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

Nature Neuroscience volume 22, pages 863–874 (2019)Cite this article

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An Author Correction to this article was published on 21 February 2023

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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.

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Fig. 1: A genetic screen revealed dPAF1C as a suppressor of (G4C2)49toxicity in the fly eye when downregulated.
Fig. 2: dPAF1C RNAi does not modify (GR)36 or TDP43 toxicity in Drosophila.
Fig. 3: Reduced expression of dPAF1C components suppresses (G4C2)49-induced toxicity in multiple contexts in the fly.
Fig. 4: Downregulation of PAF1C components selectively alters (G4C2)30+ transgene expression.
Fig. 5: Endogenous PAF1C is upregulated in response to (G4C2)30+ expression in fly and mouse brains.
Fig. 6: Upregulated hPAF1 and hLEO1 positively correlate with expression of repeat-containing C9orf72 transcripts and hLEO1 binds C9orf72.

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The authors are submitting all relevant data for publication. Any additional inquiries can be directed to the corresponding author, while any information relevant to this study will be openly shared, including all raw data, unique reagents, or unique protocols.

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Acknowledgements

The authors thank A. T. Moehlman and H. Krämer at UT Southwestern for ERG investigations and T. Gendron for GP studies in (G4C2)n animals post-screening. J. T. Lis, P. Gallant, and M. Buszczak generously shared valuable Drosophila reagents targeting dPAF1C. Additional thanks are given to E. B. Lee, T. A. Jongens, Z. Zhou, and members of the Bonini Laboratory, notably, J. Kennerdell, L. McGurk, and J. Saikumar, for helpful comments. Undergraduates D. P. Cerza and A. Chen provided minimal technical support. The authors thank the Transgenic RNAi Project at Harvard Medical School (NIH/NIGMS R01-GM084947) and the VDRC for developing transgenic RNAi fly stocks used in this study. Appreciation is also given to the NINDS Human Cell and Data Repository at Rutgers University for fibroblast cells. This work was funded by the Systems and Integrative Biology NIH/NIGMS training grant T32-GM07517 (to L.D.G.), NIH/NINDS R35-NS097263 (to A.D.G.), NIH/NINDS R35-NS097273 (to L.P.), NIH/NINDS P01-NS084974 (to L.P.), NIH/NINDS P01-NS099114 (to L.P.), Mayo Clinic Foundation (to L.P.), ALS Association (to L.P. and M.P.), Robert Packard Center for ALS Research at Johns Hopkins (to L.P.), Target ALS Foundation (to L.P.), NIH/NINDS R01-NS078283 (to N.M.B.), and NIH/NINDS R35-NS09727 (to N.M.B.).

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Authors and Affiliations

  1. Neuroscience Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Lindsey D. Goodman & Nancy M. Bonini

  2. Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA

    Mercedes Prudencio, Jeannie Chew, Casey N. Cook & Leonard Petrucelli

  3. Neuroscience Graduate Program, Stanford University School of Medicine, Stanford, CA, USA

    Nicholas J. Kramer

  4. Department of Biology, University of Pennsylvania, Philadelphia, PA, USA

    Luis F. Martinez-Ramirez, Ananth R. Srinivasan, Matthews Lan, Michael J. Parisi, Yongqing Zhu, Amit Berson & Nancy M. Bonini

  5. Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA

    Aaron D. Gitler

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Contributions

This work was performed and written by L.D.G. under the mentorship of N.M.B. M.P. contributed postmortem patient studies and analyses under the mentorship of L.P. N.J.K. contributed yeast studies under the mentorship of A.D.G. N.J.K. and A.D.G. also provided iPS cell lysates for western immunoblots performed by L.D.G. L.F.R.-M. provided technical support during dPAF1C-focused studies, including the qPCR and lifespan experiments, under the direction of L.D.G. A.R.S. performed the ChIP experiments under the guidance of L.D.G. M.L. provided technical support during fly screening and performed initial control GAL4/UAS-LacZ western blots post-screening under the direction of L.D.G. M.J.P. provided technical support for fly-based studies, including paraffin sectioning for vacuoles and internal eyes under the direction of L.D.G. Y.Z. made the G4C2 fly constructs under the direction of N.M.B. A.B. performed paraffin sectioning for fly internal eyes under the direction of L.D.G. J.C. generated the mouse models for G4C2 under the mentorship of L.P. C.N.C. provided technical support for mammalian western blots under the mentorship of L.P.

Corresponding author

Correspondence to Nancy M. Bonini.

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Supplementary Figure 1 Screen extended data.

