TDP-43 Promotes Neurodegeneration by Impairing Chromatin Remodeling Amit Berson, Ashley Sartoris, Raffaella Nativio, Vivianna Van Deerlin, Jon B. Toledo, Sílvia Porta, Shichong Liu, Chia-Yu Chung, Benjamin A. Garcia, Virginia M.-Y. Lee, John Q. Trojanowski, F. Brad Johnson, Shelley L. Berger, Nancy M. Bonini  Current Biology  Volume 27, Issue 23, Pages 3579-3590.e6 (December 2017) DOI: 10.1016/j.cub.2017.10.024 Copyright © 2017 Elsevier Ltd Terms and Conditions

Current Biology 2017 27, 3579-3590.e6DOI: (10.1016/j.cub.2017.10.024) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 H3K4me3-Related Chromatin Factors Modify TDP-43 Toxicity (A) Heatmap of TDP-43 modifiers, ranked by their impact on the external eye of YH3-Gal4>UAS-TDP-43 flies. Asterisks denote modifiers with functions related to H3K4me3. (B) Chd1 RNAi enhances and lid RNAi suppresses TDP-43-mediated neurodegeneration. Top: external eye images. Bottom: internal retinal structure. Arrows indicate retinal width. (C) Quantification of intact retinal tissue. Control, n = 5 animals; TDP-43, n = 14; lid, n = 6; Chd1, n = 8. F(3, 19) = 111.8, p < 0.0001, one-way ANOVA. n.s., p > 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, Tukey post hoc test. (D) Lifespan analysis of flies expressing TDP-43 and the indicated RNAi with the neuronal driver elav3A-Gal4. Lifespan was carried out at 29°C. TDP-43 expression shortens lifespan compared to flies expressing only control RNAi. Chd1 RNAi further shortens lifespan whereas lid RNAi prolongs lifespan. n = 200 flies per group. p < 0.001, log-rank test. (E) Knockdown of Chd1 or lid in the nervous system without TDP-43 expression has minimal effects on lifespan. Conditions were as in (D). (F) Lid and Set1 robustly modulate, but TDP-43 does not affect, brain H3K4me3 levels. F(3, 20) = 26.91, p < 0.0001, one-way ANOVA. n.s., p > 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, Tukey post hoc test. Graphs denote mean ± SEM. Full genotypes are detailed in Table S2. See also Figure S1 and Table S1. Current Biology 2017 27, 3579-3590.e6DOI: (10.1016/j.cub.2017.10.024) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Chd1 Depletion Promotes SG Formation (A) Chd1 knockdown efficiency in S2R+ cells. (B) Experimental flow. S2R+ cells were treated with double-stranded RNA (dsRNA) for 4 days, stressed with 0.2 mM sodium arsenite (SA) for 2 hr, fixed, immunostained, and imaged. For automated image analysis, images were deconvolved and max projected, and automated detection of cells and SGs was performed with CellProfiler. IF, immunofluorescence. Arrowheads indicate cells with SGs and arrows indicate cells without visible SGs. (C) Chd1 knockdown facilitates SG formation upon arsenite stress but does not induce SGs on its own. Red; FMR1; blue, DAPI. Scale bars, 7.5 μm. Dashed boxes indicate magnified area shown in the panel below. (D) Chd1 knockdown enhances SG number per cell, SG size, and the percentage of cells that stain positive for SGs. Control dsRNA, n = 183 cells; Chd1 dsRNA, n = 193 cells; 14 images per condition. ∗p < 0.05, ∗∗p < 0.01, Mann-Whitney test. SG, stress granule. See also Figure S3. Current Biology 2017 27, 3579-3590.e6DOI: (10.1016/j.cub.2017.10.024) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Chd1 Modulates Stress Response Impairments Caused by TDP-43 (A) Stress sensitivity assay. Flies were heat shocked for 2 hr at 40°C and