PARP1-dependent recruitment of the FBXL10-RNF68-RNF2 ubiquitin ligase to sites of DNA damage controls H2A.Z loading
- New York University School of Medicine, United States
- Hungarian Academy of Sciences, Hungary
- Howard Hughes Medical Institute, New York University School of Medicine, United States
Figures
Figure 1 with 3 supplements
FBXL10, RNF68, and RNF2 are recruited to sites of DNA damage in a PARP1- and TIMELESS- dependent manner.
(A) Super-resolution imaging was performed in U-2OS cells to analyze the colocalization between XRCC5 (magenta) and FBXL10, RNF68 and RNF2 (green), with or without 20 min of NCS treatment. …
Figure 1—figure supplement 1
The trimeric FRUCC recruits to sites of DNA damage.
(A) HEK293T cells were transfected with FLAG-RNF68, HA-RNF2, and Myc-FBXL10. Cell lysates were immunoprecipitated with an anti-FLAG resin, followed by elution using 3x FLAG peptide. The eluate was …
Figure 1—figure supplement 2
Recruitment of the FRUCC to DNA damage sites.
(A) Representative images of the kinetic plots showed in Figure 1C. U-2OS cells stably expressing either GFP-FBXL10, mCherry-RNF68 and GFP-RNF2 and transfected with the indicated siRNAs, were …
Figure 1—figure supplement 3
Extended kinetics of FBXL10 recruitment, and TIMELESS-independent recruitment of XRCC1 and Ligase 3.
(A) U-2OS cells stably expressing either GFP-FBXL10 were pre-sensitized with BrdU (10 μM) for 36 hr and subjected to 405 nm laser induced damage. DNA damage recruitment dynamics were captured by …
Figure 2 with 2 supplements
PARP1-, TIMELESS-, and RNF68-dependent recruitment of the cPRC1 subunits, RNF51 and RNF110.
(A) U-2OS cells stably expressing GFP-FBXL10 (left), mCherry-RNF68 (middle) or GFP-RNF2 (right) were transfected with siRNAs targeting RNF51, RNF110 or a non-targeting control (CTRL). Cells were …
Figure 2—figure supplement 1
Representative images for Figure 2.
(A) Representative images of the kinetic plots showed in Figure 2A. U-2OS cells stably expressing either GFP-FBXL10, mCherry-RNF68 and GFP-RNF2 and transfected with the indicated siRNAs, were …
Figure 2—figure supplement 2
Mapping of FBXL10 and RNF2 binding domains in RNF68.
Left, Schematic representation of RNF68 mutants used in mapping experiments. RNF68 mutants found to interact with FBXL10 or RNF2 are indicated by the symbol (+). Right, HEK293T cells were …
Figure 3 with 1 supplement
The FRRUC is required for H2A-ubiquitylation at sites of DNA damage.
(A) U-2OS cells were transfected with the indicated siRNAs. Cells were fixed 30 min after laser microirradiation and stained with antibodies to γH2A.X (orange) and H2AK119Ub1 (green). Scale bar …
Figure 3—figure supplement 1
Representative efficiency of siRNA silencing for all oligos used in this study.
(A) U-2OS cells were transfected with the indicated siRNA sequences. The graph shows representative knockdown efficiencies of the indicated genes. Corresponding mRNA levels were analyzed using …
Figure 4 with 1 supplement
The FRUCC is required for transcriptional repression at sites of DNA damage.
(A) Schematic of the U-2OS reporter system that allows monitoring transcription following induction of DSBs. DSBs are induced in the LacO repeats by activating the ER-mCherry-LacI-FokI-DD fusion …
Figure 4—figure supplement 1
Top, representative immunoblots corresponding to Figure 4B; bottom, mode of quantification of experiments shown in Figure 4D–E.
(A) U-2OS FokI-YFP-MS2 reporter cells used to detect transcriptional repression upon DNA damage were transfected with either the indicated siRNAs or a non-targeting control (siCTRL). 48 hr later, …
Figure 5 with 2 supplements
The FRUCC promotes the DNA damage response and Homologous Recombination Repair.
(A) U-2OS cells were transfected with siRNA oligos targeting FBXL10, RNF68, RNF2, RNF8 or a non-targeting control (siCTRL). Cells were treated for 3 hr (BRCA1, Rad51) or 1 hr (FK2, pRPA S33) with …
Figure 5—figure supplement 1
Representative images for Figure 5A-B.
(A) U-2OS cells were transfected with siRNAs targeting FBXL10, RNF68, RNF2, RNF8, or a non-targeting control (siCTRL). Cells were treated with 0.1 μg/ml NCS (+) for 1 hr (FK2, pRPA S33, MDC1, …
Figure 5—figure supplement 2
The FRUCC promotes a proper DNA damage response and is needed for cell survival upon genotoxic stress.
(A) U-2OS cells were transfected with siRNAs targeting FBXL10, RNF68, or RNF2, or a non-targeting control (siCTRL). Cells were treated with 0.1 μg/mL NCS for 30 min or 1 μM CPT for 3 hr. Cells were …
Figure 6 with 1 supplement
The recruitment of H2A.Z to DSBs depends on the FRUCC.
(A) qChIP was performed in the DSB reporter U-2OS cells (see Figure 3C) with and without FokI-induced DSBs using the indicated antibodies. The results are shown as a ratio of H2A.Z to H2A levels. …
Figure 6—figure supplement 1
The recruitment to DSBs of H2A.Z, but not TIP48 and TIP49, depends on the FRUCC.
(A) U-2OS FokI reporter cells were used in qChIP experiments. The ER-mCherry-LacI-FokI-DD nuclease was induced for 1 hr using Shield-1 and 4-OHT. qChIP was performed with and without …
Figure 7 with 1 supplement
H2A.Z is involved in transcriptional repression at sites of DNA breaks and is required for efficient DSB signaling.
(A) U-2OS cells were transfected with H2A.Z siRNA or CTRL siRNA. Cells were fixed 30 min after laser micro-irradiation and stained with antibodies to γH2A.X (orange) and H2AK119Ub1 (green). Scale …
Figure 7—figure supplement 1
FokI levels in H2A.Z depleted cells.
U-2OS FokI-YFP-MS2 reporter cells used to measure transcription near DSB sites were transfected with siRNA against H2A.Z or a non-targeting control (siCTRL).
48 hr later cells were treated with Shield-1 ligand (250 nM) and 4-OHT (500 nM) for 4 hr. Cells were lysed, and immunoblotting was performed as indicated.
Author response image 1
U2OS cells were transfected with the indicated siRNAs.
After 72 h, cells were treated with NCS for 20 minutes. After harvesting, cells were fractionated into soluble and chromatin fractions, and lysates were immunoblotted as indicated. For chromatin …
Author response image 2
U-2OS cells stably expressing GFP-RNF8 (a kind gift from Dr D Durocher) were transfected with siRNAs targeting FBXL10, RNF68, RNF2 or H2A.Z, or a non-targeting control (CTRL).
Cells were pre-sensitized with BrdU (10 μM) for 36 hours and subjected to 405 nm laser induced damage. DNA damage recruitment dynamics were captured by live cell imaging. Relative fluorescence …
Additional files
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Source data 1
Raw data for all graphs in main figures and figure supplements.
- https://doi.org/10.7554/eLife.38771.020
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Transparent reporting form
- https://doi.org/10.7554/eLife.38771.021