(a) Representative images for the categories identified during the RNAi-based screen in Gmr-GAL4 > (G4C2)49 expressing animals: “suppressors” (strong recovery of ommatidial organization, eye color, and eye size), “mild suppressors” (recovery of ommatidial organization, eye color, and eye size while degenerative effects were still present), “no effect” (indistinguishable from controls), “mild enhancers” (increased disruptions in ommatidial organization and pigment loss), “enhancers” (strongly increased disruptions in ommatidial organization, pigment loss, and eye size), and “lethal enhancers” (lethality in the late pupal stage ; rare (n < 2) escapers’ eyes looked like “enhancers”). All hits were independently tested 2+ times to confirm reproducibility (n > 5 flies/cross). (b) Mild enhancers, enhancers, and lethal enhancers were expressed alone or in control (G4C2)8 expressing animals to define those that were toxic in control scenarios. Shown are representative images for an “unspecific enhancer” (RNAi line that caused toxicity in controls) and a “true enhancer” (RNAi line that had no effect in controls while enhancing (G4C2)49-toxicity). Unspecific enhancers were excluded. (a-b) Scale bars: 100μm (c) To identify RNAi lines that altered (G4C2)49-toxicity indirectly by effecting the GAL4/UAS expression system, RNAi lines were co-expressed with a control LacZ transgene. β-Galactosidase protein levels were quantified by western immunoblot. RNAi lines that significantly increased or decreased LacZ expression in a manner consistent with its effect on (G4C2)49-toxicity were reclassified as either “LacZ-suppressors” or “LacZ-enhancers” and excluded. Shown is a representative western immunoblot. All lines with potential effects were independently tested twice with biological duplicates to confirm reproducibility. (d) Gene ontology analyses revealed GO terms enriched in the panel of modifiers identified in the screen that alter (G4C2)49-toxicity (119/3582 genes). To compensate for any biases introduced in the RNAi library, the genes screened were used as the background. RNAi lines that failed control scenarios (b-c) were excluded. Plotted: significant (p-value ≤ 10^−3) enrichment scores of > 3.00. Additional details for this and subsequent figures: Supplemental Data (all screen data and significant GO-terms), Supplementary Table 1 (detailed sampling, reproducibility and statistics), methods (sampling methods).

Supplementary Figure 2 Downregulation of PAF1C and dSpt4 does not suppress toxicity caused by expression of (GR)30+ or (PR)50 dipeptides.

(a-b) RNAi against dPAF1C components and dSpt4 were examined for modification of (GR)36-toxicity in the fly optic system (Gmr-GAL4). Internally, there was no significant recovery in retinal tissue loss with multiple RNAi lines targeting dPaf1, dLeo1, dCtr9, and dRtf1. dCDC73 RNAi enhanced toxicity both externally and internally while this effect was not reproducible with dCDC73 RNAi-2. Internal retina depth (arrows) was quantified for individual animals. N flies for RNAi: control = 9, dPaf1 = 10, dLeo1 = 9, dCDC73 = 4, dCtr9 = 8, dRtf1 = 6, dSpt4 = 14. N flies for RNAi-2 = 4 for all genotypes. Shown: individual data points (each representing 1 animal) with mean ± SD. Statistics: ANOVAs with Dunnett’s correction (RNAi), ANOVA with Tukey’s correction (RNAi-2), p-values: * = 0.03, no significance (n.s.) > 0.05. Scale bars: external eye = 100 μm, internal eye = 35 μm. (c-d) To further assess the specificity of PAF1C to (G4C2)30+ RNA models, yeast spotting assays were used. Control (CCDB), (PR)50 or (GR)100 were expressed in yeast using galactose-inducible transgenes (see methods). Expression of (PR)50 and (GR)100 reduced colony formation in WT cells. This effect was unaltered in leo1Δ yeast. Interestingly, cdc73Δ caused further reduced growth in (GR)100 expressing yeast but had no effect on yeast expressing (PR)50. This is consistent with dCDC73 RNAi data in (GR)36-expressing flies where loss of this component, in one RNAi line, increased GR-associated toxicity. Note that any mild effects observed with downregulation of individual PAF1C components and dSpt4 can be explained by the fact that a GR-producing mRNA transcript, despite being G4C2-independent, will still be GC-rich based on the standard codon table. PAF1C and Spt4 are important for transcription of GC-rich DNA (Rondón, A. G. et al, 2004; Rondón, A. G. et al, 2003). Shown: data from one experiment; all experiments were independently repeated with similar results.