allowed to recover overnight (O/N), after which the percentage of dead animals was scored. (B) Chd1-deficient flies are heat stress sensitive. Heteroallelic Chd1 null flies (Chd1[4]/Chd1[5]), ubiquitous Chd1 knockdown flies, and controls were heat shocked at 40°C for 2 hr. Wild-type, n = 6; Chd1 null, n = 6; control RNAi, n = 5; Chd1 RNAi, n = 5 biological replicates of 20–30 flies per replicate. ∗∗p < 0.01, ∗∗∗p < 0.001, two-tailed t test. (C) Ubiquitous expression of TDP-43 in adult flies induces heat stress hypersensitivity. Chd1 RNAi increases and overexpression of Chd1 reduces mortality of TDP-43 animals following stress. Control, n = 13; TDP-43, n = 23; TDP-43 + Chd1 RNAi, n = 11; TDP-43 + Chd1-OE, n = 13 biological replicates. F(3, 56) = 229.1, p < 0.001, one-way ANOVA. ∗∗p < 0.01, ∗∗∗p < 0.001, Tukey post hoc test. (D) Ubiquitous expression of wild-type or R406W Tau does not promote heat stress sensitivity. n = 3 biological replicates. n.s., p > 0.05, two-tailed t test. (E) Western immunoblot analysis of TDP-43 and Tau expression in whole flies. (F) Without additional stress, TDP-43 induces chaperone gene expression. qRT-PCR analysis of mRNA levels in whole flies. Following a 10-min heat stress at 35°C, TDP-43 impairs the heat shock response. mRNA levels are shown as fold induction from no stress, normalized to RpS20. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, two-tailed t test. n = 5 biological replicates. HS, heat shock; fc, fold change. (G) TDP-43 impairs and Chd1 rescues heat shock gene expression. Flies were heat shocked for 30 min at 35°C. Hsp mRNA levels are shown as fold induction from no stress, normalized to RpS20. Hsp40 F(2, 11) = 25.55, p < 0.001; Hsp68 F(2, 12) = 23.9, p < 0.001; Hsp70 loci F(2, 12) = 25, p < 0.00; Hsp83 F(2, 12) = 21.35, p < 0.001. ∗p < 0.05, one-way ANOVA followed by Tukey post hoc test. (H) TDP-43 impairs heat shock gene induction in mammalian cells. TetON-GFP and TetON-GFP-TDP-43 cells were induced with doxycycline for 6 days, after which heat shock was applied for 1 hr at 46°C, and RNA was extracted for qRT-PCR analysis at 6 and 8 hr of recovery. ∗p < 0.05, ∗∗∗p < 0.001, two-tailed t test. n = 6 biological replicates. Expression data are relative to the control non-heat shock samples (defined as 1) to allow direct comparison between control and TDP-43 samples between the different stress conditions. All graphs denote mean ± SEM. Full genotypes are detailed in Table S2. See also Figure S2. Current Biology 2017 27, 3579-3590.e6DOI: (10.1016/j.cub.2017.10.024) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 TDP-43 Impairs Nucleosomal Dynamics following Stress (A) MNase protection assay experimental design. (B) Control (non-heat shock) and heat shock (10 min at 35°C) flies were fixed and used for MNase protection. Results of real-time qPCR with primers spanning Hsp70 loci are shown as digested/undigested (total) chromatin. Two-way ANOVA revealed a significant effect of group F(5, 388) = 572.6, p < 0.001. ∗p < 0.05, ∗∗∗p < 0.001, Tukey post hoc test. n = 6 biological replicates. Error bars show SEM. (C) Histone H3 ChIP experimental design. (D) Control (non-heat shock) and heat shock (10 min at 35°C) flies were fixed and used for total histone H3 ChIP. Results of real-time qPCR with primers spanning Hsp70 loci are shown as IP/input ratio. Two-way ANOVA revealed a significant effect of group F(5, 231) = 