Toxic expanded GGGGCC repeat transcription is mediated by the PAF1 complex in C9orf72-associated FTD

Abstract

An expanded GGGGCC hexanucleotide of more than 30 repeats (termed (G4C2)₃₀₊) within C9orf72 is the most prominent mutation in familial frontotemporal degeneration (FTD) and amyotrophic lateral sclerosis (ALS) (termed C9⁺). Through an unbiased large-scale screen of (G4C2)₄₉-expressing Drosophila we identify the CDC73/PAF1 complex (PAF1C), a transcriptional regulator of RNA polymerase II, as a suppressor of G4C2-associated toxicity when knocked-down. Depletion of PAF1C reduces RNA and GR dipeptide production from (G4C2)₃₀₊ transgenes. Notably, in Drosophila, the PAF1C components Paf1 and Leo1 appear to be selective for the transcription of long, toxic repeat expansions, but not shorter, nontoxic expansions. In yeast, PAF1C components regulate the expression of both sense and antisense repeats. PAF1C is upregulated following (G4C2)₃₀₊ expression in flies and mice. In humans, PAF1 is also upregulated in C9⁺-derived cells, and its heterodimer partner, LEO1, binds C9⁺ repeat chromatin. In C9⁺ FTD, PAF1 and LEO1 are upregulated and their expression positively correlates with the expression of repeat-containing C9orf72 transcripts. These data indicate that PAF1C activity is an important factor for transcription of the long, toxic repeat in C9⁺ FTD.

Figure 1:. A genetic screen reveals PAF1C as a suppressor of (G4C2)49-toxicity in the fly eye.

(a) UAS-(G4C2)n transgenes were designed expressing a pure repeat. (b) PCR reactions were used to quantify of the number of repeats in individual UAS-G4C2 transgenic fly lines. Shown: individual data points with mean from 2 experiments (30 flies/experiment). (c) RNA expression of UAS-(G4C2)n transgenes using HS-GAL4 were compared by northern blots. Statistics: ANOVA with Tukey’s correction, p-value: no significance >0.05. Shown: individual data points with mean±SEM; mean value of biological triplicates (n=30 flies) from 3 independent experiments. (d) Expression of UAS-(G4C2)n transgenes in the fly eye compared to controls: (G4C2)8 had no effect, (G4C2)29 caused mild disruptions in 80% of animals, (G4C2)49 caused strong degeneration. Shown: data from one experiment; data reproduced in 3+ independent experiments. Arrows: internal tissue depth, lost tissue. (e) RNAi were co-expressed with (G4C2)49 (III) within the fly optic system. Effects of RNAi were recorded: “suppressors” reduced degeneration, “enhancers” increased degeneration. Shown: representative images. Hits were independently tested 3+ times to confirm reproducibility (>5 flies examined/cross). (f) 119 modifiers were identified. Control experiments excluded 231 RNAi lines with unspecific effects. (g-h) GO analyses revealed terms enriched in suppressors (55/3582 genes) or enhancers (64/3582 genes). Plotted: significant (p-value ≤10^−3) enrichment scores of >3.00. (d-e) Scale bars: external eye =100μm, internal eye =25μm. (a-h) Additional details for this and subsequent figures: Supplementary Fig. 1 (extended screen data), Supplementary Data (all screen results), Supplementary Table 1 (detailed sampling/reproducibility/statistics), methods.

Figure 2:. PAF1C is not a modifier of (GR)36 or TDP43 toxicity in Drosophila.

