NELF Potentiates Gene Transcription in the Drosophila Embryo

A hallmark of genes that are subject to developmental regulation of transcriptional elongation is association of the negative elongation factor NELF with the paused RNA polymerase complex. Here we use a combination of biochemical and genetic experiments to investigate the in vivo function of NELF in the Drosophila embryo. NELF associates with different gene promoter regions in correlation with the association of RNA polymerase II (Pol II) and the initial activation of gene expression during the early stages of embryogenesis. Genetic experiments reveal that maternally provided NELF is required for the activation, rather than the repression of reporter genes that emulate the expression of key developmental control genes. Furthermore, the relative requirement for NELF is dictated by attributes of the flanking cis-regulatory information. We propose that NELF-associated paused Pol II complexes provide a platform for high fidelity integration of the combinatorial spatial and temporal information that is central to the regulation of gene expression during animal development

Cellular labeling of endogenous retrovirus replication (CLEVR) reveals de novo insertions of the gypsy retrotransposable element in cell culture and in both neurons and glial cells of aging fruit flies.

Evidence is rapidly mounting that transposable element (TE) expression and replication may impact biology more widely than previously thought. This includes potential effects on normal physiology of somatic tissues and dysfunctional impacts in diseases associated with aging, such as cancer and neurodegeneration. Investigation of the biological impact of mobile elements in somatic cells will be greatly facilitated by the use of donor elements that are engineered to report de novo events in vivo. In multicellular organisms, reporter constructs demonstrating engineered long interspersed nuclear element (LINE-1; L1) mobilization have been in use for quite some time, and strategies similar to L1 retrotransposition reporter assays have been developed to report replication of Ty1 elements in yeast and mouse intracisternal A particle (IAP) long terminal repeat (LTR) retrotransposons in cultivated cells. We describe a novel approach termed cellular labeling of endogenous retrovirus replication (CLEVR), which reports replication of the gypsy element within specific cells in vivo in Drosophila. The gypsy-CLEVR reporter reveals gypsy replication both in cell culture and in individual neurons and glial cells of the aging adult fly. We also demonstrate that the gypsy-CLEVR replication rate is increased when the short interfering RNA (siRNA) silencing system is genetically disrupted. This CLEVR strategy makes use of universally conserved features of retroviruses and should be widely applicable to other LTR retrotransposons, endogenous retroviruses (ERVs), and exogenous retroviruses

Concordance between mis-regulated TE transcripts upon TDP-43 manipulation and TDP-43 bound TE transcripts.

<p>(A,B) Over-expression <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044099#pone.0044099-Shan1" target="_blank">[20]</a> of TDP-43 in transgenic mice and depletion <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044099#pone.0044099-Polymenidou1" target="_blank">[19]</a> of TDP-43 in mouse striatum each result in elevated expression of many TE derived transcripts. The majority of over-expressed TEs also were detected (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044099#pone.0044099.s004" target="_blank">Table S3</a>) as binding targets by CLIP-seq (RED). A few showed elevated expression but were not detected as binding targets (BLUE).</p

TDP-43 binding to TEs is selectively lost in FTLD patients.

<p>(<b>A</b>) In the human CLIP-seq data from FTLD versus healthy control, 38 repeat elements showed significant (<i>p</i>-value< = 1e-5 and fold changes> = 2) differential binding. Log2 fold binding differences are shown for significantly enriched/depleted elements. (<b>B,C,D</b>) Peaks are shown in genome browser for one RefGene control (<b>B</b>) and two differentially targeted TEs (<b>C,D</b>) in Healthy (<b>top</b>) versus FTLD (<b>bottom</b>). (<b>E</b>) Enrichment for the UGUGU motif relative to its prevalence in the genome is shown across a 51-nt window surrounding binding sites (−25 nt, 25 nt). Healthy samples (<b>Blue</b>) show similar enrichment for the UGUGU pentamer motif among RefGene (<b>solid</b>) and repeat (<b>dashed</b>) sequences (RefGene/repeat motif enrichment ratio ≈1.3). In contrast, motif enrichment in FTLD samples (<b>Red</b>) is significantly reduced among repeat (<b>dashed</b>) annotations relative to RefGene (<b>solid</b>; <i>p</i>-value< = 0.01; RefGene/repeat motif enrichment ratio ≈2.0).</p

TDP-43 binds broadly to transposable element (TE)-derived transcripts.

<p>Magnitude (log2-fold) of enrichments (<b>up</b>) or depletions (<b>down</b>) are shown (<b>A</b>, rat; <b>B</b>, mouse) for significantly bound repeat elements grouped by class. <b>MULTI</b> method (see text) was used for <b>A</b> and <b>B</b>. (<b>C</b>) The majority of rat TE targets identified with <b>MULTI</b> also are identified (<b>Left Panel, Rat</b>) when analysis is restricted to reads that map uniquely (<b>UNIQ</b>) or when both uniquely mapped and multi-mapped reads that map to the same TE were included (<b>UNIQ+SameEle</b>). These conclusions also hold for TE targets whose binding is reduced in FTLD samples from human tissue relative to healthy controls (<b>Left panel, Human</b>). Most rat TE targets and differentially bound human TE targets identified from uniquely mapped reads are intergenic (<b>Right panel</b>). (<b>D</b>) For TDP-43, peaks (UNIQ+SameEle) over TE targets are tall and sharp with mean peak height of 158 counts/peak. In contrast, peak heights are lower for FUS (mean peak height of 17).</p

Activation of transposable elements during aging and neuronal decline in Drosophila

We found that several transposable elements were highly active in Drosophila brain during normal aging. In addition, we found that mutations in Drosophila Argonaute 2 (Ago2) resulted in exacerbated transposon expression in the brain, progressive and age-dependent memory impairment, and shortened lifespan. These findings suggest that transposon activation may contribute to age-dependent loss of neuronal function

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

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

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

<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