DNA damage-induced cell death and <i>gypsy</i> ERV expression contribute hTDP-43 mediated toxicity.

<p>(A) Lifespan analysis shows that co-expression of <i>loki</i>(IR) (<i>Repo</i> > <i>loki</i>(IR) + hTDP-43) fully rescues the lifespan deficit exhibited by flies expressing glial hTDP-43 (<i>Repo</i> > hTDP-43). (B) Co-expression of <i>loki</i>(IR) (<i>ELAV</i> > <i>loki</i>(IR) + hTDP-43) likewise fully rescues the lifespan deficit exhibited by flies expressing neuronal hTDP-43 (<i>ELAV</i> > hTDP-43). (C) Central projections of whole-mount TUNEL stained brains reveal a noticeable reduction in the apoptotic activity induced by glial hTDP-43 expression (<i>Repo</i> > hTDP-43 + GFP(IR)) when <i>gypsy</i> expression is knocked down (<i>Repo</i> > hTDP-43 + <i>gypsy</i>(IR)), while knocking down <i>loki</i> completely alleviates the apoptosis induced by glial hTDP-43 expression (<i>Repo</i> > hTDP-43 + <i>loki</i>(IR)). (D) Quantification of (H), normalized to the positive control (<i>Repo</i> > hTDP-43 + GFP(IR)). <i>N</i> = 12 for <i>Repo</i> / +; <i>N</i> = 9 for <i>Repo</i> > hTDP-43 + GFP(IR); <i>N</i> = 7 for <i>Repo</i> > hTDP-43 + <i>gypsy</i>(IR); and <i>N</i> = 7 for <i>Repo</i> > hTDP-43 + <i>loki</i>(IR). <b>*</b>All of the lifespans with the exception of the NRTI feeding experiments shown in Figs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006635#pgen.1006635.g004" target="_blank">4</a> and 5, were performed concurrently in order to ensure comparability across groups. Therefore, appropriate controls are shared across panels.</p

Glial and neuronal hTDP-43 expression erodes siRNA-mediated silencing.

<p>(A) Representative central projections show that co-expression of the hTDP-43 transgene, but not an unrelated tdTomato control transgene, interferes with the ability of a Dcr-2 processed IR (GFP(IR)) to silence a GFP transgenic reporter in glial cells using the <i>Repo-GAL4</i> driver. Quantification of GFP signal for each group is shown in the appropriate bar graph; values are represented as relative fold change over <i>Repo</i> > GFP + GFP(IR) (mean + SEM). A two-way ANOVA reveals significant effects of both genotype (p < 0.0001) and age (p < 0.0001), and a significant age x genotype interaction (p < 0.0001). <i>N</i> = 5 for <i>Repo</i> > GFP and <i>Repo</i> > GFP + GFP(IR); <i>N</i> = 10 for all other groups. (B) An equivalent analysis demonstrates that hTDP-43 has a similar effect in the neuronal cells of the <i>Drosophila</i> mushroom body using the <i>OK107-Gal4</i> driver, but with a later age of onset than hTDP-43 expression in glial cells. Quantification of GFP signal for each group is shown in the appropriate bar graph as in (A). A two-way ANOVA reveals significant effects of genotype (p = 0.0054) and age (p < 0.0001), as well as a significant age x genotype interaction (p = 0.0021). <i>N</i> = 5 for <i>OK107</i> > GFP and <i>OK107</i> > GFP + GFP(IR); <i>N</i> = 10 for all other groups. (C) Co-expression of hTDP-43, but not GFP, in the photoreceptor neurons of the fly eye under the <i>GMR-Gal4</i> driver interrupts the ability of a Dcr-2 processed IR to silence the endogenous <i>white</i>^<i>+</i> pigment gene with an age of onset similar to that observed with neuronal expression of hTDP-43 in the CNS under <i>OK107-Gal4</i>, resulting in characteristic clusters of red-pigmented ommatidia. <i>N</i> = 5 for <i>GMR</i> > <i>w</i>(IR) + Gal80^ts <i>OFF</i> and <i>GMR</i> > <i>w</i>(IR) + Gal80^ts <i>ON</i>; <i>N</i> = 20 for all other groups.</p

Glial hTDP-43 expression results in early and dramatic de-suppression of the <i>gypsy</i> ERV.