Supplementary Figure 3 Downregulation of dPAF1C and dSpt4 does not suppress toxicity caused by expression of TDP43 in the fly eye.

(a-b) dPAF1C and dSpt4 RNAi were tested as modifiers of TDP43-toxicity in the fly optic system (Gmr-GAL4). Only dCDC73 RNAi caused significant suppression of the external and internal eye in these animals. This effect was not reproduced with a second, independent RNAi lines targeting dCDC73. Internal retina depth (arrows) was quantified for individual animals. N flies for RNAi: control = 13, dPaf1 = 9, dLeo1 = 4, dCDC73 = 5, dCtr9 = 4, dRtf1 = 4, dSpt4 = 9. N flies for RNAi-2: control = 6, dPaf1 = 4, dLeo1 = 6, dCDC73=6, dCtr9 = 6, dRtf1 = 6. Shown: individual data points (each representing 1 animal) with mean±SD. Scale bars: external eye = 100 μm, internal eye = 35 μm. (c) Expression from the TDP43 transgene was analyzed by western immunoblot when dPAF1C was downregulated revealing no change in TDP43 protein levels. Shown: individual data points with mean ± SD; mean value of biological triplicates (n = 10 flies/replicate). Statistics: ANOVAs with Tukey’s correction, p-values: **** < 0.0001, no significance (n.s.) > 0.05. Shown: data from one experiment while all experiments were independently repeated with similar results. See Supplementary Figure 11 for uncropped western images for this and subsequent figures.

Supplementary Figure 4 Characterization of dPAF1C RNAi fly lines.

(a) RNA levels of dPAF1C components were assessed by qPCR to define RNAi efficacy in flies. UAS-RNAi fly lines were ubiquitously expressed and compared to background-matched controls. For samples analyzed with Da-GAL4, expression was measured in larvae. For samples analyzed with Da-GS, expression was measured in adult animals (n = 10) after 5d or 10d of drug-induced expression. Shown: individual data points with mean ± SD; mean value of 3–6 biological replicates. (b-c) RNAi lines targeting dCDC73 or dRtf1 were analyzed for level of protein knockdown by western immunoblot. Shown: individual data points with mean ± SD; mean value of 3 biological replicates (n = 10 flies/replicate). Statistics: ANOVAs with Tukey’s correction (dPaf1 and dLeo1 RNAi only), unpaired 2-tailed student t-test; p-values: **** < 0.0001, *** < 0.001, ** < 0.01, * < 0.05. Shown: data from one experiment while all experiments were independently repeated with similar results.

Supplementary Figure 5 Extended data showing dPAF1C RNAi lines suppress (G4C2)30+ toxicity in the fly.

(a) Downregulation of each of the components of dPAF1C with a second set of RNAi lines mitigates toxicity associated with (G4C2)49 expression in the fly eye, seen externally by reduced pigment loss and reduced disruption in ommatidial organization and internally by increased integrity of the retinal tissue. All RNAi-2 lines are in a w- genetic background. Internal retina depth (arrows) was quantified for individual animals. N flies: control = 12, dPaf1 = 5, dLeo1 = 6, dCDC73=6, dCtr9 = 6, dRtf1 = 3. (b) Downregulation of components of dPAF1C with a second set of RNAi lines has no effect on control fly eyes. Internal retina depth (arrows) was quantified for individual animals. N flies: control = 4, dPaf1 = 3, dLeo1 = 4, dCDC73 = 4, dCtr9 = 3, dRtf1 = 5. (c) dPAF1C RNAi were tested in a second, independent fly model expressing expanded (G4C2)30+ in the fly eye. This model contains a sequence 5′ of the repeat (leader sequence, LDS; 114 bp of intronic sequence found upstream of the repeat in C9orf72) and a 3′ GFP tag in the GR reading frame. Toxicity in this model is also suppressed by dPAF1C RNAi co-expression, seen externally by reduced pigment loss and reduced disruption in ommatidial organization and internally by increased integrity of the retinal tissue. Internal retina depth (arrows) was quantified for individual animals. N flies: control = 6, dPaf1 = 5, dLeo1 = 5, dCDC73 = 5, dCtr9 = 6, dRtf1 = 6. (a-c) each data point represents one animal. Scale bars: external eye = 100 μm, internal eye = 35 μm. (d) At 20 d, climbing deficits caused by (G4C2)49 expression in the adult male nervous system are rescued when dPAF1C RNAi-2 lines are co-expressed. N flies: control = 51, dPaf1 = 85, dLeo1 = 73, dCDC73 = 67, dCtr9 = 87, dRtf1 = 31. Individual data points are the mean % of animals that could climb per tube. Average of 16 ± 3.6 animals per tube. Statistics: ANOVAs with Tukey’s correction, p-values: **** < 0.0001, *** < 0.001, ** < 0.01, * < 0.05. Shown: individual data points with mean ± SD; data from one experiment; all experiments were independently repeated twice with similar results.

