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. 2012 May 30;31(11):2511-27.
doi: 10.1038/emboj.2012.104. Epub 2012 Apr 24.

A new non-catalytic role for ubiquitin ligase RNF8 in unfolding higher-order chromatin structure

Martijn S Luijsterburg  1 , Klara Acs, Leena Ackermann, Wouter W Wiegant, Simon Bekker-Jensen, Dorthe H Larsen, Kum Kum Khanna, Haico van Attikum, Niels Mailand, Nico P Dantuma
Affiliations

A new non-catalytic role for ubiquitin ligase RNF8 in unfolding higher-order chromatin structure

Martijn S Luijsterburg et al. EMBO J. .

Abstract

The ubiquitin ligases RNF8 and RNF168 orchestrate DNA damage signalling through the ubiquitylation of histone H2A and the recruitment of downstream repair factors. Here, we demonstrate that RNF8, but not RNF168 or the canonical H2A ubiquitin ligase RNF2, mediates extensive chromatin decondensation. Our data show that CHD4, the catalytic subunit of the NuRD complex, interacts with RNF8 and is essential for RNF8-mediated chromatin unfolding. The chromatin remodelling activity of CHD4 promotes efficient ubiquitin conjugation and assembly of RNF168 and BRCA1 at DNA double-strand breaks. Interestingly, RNF8-mediated recruitment of CHD4 and subsequent chromatin remodelling were independent of the ubiquitin-ligase activity of RNF8, but involved a non-canonical interaction with the forkhead-associated (FHA) domain. Our study reveals a new mechanism of chromatin remodelling-assisted ubiquitylation, which involves the cooperation between CHD4 and RNF8 to create a local chromatin environment that is permissive to the assembly of checkpoint and repair machineries at DNA lesions.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Tethering histone ubiquitin ligases triggers localized ubiquitylation of histone H2A. (A) Schematic representation of the targeting approach. The white circles represent nucleosomes some of which contain LacO repeats. (B) Schematic representation of the mCherry-LacR-RNF fusion proteins. Mutations in the MIU domains of RNF168 are indicated in red. (C–E) Tethering of ubiquitin ligases (red) in NIH2/4 cells and analysis of (C) uH2A, (D) poly-ubiquitin conjugates (FK1) and (E) Lys63-linked poly-ubiquitin signal at the array (green). (F) Cross-recruitment of ubiquitin ligases. (G) Quantification of the percentage of cells with positive signals at the array after tethering the indicated fusion proteins. Catalytic inactive ubiquitin ligases are indicated with an asterisk. Note that in the RNF168 panel, we have tethered either LacR-RNF8, LacR-RNF8*RING or LacR-RNF168 as indicated on the x-axis, while we monitored the recruitment of GFP-RNF168WT or GFP-RNF168*MIU as indicated above the bars. Values represent the mean of two independent experiments (n=50 cells). The scale bars are 5 μm.
Figure 2
Figure 2
Higher-order chromatin alterations by tethering histone ubiquitin ligases in AO3 cells. (A–H) AO3 cells containing a 90-Mbp heterochromatic array were transfected with (A) mCherry-LacR, (B) mCherry-LacR-RNF2, (C) mCherry-LacR-RNF168, (D) mCherry-LacR-RNF8, (E) mCherry-LacR-RNF8*RING, (F) mCherry-LacR-RNF8*FHA, (G) mCherry-LacR-RNF8*FHA*RING and (H) mCherry-LacR-RNF168*RING fusion protein. Images show confocal slices of the nuclear distribution of the mCherry-LacR fusion proteins. The LUT for the colour coding is shown left of all panels. Cells were co-transfected with GFP-NLS (not shown) to measure the surface area of the nucleus. Quantitative measurements on the relative size of the array (area of the array/area of the nucleus) on a large numbers of cells (∼100 cells for each condition) are shown next the images as histograms. The mean size of the array (in % of nuclear area) for each condition is shown in red in the histograms. The scale bar is 2 μm. NIH2/4 cells containing a 256 × LacO array (10 kbp) were transfected with (I) mCherry-LacR, (J) mCherry-LacR-RNF8 or (K) mCherry-LacR-RNF168 (red). An overlay of the mCherry-LacR signal and counterstaining with Hoechst 33258 (white) is shown. Quantitative measurements on the relative size of the array (in ‰ of nuclear area) are shown next to the images as histograms. Values represent the mean of ∼50 cells from two independent experiments. The scale bar is 5 μm. Note that the array in NIH2/4 cells (∼10 kbp) is about 9000 times smaller than the array in AO3 cells (∼90 Mbp). (L) The relative increase in the array size induced by RNF8 in NIH2/4 and AO3 cells, about five-fold, is very similar between these two cell types. Thus, RNF8 unfolds chromatin structure ∼5-fold irrespective of cell type.