<p>(A) Transcript levels of <i>gypsy ORF2</i> (<i>Pol</i>) as detected by qPCR in whole head tissue of flies expressing hTDP-43 in neurons (<i>ELAV</i> > hTDP-43) versus glia (<i>Repo</i> > hTDP-43) at a young (2–4 Day) or aged (8–10 Day) time point. Transcript levels normalized to <i>Actin</i> and displayed as fold change relative to flies carrying the hTDP-43 transgene with no Gal4 driver (hTDP-43 / +) at 2–4 Days (means + SEM). A two-way ANOVA reveals a significant effect of genotype (p < 0.0001) but no effect of age (p = 0.5414). <i>N</i> = 8 for all groups. (B) An equivalent analysis shows that <i>gypsy ORF3</i> (<i>Env</i>) likewise displays a significant effect of genotype (p < 0.0001) and no effect of age (p = 0.6530). <i>N</i> = 4 for the 2–4 Day cohort and <i>N</i> = 5 for the 8–10 Day cohort. (C) Central projections of whole mount brains immunostained with a monoclonal antibody directed against <i>gypsy</i> ENV protein reveals dramatic, early accumulation of ENV immunoreactive puncta in brains expressing glial hTDP-43 (5–8 Days) in comparison to both age-matched genetic controls (<i>ELAV</i> / +; <i>Repo</i> / +; hTDP-43 / +) and flies expressing neuronal hTDP-43. This effect persists out to 19–25 Days post-eclosion. <i>ELAV</i> / +, 5–8 Day (<i>N</i> = 3), 19–25 Day (<i>N</i> = 4); <i>Repo</i> / +, 5–8 Day (<i>N</i> = 3), 19–25 Day (<i>N</i> = 3); hTDP-43 / +, 5–8 Day (<i>N</i> = 5), 19–25 Day (<i>N</i> = 2); <i>ELAV</i> > hTDP-43, 5–8 Day (<i>N</i> = 2), 19–25 Day (<i>N</i> = 4); <i>Repo</i> > hTDP-43, 5–8 Day (<i>N</i> = 7), 19–25 Day (<i>N</i> = 8).</p

Neuronal and glial hTDP-43 expression induces physiological impairment and toxicity with varying severity.

<p>(A) Flies expressing glial hTDP-43 display extreme locomotor impairment at 1–5 days post-eclosion in the Benzer fast phototaxis assay, while flies expressing neuronal hTDP-43 demonstrate a slight locomotor deficit in comparison to genetic controls (one-way ANOVA, p < 0.0001). This trend continues and is exacerbated by 5–10 days post-eclosion (one-way ANOVA, p < 0.0001). Four biological replicates performed for each experiment. (B) Lifespan analysis of flies expressing neuronal versus glial hTDP-43 in comparison to genetic controls. (C) Central projections of whole-mount brains reveals a stark increase in TUNEL-positive cells in flies expressing glial hTDP-43 in comparison to genetic controls at 5 days post-eclosion. <i>N</i> = 16 for <i>Repo</i> / + and <i>N</i> = 18 for <i>Repo</i> > hTDP-43. (D) TEM likewise reveals rampant apoptosis in the neuropil of flies expressing glial hTDP-43 at 12 days post-eclosion. Arrowheads indicate pro-apoptotic nuclei, as identified by morphology.</p

<i>gypsy</i> ERV expression contributes to hTDP-43 mediated toxicity.

<p>(A) Lifespan analysis shows that co-expression of <i>gypsy</i>(IR) (<i>Repo</i> > <i>gypsy</i>(IR) + hTDP-43) partially rescues the lifespan deficit exhibited by flies expressing glial hTDP-43 (<i>Repo</i> > hTDP-43). (B) Co-expression of an unrelated GFP(IR) control transgene (<i>Repo</i> > GFP(IR) + hTDP-43) does not effect the lifespan of flies expressing glial hTDP-43 (<i>Repo</i> > hTDP-43). (C) Co-expression of <i>gypsy</i>(IR) (<i>ELAV</i> > <i>gypsy</i>(IR) + hTDP-43) has no effect on lifespan in flies expressing neuronal hTDP-43 (<i>ELAV</i> > hTDP-43).</p

Neuronal and glial hTDP-43 expression results in induction of RTE expression.

