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Interaction of FUS and HDAC1 regulates DNA damage response and repair in neurons

Abstract

Defects in DNA repair have been extensively linked to neurodegenerative diseases, but the exact mechanisms remain poorly understood. We found that FUS, an RNA/DNA-binding protein that has been linked to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration, is important for the DNA damage response (DDR). The function of FUS in DDR involved a direct interaction with histone deacetylase 1 (HDAC1), and the recruitment of FUS to double-stranded break sites was important for proper DDR signaling. Notably, FUS proteins carrying familial ALS mutations were defective in DDR and DNA repair and showed a diminished interaction with HDAC1. Moreover, we observed increased DNA damage in human ALS patients harboring FUS mutations. Our findings suggest that an impaired DDR and DNA repair may contribute to the pathogenesis of neurodegenerative diseases linked to FUS mutations.

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Figure 1: FUS is important for DDR and repair in proliferative cells and in postmitotic neurons.
Figure 2: FUS is rapidly recruited to DSBs and is one of the earliest proteins to respond to DNA damage.
Figure 3: FUS directly interacts with HDAC1 and both proteins are necessary for successful DNA repair.
Figure 4: The G-rich and C-terminal domains of FUS directly interact with HDAC1, and this interaction is important for successful DSB repair.
Figure 5: Cells expressing human fALS FUS mutations exhibit impaired DNA repair efficiency and a diminished FUS-HDAC1 interaction.
Figure 6: fALS patients harboring FUS mutations exhibit increased DNA damage.

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Acknowledgements

We thank T. Misteli (National Cancer Institute) for kindly providing the NIH2/4 cell line and Lac-R constructs, M. Dobbin (Massachusetts Institute of Technology) for technical assistance on microirradiation experiments, A. Mungenast, R. Madabhushi, A. Bero, J. Penney and A. Nott for critical reading of the manuscript, and other members of the Tsai and Huang laboratories for helpful discussions. We also thank R.H. Brown Jr and L.J. Hayward for insightful discussions. W.-Y.W. was supported by a Postdoctoral Fellowship from the Simons Foundation, J.C.J. was supported by the Howard Hughes Medical Institute Exceptional Research Opportunities Program. This work is also supported by grants from National Institute of Neurological Disorders and Stroke (NS078839 to L.-H.T.), the Department of Veterans Affairs (BX001108 to E.J.H.) and the Muscular Dystrophy Association (MDA217592 to E.J.H.). L.-H.T. is a member of the Neurodegeneration Consortium. E.J.H. is a member of the Consortium for Frontotemporal Dementia Research. L.-H.T. receives funding from the Howard Hughes Medical Institute.

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W.-Y.W. performed the DNA repair assay, in vitro protein interaction, domain mapping assay, created mutant cell lines, and completed the immunoprecipitation, ChIP and qPCR assays. L.P. performed the micro-irradiation and comet assays and evaluated DNA damage response in cultured neurons. S.C.S. purified full-length and fragment GST-tagged proteins. E.J.Q. and M.S. helped with neuronal culture. J.C.J. helped with the in vitro GST pulldown assay. E.J.H. and I.R.A.M. provided brain sections from control individuals and human fALS patients. L.-H.T. and E.J.H. supervised the project, and W.-Y.W. and L.-H.T. wrote the manuscript.

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Correspondence to Li-Huei Tsai.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Schematic figures of HR and NHEJ reporter systems used in this study.

(a). The I-SceI mediated DSB induction and repair by HR. A I-SceI endonuclease recognition site was inserted into the GFP coding sequence to create a nonfunctional GFP transgene, and a wild type GFP fragment was inserted downstream of the GFP tansgene. Transfection of I-SceI expressing construct will induce a DSB at the I-SecI site, and repair of this DSB by HR will successfully restore a functional GFP, which could be detected by fluorescence microscope or by FACS. Arrows in up panel (red) represent the primers used for ChIP-qPCR analysis of the repair proteins accumulted at DSBs. (b). Reporter construct for NHEJ. The GFP coding sequence was interrupted by inserting an adenoviral exon flanked by artificial introns. The GFP is inactive due to the expression of the adeno exon in GFP gene; however, once digested with HindIII or I-SceI, the exon will be cutted out and a DSB will be created. Successful repair of this DSB using NHEJ will restore the expression of GFP. (Adapted from Oberdoerffer et al, cell, 2008; Seluanov et. al. PNAS. 2004).

Supplementary Figure 2 Verification of the shRNAs and siRNA used in this study.