(a) The 119 (G4C2)49 modifiers were analyzed in (GR)36 and TDP43 models to determine if they could act on GR-dipeptide toxicity or had overlapping effects on TDP43-toxicity. All were independently tested 3+ times to confirm reproducibility of results (>5 flies examined/cross). Scale bars: 100μm. (b) Of the 119 modifiers of (G4C2)49 toxicity, 71 (59.7%) similarly modified (GR)36, arguing they may be acting on toxic DPR. 63 (52.9%) similarly affected TDP-43 toxicity, arguing overlap between these disease models. (c) GO analyses revealed terms enriched in the modifiers that did not similarly alter (GR)36 toxicity (48/3582 genes), revealing those acting selectively on the (G4C2)49 RNA model. (d) GO analyses revealed terms enriched in the modifiers that did not similarly alter TDP43 toxicity (56/3582 genes), revealing those specific to the expanded G4C2 repeat in C9+ FTD/ALS. (c-d) Plotted: GO-terms with significant (p-value ≤ 10^−3) enrichment scores of >3.00.

Figure 3:. Reduced expression of components of PAF1C suppress (G4C2)49-induced toxicity in multiple contexts in the fly.

(a-b) dPAF1C RNAi mitigates toxicity associated with (G4C2)49 expression in the eye. N flies: control=9, dPaf1=7, dLeo1=6, dCDC73=8, dCtr9=6, dRtf1=5. (c-d) dPAF1C RNAi has no effect on control fly eyes. N flies: control=9, dPaf1=5, dLeo1=9, dCDC73=9, dCtr9=5, dRtf1=4. (a-d) Internal retina depth (arrows) quantified for individual animals. Scale bars: external eye =100μm, internal eye =45μm. (e) Climbing deficits caused by (G4C2)49 expression in the adult nervous system (ElavGS, 14d) are rescued by dPAF1C RNAi. N flies: control=117, dPaf1=98, dLeo1=108, dCDC73=103, dCtr9=120, dRtf1=115. Individual data points are mean % of animals that could climb per tube; average of 19±2 animals per tube. (f) dPAF1C RNAi mitigated vacuole formation (arrowheads) in the brain with ElavGS driven expression of (G4C2)49. For quantification, a vacuole severity scoring system was developed where 0=no vacuoles and 4=medium/large, frequent (>5) vacuoles (see Sup. Fig. 6a). N flies: (G4C2)0=9, (G4C2)49: control=10, dPaf1=10, dLeo1=7, dCDC73=10, dCtr9=8, dRtf1=9. Scale bars: 50μm. (g) Knockdown of dCDC73 in adult flies ubiquitously expressing (G4C2)49 results in lifespan extension. N flies: control=198, dCDC73=197. (b,d,f) each data point represents one animal. Statistics: (a-f) ANOVAs with Tukey’s correction, (g) log-rank; p-values: ****<0.0001, ***<0.001, **<0.01, *<0.05, no significance (n.s.) >0.05. Shown on graphs: individual data points with mean±SD; data from one experiment; all experiments were repeated twice with similar results.

Figure 4:. Downregulation of components of PAF1C selectively alter (G4C2)30+ transgene expression.

(a) (G4C2)n transgenes were co-expressed with dPAF1C RNAi lines in the adult brain using a drug inducible, neuronal driver (ElavGS, 16d). Transgene expression levels in heads measured by qPCR. dPaf1 and dLeo1 RNAi did not affect RNA levels of (G4C2)8 and (G4C2)29 but significantly reduced (G4C2)49 expression. dSpt4, dCDC73, dCtr9, dRtf1 RNAi altered expression of all (G4C2)n transgenes. Shown: individual data points with mean±SEM; mean value of 3 biological replicates (n=25 flies/replicate) from 2–3 independent experiments. (b) dPAF1C RNAi did not alter TDP43 (ElavGS, 16d, heads) RNA levels by qPCR. Shown: individual data points with mean±SEM; mean value of 3 biological replicates (n=25 flies/replicate) from 2 independent experiments. (c) Downregulation of dPAF1C components reduces GR-GFP signal in LDS-(G4C2)44^GR-GFP animals. Quantification of total GFP fluorescence relative to control animals. N flies: control=8, dPaf1=7, dLeo1=4, dCDC73=6, dCtr9=6, dRtf1=6. Each data point represents one eye of one animal. Shown: data from one experiment; data reproduced in two independent experiments. Scale bars: 100μm. (d) Effects of deleting scCDC73 (cdc73Δ) or scLeo1 (leo1Δ) in S. cerevisiae on RNA levels from transgenes assessed by qPCR. Transgenes included eYFP (control), sense-(G4C2)66 (disease) and antisense-(G2C4)66 (disease). Shown: individual data points with mean±SD; mean value of biological duplicates from 2 independent experiments. Statistics: ANOVAs with Tukey’s correction, p-values: ****<0.0001, ***<0.001, **<0.01, *<0.05, no significance (n.s.) >0.05.

