruitment of Af1521(WT), Af1521(K35E/Y145R) and Af1521(G42E) expressed in U2OS cells to sites of laser micro-irradiation (405nm) following BrdU sensitization (10μM, 24 hours), N≥25 cells, recruitment intensity normalized to first frame; (E) Relative peak intensity for recruitment of Af1521(K35E/Y145R)-EGFP expressed in U2OS cells following pre-treatment with vehicle (DMSO, 0.1%, 60 minutes) or the PARGi PDD00017273 (10μM, 30 minutes). Each point represents a single cell recruitment focus, graph shows mean ± SEM; (F) Relative peak intensity for recruitment of RNF146(100–182)-EGFP expressed in LN428 cells following pre-treatment with vehicle (DMSO, 0.1%, 60 minutes) or the PARGi PDD00017273 (10μM, 30 minutes). Each point represents a single cell recruitment focus, graph shows mean ± SEM; (G) Peak recruitment time of Af1521(K35E/Y145R)-EGFP foci, in U2OS cells Each point represents a single cell recruitment focus, graph shows mean ± SEM; (H) Peak recruitment time of RNF146(100–182)-EGFP foci in LN428 cells. Each point represents a single cell recruitment focus, graph shows mean ± SEM; (I) Plot depicting the dissociation dynamics of Af1521(K35E/Y145R) foci during 20 minutes time span following laser-induced DNA damage in U2OS cells. N ≥ 20 cells.; (J) Plot depicting the dissociation dynamics of RNF146(100–182) foci in LN428 cells during 20 minutes time span following laser-induced DNA damage in LN428 cells. N ≥ 20 cells. Recruitment foci having a relative peak intensity below 1.15 of first frame were excluded from the experiment and from statistical analysis in graphs (E-J). Exclusion percentages were 10% (2 foci) for DMSO treated RNF146(100–182) and 15% for (3 foci) PARGi treated RNF146(100–182). No exclusion was done in DMSO and PARGi- treated Af1521(K35E/Y145R). NS: no significance, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; a t-test was used for panel E, a Mann-Whitney test was used for Panels F, G, and H and a Kaplan-Meier test for panels I and J.

Figure 3.

Recruitment dynamics of RNF146(100–182)-EGFP and of Af1521(K35E/Y145R)-EGFP to sites of laser micro-irradiation. (A) Confocal micrograph images of cells expressing EGFP fusions with RNF146(100–182), Af1521(K35E/Y145R), and Af1521(WT) in U2OS cells, following laser micro-irradiation; (B) Recruitment of RNF146(100–182), Af1521(WT), and Af1521(K35E/Y145R), to sites of laser micro-irradiation (405nm) following BrdU sensitization (10μM, 24 hours), N≥25 cells, recruitment intensity normalized to first frame (F/F₀: Maximum Fluorescence intensity / Fluorescence intensity at t₀); (C) Relative peak intensity of recruitment for RNF146(100–182), Af1521(WT), and Af1521(K35E/Y145R), in U2OS cells. Each point represents a single cell recruitment focus, graph shows mean ± SEM. (F/F₀: Maximum Fluorescence intensity / Fluorescence intensity at t₀); (D) Peak recruitment time for RNF146(100–182), Af1521(WT), and Af1521(K35E/Y145R) expressed in U2OS cells. Each point represents a single cell recruitment focus, graph shows mean ± SEM; (E) Plot depicting the dissociation dynamics of RNF146(100–182), Af1521(K35E/Y145R) and Af1521(WT) foci in U2OS cells during 20 minutes following laser-induced DNA damage, N ≥ 40 cells. Recruitment foci having a relative peak intensity below 1.15 of the first frame were excluded from the experiment and from statistical analysis in graphs (C-E). Exclusion percentages were 5% (2 foci) for Af1521(K35E/Y145R) and 37.5% (15 foci) for Af1521(WT). No exclusion was made in RNF146(100–182). (F) Immunoblots of pull-down experiments using purified GST-tagged Af1521(K35E/Y145R) (100µg) or GST-tagged RNF146(100–182) (100µg), bound to glutathione agarose beads of PAR-containing ES-2 cell lysate under increasing salt concentration, as indicated on the left side of the blot (4°C, overnight). Eluates were transferred to nitrocellulose membranes and probed by PAR primary antibody. Graph shows mean ± SEM. NS: no significance, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; a Kruskal-Wallis test was used for panels C and D and a Kaplan-Meier test for panel E.

Figure 4.