289.0, p < 0.001, ∗∗∗p < 0.001, Tukey post hoc test. n = 6 biological replicates. Error bars show SEM. Chd1-OE, Chd1 overexpression. For (B) and (D), shaded areas show the range of nucleosome-occupied gene body without stress, for comparison with heat shock graphs. (E) Flies expressing tagged Chd1-HA, with or without TDP-43, were subjected to heat shock at 35°C or control temperatures for 10 min, fixed, and used for ChIP analysis. n = 6 biological replicates. Results are shown as IP/input ratio. Hsp70(−190) F(3, 20) = 13.35, p < 0.001; Hsp70(−140) F(3, 20) = 6.79, p < 0.01; Hsp70(−25) F(3, 20) = 45.18, p < 0.001; Hsp70(+247) F(3, 20) = 57.51, p < 0.001; Hsp70(+280) F(3, 20) = 63.97, p < 0.001; Hsp70(583) F(3, 20) = 53.1, p < 0.001; Hsp70(872) F(3, 20) = 14.89, p < 0.001; Hsp70(1275) F(3, 20) = 44.84, p < 0.001. One-way ANOVA followed by Tukey post hoc test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n.s., not significant. Error bars show SEM. x axis labels describe the location of the reverse primer relative to the transcription start site. Full genotypes are detailed in Table S2. See also Figures S4 and S5. Current Biology 2017 27, 3579-3590.e6DOI: (10.1016/j.cub.2017.10.024) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 TDP-43 Physically Interacts with Drosophila Chd1 and Human CHD2 (A) Human CHD1 and CHD2 are orthologs of Drosophila Chd1. (B) Adult Drosophila soluble and chromatin fractions were used for IP. IP samples were analyzed using western immunoblot with an anti-HA antibody to detect Chd1-HA. IgG IP serves as control. β-tubulin western immunoblot shows no significant interaction with TDP-43. (C and D) Human HEK293 cells were fractionated to soluble and chromatin fractions, and IP was performed with anti-TDP-43 antibodies (C), anti-CHD2 antibodies (D), or rabbit IgG as negative control. Note the selective interactions of CHD2 and TDP-43 primarily in the soluble fraction. Full genotypes are detailed in Table S2. See also Figure S5. Current Biology 2017 27, 3579-3590.e6DOI: (10.1016/j.cub.2017.10.024) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 Abnormal TDP-43 Accumulations Promote Loss of CHD2 in Cultured Cells and Human FTD Cortex (A) CHD2 immunostaining in TetOn-GFP and TetOn-GFP-TDP-43 cells induced with doxycycline for 6 days. (B) Quantification of (A); ∗∗∗p < 0.001, two-tailed t test; n = 412 GFP cells, n = 332 GFP-TDP-43 cells. CHD2 intensity is decreased. (C) Reduced CHD2 intensity in cells bearing abnormal cytoplasmic TDP-43 in TetOn-GFP-TDP-43 cells. (D) Quantification of (C); ∗∗∗p < 0.001, two-tailed t test; n = 333 cells with nuclear TDP-43 localization, n = 54 cells with cytoplasmic TDP-43 localization. (E) Double immunolabeling of TDP-43 (green) and CHD2 (red) reveals reduced levels of CHD2 in FTD temporal cortex. (F) Quantification of CHD2 intensity in human post-mortem FTD temporal cortex. n = 6 controls, n = 8 FTD; ∗∗∗p < 0.001, two-tailed t test. See also Table S4. (G) Quantification of CHD2 intensity in FTD temporal cortex cells with nuclear or cytoplasmic TDP-43. ∗∗∗p < 0.001, two-tailed t test. (H) Model of TDP-43-mediated impairments in chromatin remodeling. See text for details. Scale bars, 10 μm. Arrowheads in graphs show mean values. See also Figures S5 and S6 and Table S4. Current Biology 2017 27, 3579-3590.e6DOI: (10.1016/j.cub.2017.10.024) Copyright © 2017 Elsevier Ltd Terms and Conditions