Figure 5:. Endogenous PAF1C is upregulated in response to (G4C2)49 expression in the brain in flies and mice.

(a) Endogenous dPAF1C RNA expression is upregulated in (G4C2)49 expressing flies compared to control or (G4C2)8 expressing flies (qPCR). Transgenes expressed in the adult fly nervous system (ElavGS, 16d). Differences in expression are likely underestimated as RNA was extracted from whole head tissue, while transgenes were expressed selectively in neurons. (b) A non-G4C2 disease transgene, TDP43, was expressed with ElavGS (16d). Whole head analysis showed no upregulation of PAF1C components. (a-b) Shown: individual data points with mean±SEM; mean value of biological triplicates (n=25 flies/replicate) from 2 independent experiments. (c) Mouse endogenous mLeo1 protein levels measured in cortical tissue by western immunoblot using lysates from mice injected intracerebroventricularly with AAV2/9-(G4C2)2 or -(G4C2)149 at postnatal day 0. mLeo1 is upregulated by 6mo in response to expression of expanded (G4C2)149. Differences in expression are likely underestimated as protein was extracted from total cortical tissue while transgenes were expressed using AAV2/9 which predominantly transduces neurons. 3mo N animals: (G4C2)2=6, (G4C2)149=6. 6mo N animals: (G4C2)2=6, (G4C2)149=7. Shown: individual data points (each representing 1 animal) with mean±SEM. Data reproduced in two independent experiments. Statistics: (a) ANOVAs with Tukey’s correction, (b-c) unpaired 2-tailed student t-test; p-values: ****<0.0001, ***<0.001, **<0.01, *<0.05, no significance (n.s.) >0.05. See Supplementary Figure 11 for uncropped western images for this and subsequent figures.

Figure 6:. Upregulated hPAF1 and hLEO1 positively correlate to expression of repeat-containing C9orf72 transcripts and hLeo1 binds C9orf72.

(a) Western immunoblots for hPAF1C components in iPS cells. hPAF1C band densities were normalized to the mean of loading controls: hTubulin, hGAPDH. Shown: individual data points with mean±SEM; mean value of 2 biological replicates from 2 independent experiments per cell line relative to the mean signal in controls. (b) Chromatin immunoprecipitation studies using a hLeo1 antibody on 4 independent C9+-derived fibroblast lines. Data: relative to IgG controls after normalizing by input. Shown: individual data points (each representing 1 cell line) with mean±SEM; mean value of technical quadruplicates from 1 experiment. Data reproduced in 2 independent experiments per line. (c) qPCR analysis of hPAF1 and hLEO1 expression from healthy control (n=27), C9- (n=56), and C9+ patients (n=67) frontal cortex tissue. Shown: individual data points (each representing 1 individual) with mean±SEM. (d) Spearman r coefficients defined correlations in expression from hPAF1 or hLEO1 expression with C9orf72 transcripts in individuals. r values: 0=no correlation, 1.0=100% correlated. Shown: individual data points (each representing 1 individual) with linear regression±SE. Statistics: (a) unpaired 2-tailed student t-test, (b) ANOVA with Sidak’s correction, (c) Kruskal-Wallis ANOVA with Dunn’s correction, (d) Spearman R correlation; p-values: ****<0.0001, ***<0.001, **<0.01, *<0.05, no significance >0.05. C9orf72 intron 1: intronic region immediately 3’ of the G4C2 repeat in the C9orf72 gene. See Supplementary Figure 10 for cell line and patient characteristics.