Figure 3
Figure 3
RNF8 recruits chromatin remodelling ATPase CHD4. Tethering of mCherry-LacR-RNF8 (red) to the LacO array in NIH2/4 cells triggers recruitment of (A) endogenous CHD4 (green), and (B) GFP-HDAC1 (green), while (C) mCherry-LacR-RNF168 (red) immobilization does not trigger endogenous CHD4 recruitment (green) to the array. Tethering of (D) mCherry-LacR-RNF8*FHA/*RING and (E) mCherry-LacR-RNF8 following treatment with PARP inhibitors also results in endogenous CHD4 recruitment (green). (F) mCherry-LacR-RNF8-mediated unfolded chromatin structures (red) in AO3 cells are decorated with endogenous CHD4 (green). The merge also shows staining with Hoechst 33258 (2 μg/ml). (G) Quantification of the percentage of cells with positive signal (CHD4 staining) at the array after tethering the indicated mCherry-LacR fusion proteins. Values represent the mean of two independent experiments (n=50 cells). (H) GFP-RNF8WT or GFP-RNF8*FHA/*RING was immunoprecipitated from U2OS cells followed by immunoblotting with CHD4 and GFP antibodies. WCE; whole-cell lysates. (I) HEK293T cells were transfected with plasmids encoding GFP or GFP-CHD4 for 24 h, and cells were lysed under denaturing conditions and subjected to GFP immunoprecipitation (IP). The beads were then washed extensively in denaturing buffer, re-equilibrated in native buffer and incubated or not with 100 ng bacterially purified, recombinant His6-RNF8 as indicated. After thorough washing, the bound complexes were resolved on SDS–PAGE and immunoblotted with RNF8 and GFP antibodies. (J) A series of overlapping GST-tagged CHD4 fragments were immobilized on GSH Sepharose and incubated with purified, recombinant His6-RNF8. Bound complexes were analysed by immunoblotting with RNF8 antibody. Figure source data can be found with the Supplementary data.
Figure 4
Figure 4
The FHA domain of RNF8 regulates chromatin decondensation and CHD4 recruitment in a phospho-independent manner. (A) Schematic representation of the RNF8 deletion mutants used in (A–J). The mutations to inactive the FHA and RING domain are indicated in red. (B–F) Chromatin decondensation in AO3 cells upon tethering the indicated RNF8 deletion mutants to the array. (G) Quantification of the percentage of cells with positive signal (CHD4 staining) at the array after tethering the indicated RNF8 fusion proteins. Values represent the mean of two independent experiments (n=30 cells). (H–L) Recruitment of endogenous CHD4 in NIH2/4 cells induced by tethering of the indicated RNF8 deletion mutants.
Figure 5
Figure 5
RNF8-dependent chromatin decondensation depends on the ATPase activity of CHD4. (A) Schematic representation of the CHD4 fusion proteins. (B–F) Chromatin decondensation in AO3 cells upon tethering the indicated CHD4 fusion proteins or LacR to the array. (G) Western blot analysis of AO3 cells transfected with control siRNA or CHD4 siRNA. (H) Immunofluorescence microscopy on cells transfected with control siRNA or CHD4 siRNA. Cells were pre-extracted, fixed and stained with antibodies against CHD4. (I–L) Chromatin decondensation upon tethering mCherry-LacR-RNF8 to the array in AO3 transfected with (I) siNeg, (J) siCHD4, (K) GFP-CHD4WT or (L) dominant-negative GFP-CHD4K757A. Quantitative measurements on the relative size of the array (in % of nuclear area) are shown next to the images as histograms. The values represent the mean of ∼50 cells from two independent experiments. The mean size of the array (in % of nuclear area) for each condition is shown in red in the histograms. The LUT for the colour coding is shown left of the panel. Figure source data can be found with the Supplementary data.
Figure 6
Figure 6