Overexpression of the RNF146(100–182) WWE domain modulates PAR levels and dynamics at sites of laser micro-irradiation. (A) Schematic representation of the RNF146(100–182) WWE domain (fused to a myc-tag) and the engineered macrodomain Af1521(K35E/Y145R) (fused to EGFP). Af1521(K35E/Y145R)-EGFP is used for tracking PAR levels, and the modulation of PAR dynamics, impacted by the expression of the RNF146(100–182)-myc; (B) Recruitment of Af1521(K35E/Y145R)-EGFP in U2OS cells, after overexpression of RNF146(100–182)-myc, to sites of laser micro-irradiation (405nm) following BrdU sensitization (10μM, 24 hours), N≥49 cells, recruitment intensity normalized to first frame, (F/F₀: Maximum Fluorescence intensity / Fluorescence intensity at t₀); (C) Relative peak intensity of recruitment for Af1521(K35E/Y145R)-EGFP, after overexpression of RNF146(100–182)-myc, in U2OS cells. Each point represents a single cell recruitment focus. Graph shows mean ± SEM (F/F₀: Maximum Fluorescence intensity/ Fluorescence intensity at t₀); (D) Peak recruitment time for Af1521(K35E/Y145R)-EGFP, after overexpression of RNF146(100–182)-myc, in U2OS cells. Each point represents a single cell recruitment focus, graph shows mean ± SEM; (E) Plot depicting the dissociation dynamics of Af1521(K35E/Y145R) foci after overexpression of RNF146(100–182) in U2OS cells during 20 minutes following laser-induced DNA damage. N ≥ 48 cells. Af1521(K35E/Y145R) EGFP foci having a relative peak intensity below 1.15 of first frame were excluded from the experiment and from statistical analysis in graphs (C-E). Exclusion percentages were 4% (2 foci) for Af1521(K35E/Y145R) and no exclusion for Af1521(K35E/Y145R) foci after overexpression of RNF146(100–182). (F) Immunoblot probing PAR levels in cells expressing Af1521(K35E/Y145R) and/or the RNF146(100–182) and after treatment with the PARGi PDD00017273 (10μM, 8 hours) and MNNG (20μM, 1 hour); graph shows mean ± SEM. Af1521(K35E/Y145R)-EGFP foci having a relative peak intensity below 1.15 of first frame were excluded from the experiment and from statistical analysis in graphs (C-E). NS: no significance, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; a Kruskal-Wallis test was used for panel C, a Mann-Whitney test for panel D and a Kaplan-Meier test for panel E.

Figure 5.

Overexpression of the engineered macrodomain Af1521(K35E/Y145R) does not modulate PAR levels or dynamics at sites of laser micro-irradiation. (A) Schematic representation of the engineered macrodomain Af1521(K35E/Y145R) (fused to a myc-tag) and the RNF146(100–182) WWE domain (fused to EGFP). RNF146(100–182)-EGFP is used for tracking PAR levels, and the modulation of PAR dynamics, impacted by the expression of Af1521(K35E/Y145R)-myc; (B) Recruitment of RNF146(100–182)-EGFP in U2OS cells, after overexpression of Af1521(K35E/Y145R)-myc, to sites of laser micro-irradiation (405nm), N≥48 cells, recruitment intensity normalized to first frame, (F/F₀: Maximum Fluorescence intensity / Fluorescence intensity at t₀); (C) Relative peak intensity of recruitment for RNF146(100–182)-EGFP, after overexpression of Af1521(K35E/Y145R)-myc, in U2OS cells at sites of laser micro-irradiation (405nm). Each point represents a single cell recruitment focus, graph shows mean ± SEM (F/F₀: Maximum Fluorescence intensity / Fluorescence intensity at t₀); (D) Peak recruitment time RNF146(100–182)-EGFP, after overexpression of Af1521(K35E/Y145R)-myc, in U2OS cells at sites of laser micro-irradiation (405nm). Each point represents a single cell recruitment focus, graph shows mean ± SEM; (E) Plot depicting the dissociation dynamics of RNF146(100–182) foci after overexpression of Af1521(K35E/Y145R) in U2OS cells during 20 minutes following laser-induced DNA damage; N ≥ 48 cells. RNF146(100–182)-EGFP foci having a relative peak intensity below 1.15 of first frame were excluded from the experiment and from statistical analysis in graphs (C-E). Exclusion percentages were 4% (2 foci) for RNF146(100–182) and 2% (1 foci) for RNF146(100–182) foci after overexpression of Af1521(K35E/Y145R). NS: no significance, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; a Mann-Whitney test was used for panels C and D and a Kaplan-Meier test for panel E.

Figure 6.

WWE domain binding to sites of laser micro-irradiation is not influenced by the overexpression of RNF146(100–182)-myc. (A) Schematic representation of the RNF146(100–182) WWE domain (fused to a myc tag) and the RNF146(100–182) WWE domain (fused to EGFP). RNF146(100–182)-EGFP is used for tracking PAR levels, and the modulation of PAR dynamics, while competing for available binding sites with RNF146(100–182)-myc; (B) Recruitment of RNF146(100–182)-EGFP in U2OS cells, after overexpression of RNF146(100–182)-myc, to sites of laser micro-irradiation (405nm, N≥48 cells), recruitment intensity normalized to the first frame, (F/F₀: Maximum Fluorescence intensity / Fluorescence intensity at t₀); (C) Relative peak intensity of recruitment of RNF146(100–182)-EGFP, after overexpression of RNF146(100–182)-myc, in U2OS cells at sites of laser micro-irradiation (405nm). Each point represents a single cell recruitment focus, graph shows mean ± SEM (F/F₀: Maximum Fluorescence intensity / Fluorescence intensity at t₀); (D) Peak recruitment time for RNF146(100–182)-EGFP, after overexpression of RNF146(100–182)-myc, in U2OS cells at sites of laser micro-irradiation (405nm). Each point represents a single cell recruitment focus, graph shows mean ± SEM; (E) Plot depicting the dissociation dynamics of RNF146(100–182) foci after overexpression of RNF146(100–182)-myc in U2OS cells during 20 minutes following laser-induced DNA damage, N ≥ 48 cells. RNF146(100–182)-EGFP foci having a relative peak intensity below 1.15 of first frame were excluded from the experiment and from statistical analysis in graphs (C-E). Exclusion percentages were 2% (1 foci) for RNF146(100–182) and 4% (2 foci) for RNF146(100–182) foci after overexpression of RNF146(100–182)-myc. NS: no significance, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; a Mann-Whitney test was used for panels C and D and a Kaplan-Meier test for panel E.