(a-c). Different siRNAs targeting human BRCA2, LIG4, FUS and shRNAs targeting mouse Fus (shRNA2 and 3) or human FUS (shRNA 2, 4-6) were transfected into 293T (a,c) cells and N2A cells (b) for 72 hours and cell lysate was collected for western blotting.

Supplementary Figure 3 Immunofluorescence staining of 53BP1 and γH2AX in vehicle treated neurons following Fus knockdown.

(a). Primary cortical neurons were transfected with plasmids expressing scrambled shRNA or Fus shRNAs together with mCherry, treated with vechile for 1 hour fixed and labeled for yH2AX. No yH2AX immunoreactivity was observed in both transfected (white arrows) and non-transfected neurons. scale bar: 8μm (b). Primary cortical neurons were transduced with lentivirus carrying shRNAs targeting Fus, or scrambled control shRNA. Neurons were treated with vehicle for 1 h and labeled with anti-53BP1 antibody. 53BP1 is uniformly distributed in the nuclei. Scale bar: 4μm

Supplementary Figure 4 Immunofluorescence staining of phospho-ChK2 (p-ChK2) in cultured primary neurons treated with ETO.

(a) Primary cortical neurons were transfected with plasmids expressing scrambled shRNA or Fus shRNAs together with mCherry. 5 AM etoposide was added to induce DNA DSBs. Following 1 h etoposide treatment, neurons were fixed and labeled for p-ChK2 immunoreactivity. Fus knockdown neurons show reduced p-ChK2 immunoreactivity compared to scrambled shRNA-expressing neurons (white arrows). (b) p-ChK2 signal intensity in transfected neurons was measured by ImageJ (NIH). Scale bar: 8um. (** p<0.01, one-way ANOVA). (c). Verification of p-ChK2 antibody used for the immunofluorescnence staining.The p-ChK2 antibody (Cell Signaling #2661) detects a major band of approximately 62 kD. When the blot was stripped and re-blotted with ChK2 antibody (Cell Signaling #2662), the same 62 kD species was recognized. The p-ChK2 signal was minimal in vehicle-treated neurons and was enhanced with etoposide treatment, whereas phosphatase treatment completely abolished the signal. Note that the ChK2 antibody (Cell Signaling #2662) can recognize both human and mouse ChK2.

Supplementary Figure 5 In vivo interaction between HDAC and FUS.

Nuclear extracts prepared from WT mouse hippocampus were immunoprecipitated with antibodies against HDAC1 and HDAC2 and blotted with anti-FUS antibody.

Supplementary Figure 6 Micro-irradiation assay for HDAC1 and γH2AX.

(a). lmmunolabeling for HDAC1 and yH2AX was conducted at the indicated times following the induction of DSBs via laser micro-irradiation of U20S cells. Scale bar: 8um. (b). Fluorescence intensity in the laser-irradiated area at each time points was first normalized to the signal of whole nucleus and then to the peak fluorescence intentsity accross time. (n=5-10)

Supplementary Figure 7 Input controls for the experiment depicted in Figure 4b.

Flag tagged FUS fragments (FG) 4, 5, 7, or 4+7 were transfected into 293T cells together with FUS-mCherry for 48 hr and processed for immunoprecipitation as shown in Figure 4b. This figure shows the input controls for these experiments.

Supplementary Figure 8 Cellular distribution of wild type and mutant FUS.

Primary cultured neurons were transfected with FUS-WT, FUS-R244C, FUS-R514S, FUS-H517Q, or FUS-R521C, and the percentage of the cells showing a cytoplasmic accumulation of FUS was analyzed for each condition (n ≥ 50, ns: no significant difference. ***p<0.001, unpaired t-test). Scale bar: 6μm

Supplementary Figure 9 Laser micro-irradiation assay in U2OS cells expressing wild type FUS or fALS FUS mutants.

Endogenous FUS was knocked down and replaced by mCherry tagged wild type or mutant FUS. Cells were fixed 10 minutes after laser irradiation and processed for immunochemistry with anti-mCherry antibody.Scale bar: 4μm.

Supplementary Figure 10 Full-length pictures of the blots presented in the main figures.

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Supplementary Figures 1–10 and Supplementary Table 1 (PDF 26595 kb)

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Wang, WY., Pan, L., Su, S. et al. Interaction of FUS and HDAC1 regulates DNA damage response and repair in neurons. Nat Neurosci 16, 1383–1391 (2013). https://doi.org/10.1038/nn.3514

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