Supplementary Figure 6 Vacuole formation scoring schematic and additional examples of genotypes reported.

(a) Scoring of vacuole formation (arrowheads) in the adult fly brain. A scale was developed from 0–4 where 0 = no vacuoles and 4 = frequent medium-large vacuoles. “rare” means ≤ 5 vacuoles and “frequent” means > 5 vacuoles. Sections through the entire brain of each animal were assessed in scoring. (G4C2)49 expression in 8d animals typically receives a score of 1. (G4C2)49 expression in 28d animals typically receives a score of 3–4. Expression is driven by the drug-inducible neuronal driver, ElavGS. (b) Additional representative images of non-G4C2, age matched controls at 28d. (c) Additional representative images of vacuoles observed in (G4C2)49 brains at 28d (see Fig. 3f). dPAF1C components are targeted by RNAi and result in suppression of vacuole formation in (G4C2)49 expressing animals. Shown: data from one experiment; experiment was repeated twice with similar results. Scale bars: 50μm.

Supplementary Figure 7 Effects of downregulating dPAF1C components in control flies.

(a) RNAi for dPAF1C components were expressed in the adult fly nervous system using the drug-inducible neuronal driver, ElavGS. Survival of animals with age was evaluated. All RNAi but dCDC73 RNAi results in reduced lifespan compared to control animals. N flies: control = 301, dPaf1 = 200, dLeo1 = 275, dCDC73 = 200, dCtr9 = 210, dRtf1 = 200. (b) Co-regulation of dPAF1C components and dSpt4 were determined by looking for reduced expression of components when dPaf1, dLeo1, or dCDC73 were downregulated by RNAi ubiquitously in adult animals (DaGS, 6d). No significance was detected except for dCtr9 showing downregulation with dCDC73 RNAi. Shown: individual data points with mean ± SD; mean value of biological triplicates (n = 10 flies/replicate). Statistics: (a) log-rank tests, (b) ANOVA with Tukey’s correction; p-values: **** < 0.0001, *** < 0.001, **<0.01, * < 0.05, no significance (n.s.) > 0.05. Shown: data from one experiment; all experiments were independently repeated with similar results.

Supplementary Figure 8 Comparisons of w- vs w+ genetic background controls in (G4C2)30+ expressing flies show no significant differences.

To address potential concerns of different RNAi genetic backgrounds of the lines used in this study, multiple controls with different genetic backgrounds were examined. Here we show representative data. (a) Toxicity associated with (G4C2)49 expression looks similar both externally and internally in the fly eye when the transgene is expressed in the optic system (Gmr-GAL4) in w- vs w+ genetic backgrounds. Internal retina depth (arrows) was quantified for individual animals. N flies: w = 12, w+ = 9. Shown: individual data points (each representing 1 animal) with mean±SD. Scale bars: external eye = 100 μm, internal eye = 35μm. (b) Ubiquitous expression of (G4C2)49 in adult flies results in similar lifespans in w- vs w+ genetic backgrounds. N flies: w = 88, w+ = 106. (c) Climbing deficits develop with age in animals expressing (G4C2)49 in the adult fly nervous system, ElavGS. The % flies that can climb is comparable in w- and w+ controls (20d, males). N flies: w = 51, w+ = 72. Shown: Individual data points are mean % of animals that could climb per tube; individual data points with mean±SD. (d) qPCR analysis of (G4C2)49 RNA levels in the adult fly nervous system is statistically similar in multiple genetic backgrounds. Shown: individual data points with mean±SD; mean value of biological triplicates (n = 25 flies/replicate) from 2 independent experiments. (e) External eye imaging of GR-GFP in Gmr-GAL4 > LDS-(G4C2)44GR-GFP animals results in similar GFP fluorescence independent of genetic background. N flies: w = 8, w+ = 8. Shown: individual data points (each representing 1 animal) with mean ± SD. Scale bars: 100 μm. Statistics: (a,c,e) unpaired 2-tailed student t-tests, (b) log-rank test, (d) ANOVA with Tukey’s correction; p-value: no significance > 0.05. See Supplementary Figure 12 for optimization of fly G4C2 qPCR reactions for this and subsequent figures.