RNF8 regulates recruitment of CHD4 to DSBs independently of its ubiquitin-ligase activity. (A) U2OS cells stably expressing a doxycycline (dox)-inducible shRNA targeting RNF8 (shRNF8) were induced (lower panel) or not (upper panel) with dox for 48–72 h, exposed to laser micro-irradiation and 15 min later co-immunostained with antibodies to γH2AX (red) and CHD4 (green). (B) U2OS/shRNF8 cells engineered to induce WT or *RING alleles of FLAG-tagged, shRNA-resistant RNF8 were incubated with dox (48–72 h), exposed to laser micro-irradiation and 15 min later co-immunostained with antibodies to γH2AX (red) and CHD4 (green). Quantification of the accumulation of (C) endogenous CHD4 or (D) endogenous 53BP1 accumulation at laser-generated DSBs marked by γH2AX in the indicated cell lines. (E) U2OS/shRNF8 cells were treated with PARP inhibitor (10 μM) for 1–2 h and the expression of an shRNA targeting RNF8 was either induced with dox (48–72 h; lower panel) or not (upper panel). Cells were subsequently exposed to laser micro-irradiation and 15 min later co-immunostained with antibodies to γH2AX (red) and CHD4 (green). (F) Quantification of the accumulation of endogenous CHD4 at laser-generated DSBs using the indicated conditions. Graphs represent the relative fluorescence increase in the micro-irradiated area calculated by subtracting the background intensity in the undamaged parts of the nucleus from the intensity in the micro-irradiated area. The relative accumulation of CHD4 (C, F) or 53BP1 (D) in the absence of dox was normalized to 1 for comparison. Values represent the mean of ∼50 cells from at least two independent experiments. Error bars indicate the s.d. Stars (**) indicate highly significant differences (P=3.8 × 10−6 for panel (C), P=9.4 × 10−13 and 8.1 × 10−17 for panel (D) and P=2.8 × 10−4 and 1.9 × 10−4 for panel (F)) based on a t-test.
Figure 7
Figure 7
The ATPase activity of CHD4 regulates DNA damage-induced ubiquitin conjugation and efficient BRCA1 assembly. (A) U2OS cells expressing either GFP-CHD4WT or dominant-negative GFP-CHD4K757A were micro-irradiated and 15 min later co-immunostained with antibodies to MDC1 (red) and conjugated ubiquitin (FK2; white). Non-transfected cells were also micro-irradiated to serve as an internal control. (B) Similar to (A) except that cells were co-immunostained with antibodies to MDC1 (red) and BRCA1 (white). Stars (**) indicate highly significant differences (P=5.5 × 10−8 for panel (A), P=5.0 × 10−5 for panel (B) based on a t-test. (C) Western blot analysis of U2OS cells transfected with the indicated siRNAs and GFP constructs. Membranes were probed with antibodies to CHD4, GFP and α-tubulin. (D–F) U2OS cells were transfected with the indicated siRNAs and GFP constructs, irradiated with IR (2 Gy) and stained for (D) RNF168, (E) FK2 or (F) BRCA1. Note that cells stained for RNF168 were Triton X-100 extracted before fixation, which resulted in loss of the GFP-NLS signal. Quantification of the percentage of cells with >10 foci per nucleus is shown below the images. Values represent the mean of >100 cells from at least two independent experiments. Error bars indicate the s.d. Figure source data can be found with the Supplementary data.
Figure 8
Figure 8
The PARP-dependent recruitment of CHD4 does not contribute to BRCA1 assembly. (A) U2OS cells were treated with PARP inhibitor (10 μM) for 1–2 h (lower panel) or not (upper panel), exposed to laser micro-irradiation and 15 min later co-immunostained with antibodies to MDC1 (red) and BRCA1 (green). Quantification of the accumulation of DNA damage-induced ubiquitin conjugation or endogenous BRCA1 at laser-generated DSBs using the indicated conditions are shown next to the micrographs. Graphs represent the relative fluorescence increase in the micro-irradiated area calculated by subtracting the background intensity in the undamaged parts of the nucleus from the intensity in the micro-irradiated area. The relative accumulation of BRCA1 in the absence of PARP inhibitors was normalized to 1 for comparison. Values represent the mean of ∼50 cells. Error bars indicate the s.d. (B, C) U2OS cells transfected with (B) siLUC or (C) siRNF8 were treated with PARP inhibitor (10 μM) for 1–2 h or not, irradiated with IR (2 Gy) and stained for BRCA1. Quantification of the percentage of cells with >10 foci per nucleus is shown below the images. Values represent the mean of >300 cells from at least two independent experiments. Error bars indicate the s.d.
Figure 9
Figure 9
Model for chromatin remodelling-assisted ubiquitylation in the DDR. An RNF8 dimer (orange oval) is recruited to a DSB by binding to phosphorylated MDC1. The recruited RNF8 dimer binds CHD4 (blue oval) through its FHA domain in a phospho-independent manner, resulting in local chromatin decondensation, which subsequently enhances ubiquitin conjugation at DSBs and association of RNF168 and BRCA1.
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