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

Abstract

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.

The Gypsy Endogenous Retrovirus Drives Non-Cell-Autonomous Propagation in a Drosophila TDP-43 Model of Neurodegeneration

“…Consistent with the observations from constitutive nls-LacZ expression described above ( Figure S1), there is a gradual age-dependent increase (from days 2 to 5 post-induction) in numbers of gypsy-CLEVR-labeled glia with post-development induction of nls-LacZ (repo ts > nls-LacZ+gypsy-CLEVR) ( Figures 1D, 1E, and S2A). The age-dependent increase of gypsy ERV expression and replication is also consistent with our prior reports for neural tissue [32,36,40] and similar studies of other tissues [41][42][43][44]. In contrast with this gradual and modest increase in the number of gypsy-CLEVR-labeled glia with induced expression of nls-LacZ, we detected a dramatic increase in the numbers of glia labeled with this same reporter in response to induction of hTDP-43 ( Figures 1D, 1E, and S2A).…”

“…In the presence of pan-glial hTDP43 expression (repo > hTDP-43+gypsy-CLEVR), by contrast, we observed a striking increase in the number of H2B-mCherrylabeled glia either at 2 or 5-7 days post-eclosion ( Figure S1). This gypsy-CLEVR reporter expression was reduced in glia when we used an RNAi transgene (gypsy-IR) targeting gypsy transcripts to disrupt gypsy ERV expression [32,36,38], consistent with the conclusion that the appearance of the H2B-mCherry label was dependent upon prior gypsy expression (Figure S1; see also [36]). A small decrease in total number of glia was detected in these pan-glial hTDP-43-expressing animals (repo>hTDP-43) at day 2, consistent with the possibility that constitutive expression of hTDP-43 throughout development might impact glial differentiation or survival ( Figure S4B).…”

“…To investigate the mechanisms by which gypsy ERV contributes to TDP-43 toxicity in glia, we first monitored the replication of a transgenic copy of this ERV in response to pan-glial expression in the Drosophila model. To accomplish this, we developed Cellular Labeling of Endogenous Retrovirus Replication of gypsy (gypsy-CLEVR in Figure 1A; see also [36]), a novel approach to mark cells in which the gypsy ERV has replicated through an RNA intermediate to cDNA and been integrated back into host's genome. The gypsy-CLEVR construct commandeers the gypsy ERV's own replication machinery to fuse a Gal4-responsive UAS enhancer element to a dual fluorescent reporter, Watermelon (WM, a membrane myr-GFP and nuclear H2B-mCherry fusion gene with P2A self-cleaving peptide).…”

“…We compared the number of glial cells expressing the of gypsy-CLEVR replication reporter expression in glial cells in the presence of either hTDP-43 [32,37] or a nls-LacZ control transgene. Glia in nls-LacZ control (repo > nls-LacZ+gypsy-CLEVR) animals exhibited a modest age-dependent increase in the numbers of glia showing gypsy ERV replication, with a relatively small fraction of glia in the central brain labeled with the H2B-mCherry nuclear reporter at 2 or 5-7 days post-eclosion ( Figure S1; see also [36]). In the presence of pan-glial hTDP43 expression (repo > hTDP-43+gypsy-CLEVR), by contrast, we observed a striking increase in the number of H2B-mCherrylabeled glia either at 2 or 5-7 days post-eclosion ( Figure S1).…”

Somatic transposition in theDrosophilaintestine occurs in active chromatin and is associated with tumor suppressor gene inactivation

“…Nonetheless, evidence for active somatic transposition has been recently mounting. Reporters of transposon activity suggested TE mobility in neuronal lineages in human, mouse and Drosophila (Muotri et al 2005;Coufal et al 2009;Li et al 2013;Macia et al 2017;Chang et al 2019). Additionally, recent use of an engineered gypsy retrotransposon trapping cassette in flies suggested that somatic transposition could also occur in non-neuronal tissues such as the fat body (Jones et al 2016; or the intestine (Sousa-Victor et al 2017).…”