<p>Differential expression of many genes and RTEs are detected in response to either neuronal or glial expression of hTDP-43 in head tissue of 8–10 day old flies (<i>N</i> = 2 biological replicates per genotype). (A) Neuronal (<i>Elav</i> > hTDP-43) expression of hTDP-43 results in both increases and decreases in expression of a broad variety of cellular transcripts (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006635#pgen.1006635.s009" target="_blank">S2A Table</a>). (B) A panel of transposons, including many RTEs, also are impacted, with most exhibiting elevated expression (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006635#pgen.1006635.s009" target="_blank">S2B Table</a>). (C) Glial expression of hTDP-43 (<i>Repo</i> > hTDP-43) also results in numerous transcriptome alterations, with many transcripts either increasing or decreasing in abundance (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006635#pgen.1006635.s010" target="_blank">S3A Table</a>). (D) Many transposons, most of which are RTEs, exhibit elevated expression levels in response to glial hTDP-43 expression (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006635#pgen.1006635.s010" target="_blank">S3B Table</a>). Several RTEs display elevated expression in response to both glial and neuronal hTDP-43 expression, however a number also exhibit specificity in response to either glial or neuronal hTDP-43 expression (compare <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006635#pgen.1006635.g001" target="_blank">Fig 1B and 1D</a>). (E) The <i>gypsy</i> ERV exhibits elevated expression only in response to glial, but not neuronal, hTDP-43 expression. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006635#sec008" target="_blank">methods</a> for details regarding analysis pipeline, including statistical analysis.</p

Loss of Fun30 leads to increased Transcription at centromere regions.

<p>Analysis of transcript levels at <i>CEN3</i> region by RT-qPCR in wildtype yeast (wt), the corresponding <i>Δfun30</i> mutant, <i>Δtrf4</i> mutant and the double mutant <i>Δfun30 Δtrf4</i> strains. Primers PM22/48 detecting transcripts directly over <i>CEN3</i> were used to amplify cDNA. The graph reports the relative amount of transcript compared to a control gene that is not regulated by Fun30. Similar results were obtained when we examined absolute amounts.</p

Fun30 is required for normal <i>CEN</i>-flanking nucleosome positioning and/or <i>CEN</i> core particle structure.

<p>A) Genome browser trace of Fun30 ChIP enrichment and nucleosome dyad frequency centred on and surrounding yeast <i>CEN1</i>. The upper trace shows Log₂ Fun30 ChIP-seq enrichment values binned at 10 bp intervals and smoothed with a 3 bin moving average. Wildtype (WT) and <i>Δfun30</i> chromatin was digested with MNase and nuclease-protected DNA species sequenced using paired-end mode Illumina technology. Nucleosome sequencing data (nuc) traces were plotted as mirror images in the lower panel. The graph shows a map of the centre point positions of paired sequence reads with end-to-end distances of 150 bp+/−20% wild-type and <i>Δfun30</i> mutant chromatin samples surrounding <i>CEN1</i>. The frequency distributions, which effectively map chromatin particle dyads, were binned at 10 bp intervals, and smoothed by applying a 3 bin moving average. Peaks in the dyad distributions correspond to translationally-positioned nucleosomes in the original genome. The <i>CEN</i> core particle is also mapped using this method and can be visualised as a small peak centred on the <i>CEN</i> region marked with a grey box. Pink bars show the positions of ORFs (B–D) Genome browser plots of Fun30 ChIP-seq and nucleosome sequence distributions as described above for <i>CEN10, 11</i> and <i>12</i> respectively. Fun30-dependent changes in the height of a nucleosome dyad or <i>CEN</i> core particle peak are marked with a red asterix. Fun30-dependent changes in the position of a <i>CEN</i>-flanking nucleosome dyad peak are marked with red arrows. Genome browser plots for all yeast <i>CENs</i> are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002974#pgen.1002974.s006" target="_blank">Figure S6</a>.</p

Summary of sequence libraries.

<p>Summary of sequence libraries.</p

Fun30 is required when Cse4 function is compromised.

<p>Growth of the double mutant <i>Δfun30 cse4-1</i> is strongly affected at semi-restrictive temperatures. Fivefold dilutions of wildtype (BY4741/Y00000), <i>Δfun30</i> (Y00389), <i>cse4-1</i> (AHY666) and <i>Δfun30 cse4-1</i> (SC53) cells were plated onto YPD plates and incubated at indicated temperatures for 3 days. Lower panels: Fun30 activity is restored by expressing wildtype Fun30 <i>in trans</i>, but not Fun30 with a point mutation in the ATPase domain; Cells were spotted on media with 2% glucose and grown for 3 days at 30°C or 35°C.</p