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• W2756245919 abstract "•Piwi is induced in ISCs under regenerative pressure in a STAT-dependent manner•Piwi is required for long-term maintenance and function of ISCs•Piwi prevents retrotransposon activity, heterochromatin loss, and apoptosis in ISCs•Piwi overexpression prevents age-related ISC dysfunction Sophisticated mechanisms that preserve genome integrity are critical to ensure the maintenance of regenerative capacity while preventing transformation of somatic stem cells (SCs), yet little is known about mechanisms regulating genome maintenance in these cells. Here, we show that intestinal stem cells (ISCs) induce the Argonaute family protein Piwi in response to JAK/STAT signaling during acute proliferative episodes. Piwi function is critical to ensure heterochromatin maintenance, suppress retrotransposon activation, and prevent DNA damage in homeostasis and under regenerative pressure. Accordingly, loss of Piwi results in the loss of actively dividing ISCs and their progenies by apoptosis. We further show that Piwi expression is sufficient to allay age-related retrotransposon expression, DNA damage, apoptosis, and mis-differentiation phenotypes in the ISC lineage, improving epithelial homeostasis. Our data identify a role for Piwi in the regulation of somatic SC function, and they highlight the importance of retrotransposon control in somatic SC maintenance. Sophisticated mechanisms that preserve genome integrity are critical to ensure the maintenance of regenerative capacity while preventing transformation of somatic stem cells (SCs), yet little is known about mechanisms regulating genome maintenance in these cells. Here, we show that intestinal stem cells (ISCs) induce the Argonaute family protein Piwi in response to JAK/STAT signaling during acute proliferative episodes. Piwi function is critical to ensure heterochromatin maintenance, suppress retrotransposon activation, and prevent DNA damage in homeostasis and under regenerative pressure. Accordingly, loss of Piwi results in the loss of actively dividing ISCs and their progenies by apoptosis. We further show that Piwi expression is sufficient to allay age-related retrotransposon expression, DNA damage, apoptosis, and mis-differentiation phenotypes in the ISC lineage, improving epithelial homeostasis. Our data identify a role for Piwi in the regulation of somatic SC function, and they highlight the importance of retrotransposon control in somatic SC maintenance. Stem cells in high-turnover tissues require high precision in genome maintenance mechanisms to ensure their long-term maintenance. In vertebrate stem cell populations, such mechanisms include effective cell cycle checkpoints and DNA repair machineries (Behrens et al., 2014Behrens A. van Deursen J.M. Rudolph K.L. Schumacher B. Impact of genomic damage and ageing on stem cell function.Nat. Cell Biol. 2014; 16: 201-207Crossref PubMed Scopus (144) Google Scholar). Deficiency in these mechanisms can result in stem cell exhaustion, mis-differentiation, and cancers (Adams et al., 2015Adams P.D. Jasper H. Rudolph K.L. Aging-induced stem cell mutations as drivers for disease and cancer.Cell Stem Cell. 2015; 16: 601-612Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Especially during periods of high proliferative pressure, genome integrity becomes quickly compromised, increasing the potential for stem cell exhaustion and cancers. DNA repair pathways play a critical role in limiting stem cell failure and exhaustion during replicative pressure, and their loss contributes to the development of age-associated phenotypes (Murga et al., 2009Murga M. Bunting S. Montaña M.F. Soria R. Mulero F. Cañamero M. Lee Y. McKinnon P.J. Nussenzweig A. Fernandez-Capetillo O. A mouse model of ATR-Seckel shows embryonic replicative stress and accelerated aging.Nat. Genet. 2009; 41: 891-898Crossref PubMed Scopus (274) Google Scholar, Walter et al., 2015Walter D. Lier A. Geiselhart A. Thalheimer F.B. Huntscha S. Sobotta M.C. Moehrle B. Brocks D. Bayindir I. Kaschutnig P. et al.Exit from dormancy provokes DNA-damage-induced attrition in haematopoietic stem cells.Nature. 2015; 520: 549-552Crossref PubMed Scopus (393) Google Scholar). It can be anticipated that dynamically regulated genome maintenance strategies are employed to ensure resilience of proliferating stem cell populations. The identity and regulation of these strategies remain to be established in vivo. Potential dangers to genome integrity include replication-related DNA damage and telomere dysfunction, but also deficiencies in higher-order chromatin regulation and dysfunction in retrotransposon control. The cell-autonomous activation and integration of transposable elements (TEs) can lead to insertional mutagenesis and genome rearrangements (Burns and Boeke, 2012Burns K.H. Boeke J.D. Human transposon tectonics.Cell. 2012; 149: 740-752Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Such phenomena have recently been implicated in the aging process of multiple organisms (De Cecco et al., 2013De Cecco M. Criscione S.W. Peterson A.L. Neretti N. Sedivy J.M. Kreiling J.A. Transposable elements become active and mobile in the genomes of aging mammalian somatic tissues.Aging (Albany NY). 