Supplementary Figure 9 Extended qPCR fly and yeast data.

(a) A second, independent (G4C2)49 transgene was co-expressed with dPAF1C RNAi lines in the adult brain using a drug inducible, neuronal driver (ElavGS, 16d). Downregulation of all dPAF1C components caused significantly less RNA to be produced from the (G4C2)49 transgene by qPCR, consistent with data in Fig. 4a. Shown: individual data points with mean ± SEM; mean value of biological triplicates (n = 25 flies/replicate) from 2 independent experiments. (b) RNA levels for endogenous dRNAPII driven genes βTUB56D (β-Tubulin) and ACT5C (β-Actin) are unchanged by qPCR upon knockdown of dPAF1C. The ubiquitous, drug-inducible driver, DaGS, was used to drive RNAi expression in whole animals (6d). Shown: individual data points with mean ± SD; mean value of biological triplicates (n=10 flies/replicate); data from 1 experiment while data reproduced in a second, independent experiment. (c) leo1Δ or cdc73Δ yeast strains do not alter RNA levels produced from endogenous scRNAPII genes, TDH3 and FBA1. RNA levels were measured by qPCR. Shown: individual data points with mean±SD; mean value of biological triplicates from 1 experiment. Statistics: ANOVAs with Tukey’s correction, p-values: **** < 0.0001, *** < 0.001, ** < 0.01, * < 0.05, no significance > 0.05.

Supplementary Figure 10 Extended patient data.

(a) Summary of iPS and fibroblast cell lines used in Fig. 6a, b, respectively. (b) Summary of patient cohort used in this study. Total number, cases that were male (%) and average age at onset/death/duration: median (minimum, 25th percentile, 75th percentile, and maximum). (c) In frontal cortex tissue from FTD cases qPCR analysis of endogenous expression of hCDC73 revealed no significant change in expression in C9+ versus C9- cases. C9- cases did show a significant upregulation of hCDC73 compared to healthy controls. Shown: individual data points (each representing 1 individual) with mean ± SEM. (d) Spearman correlation coefficients for C9+ or C9- FTD cases show no correlation in hCDC73 expression and C9orf72 expression in the frontal cortex of patients. (e) Spearman correlation coefficients for C9+ or C9- ALS cases showed no correlation in expression of hPAF1 and hLEO1 and expression of C9orf72 in the frontal cortex of C9+ patients. In C9- ALS patients, there were weak correlations that were markedly lower than those in C9+ FTD patients (see Fig. 6d). C9orf72 intron 1: the intronic gene region immediately 3′ of the G4C2 repeat in the C9orf72 pre-mRNA transcript. (d,e) Shown: individual data points (each representing 1 individual) with linear regression ± SE. Statistics: (c) ANOVA with Tukey’s correction, (d,e) Spearman R correlations; p-values: **** < 0.0001, *** < 0.001, ** < 0.01, * < 0.05, no significance (n.s.) > 0.05.

Supplementary Figure 11 Full, uncropped western immunoblot images with protein standards.

Uncropped western immunoblot images for all relevant figures. (a) protein ladder shown is SuperSignal Enhanced Molecular Weight Protein Ladder (Fisher Sci #84786). (b-f) protein ladder shown is Novex Sharp Pre-stained Protein Standard (Invitrogen #LC5800).

Supplementary Figure 12 G4C2 and RP49 primer optimization for qPCR.

During optimization of primers for qPCR reactions used in this study, primer efficiencies and melt curves were closely examined to ensure validity of all reactions. (a) For measuring (G4C2)n mRNA levels in flies, qPCR primers were designed that utilized unique restriction enzyme sequences located 3′ of the repeat. (b) RP49, a common housekeeping gene, shows a similar primer efficiency to the G4C2 primers, both ~ 90%, and was used as the main reference gene. Primer efficiencies were calculated using serial dilutions of cDNA made from control samples expressing (G4C2)49 in the fly nervous system (using ElavGS). Shown: mean value of biological triplicates from 2 independent experiments and the resulting trendline is shown ± SD. Representative melt curves from one of the biological triplicates is shown. Further, qPCR products were run on an agarose gel to confirm that there was one product and that it was the expected size (data not shown).

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Goodman, L.D., Prudencio, M., Kramer, N.J. et al. Toxic expanded GGGGCC repeat transcription is mediated by the PAF1 complex in C9orf72-associated FTD. Nat Neurosci 22, 863–874 (2019). https://doi.org/10.1038/s41593-019-0396-1

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