2013; 5: 867-883Crossref PubMed Scopus (215) Google Scholar, Maxwell et al., 2011Maxwell P.H. Burhans W.C. Curcio M.J. Retrotransposition is associated with genome instability during chronological aging.Proc. Natl. Acad. Sci. USA. 2011; 108: 20376-20381Crossref PubMed Scopus (97) Google Scholar, Wang et al., 2011Wang J. Geesman G.J. Hostikka S.L. Atallah M. Blackwell B. Lee E. Cook P.J. Pasaniuc B. Shariat G. Halperin E. et al.Inhibition of activated pericentromeric SINE/Alu repeat transcription in senescent human adult stem cells reinstates self-renewal.Cell Cycle. 2011; 10: 3016-3030Crossref PubMed Scopus (77) Google Scholar). In the aging fly brain, somatic transposition increases and exacerbated TE expression results in age-associated impairment of memory and shorter lifespan (Li et al., 2013Li W. Prazak L. Chatterjee N. Grüninger S. Krug L. Theodorou D. Dubnau J. Activation of transposable elements during aging and neuronal decline in Drosophila.Nat. Neurosci. 2013; 16: 529-531Crossref PubMed Scopus (209) Google Scholar). Given the mutagenic potential and the impact at the level of genomic instability of transposition events (Wang et al., 2011Wang J. Geesman G.J. Hostikka S.L. Atallah M. Blackwell B. Lee E. Cook P.J. Pasaniuc B. Shariat G. Halperin E. et al.Inhibition of activated pericentromeric SINE/Alu repeat transcription in senescent human adult stem cells reinstates self-renewal.Cell Cycle. 2011; 10: 3016-3030Crossref PubMed Scopus (77) Google Scholar), organisms have evolved mechanisms to repress the activity of their endogenous TEs. In the germline, this is primarily achieved by the Piwi-interacting RNA pathway (Siomi et al., 2011Siomi M.C. Sato K. Pezic D. Aravin A.A. PIWI-interacting small RNAs: the vanguard of genome defence.Nat. Rev. Mol. Cell Biol. 2011; 12: 246-258Crossref PubMed Scopus (929) Google Scholar), which represses TE activity by promoting post-transcriptional processing of TE mRNAs and mediating transcriptional silencing of TE-rich regions through chromatin modifications. Piwi is the founding member of the evolutionarily conserved Ago/Piwi protein family, and it is required for the self-renewal of germline stem cells (GSCs) in flies and mice (Carmell et al., 2007Carmell M.A. Girard A. van de Kant H.J. Bourc’his D. Bestor T.H. de Rooij D.G. Hannon G.J. MIWI2 is essential for spermatogenesis and repression of transposons in the mouse male germline.Dev. Cell. 2007; 12: 503-514Abstract Full Text Full Text PDF PubMed Scopus (859) Google Scholar, Cox et al., 1998Cox D.N. Chao A. Baker J. Chang L. Qiao D. Lin H. A novel class of evolutionarily conserved genes defined by piwi are essential for stem cell self-renewal.Genes Dev. 1998; 12: 3715-3727Crossref PubMed Scopus (802) Google Scholar, Unhavaithaya et al., 2009Unhavaithaya Y. Hao Y. Beyret E. Yin H. Kuramochi-Miyagawa S. Nakano T. Lin H. MILI, a PIWI-interacting RNA-binding protein, is required for germ line stem cell self-renewal and appears to positively regulate translation.J. Biol. Chem. 2009; 284: 6507-6519Crossref PubMed Scopus (172) Google Scholar). In both species, loss of Piwi is associated with transposon desilencing and increased apoptosis (Juliano et al., 2011Juliano C. Wang J. Lin H. Uniting germline and stem cells: the function of Piwi proteins and the piRNA pathway in diverse organisms.Annu. Rev. Genet. 2011; 45: 447-469Crossref PubMed Scopus (277) Google Scholar). The functions of Piwi outside the germline are only beginning to be explored (Ross et al., 2014Ross R.J. Weiner M.M. Lin H. PIWI proteins and PIWI-interacting RNAs in the soma.Nature. 2014; 505: 353-359Crossref PubMed Scopus (301) Google Scholar). In differentiated somatic tissues, a physiologic function for the Piwi pathway has been reported in the brain and fat body of Drosophila (Janic et al., 2010Janic A. Mendizabal L. Llamazares S. Rossell D. Gonzalez C. Ectopic expression of germline genes drives malignant brain tumor growth in Drosophila.Science. 2010; 330: 1824-1827Crossref PubMed Scopus (202) Google Scholar, Jones et al., 2016Jones B.C. Wood J.G. Chang C. Tam A.D. Franklin M.J. Siegel E.R. Helfand S.L. A somatic piRNA pathway in the Drosophila fat body ensures metabolic homeostasis and normal lifespan.Nat. Commun. 2016; 7: 13856Crossref PubMed Scopus (70) Google Scholar). Piwi protein orthologs are also expressed in neoblasts of planaria (Reddien et al., 2005Reddien P.W. Oviedo N.J. Jennings J.R. Jenkin J.C. Sánchez Alvarado A. SMEDWI-2 is a PIWI-like protein that regulates planarian stem cells.Science. 2005; 310: 1327-1330Crossref PubMed Scopus (455) Google Scholar) and blastemal cells of salamander (Zhu et al., 2012Zhu W. Pao G.M. Satoh A. Cummings G. Monaghan J.R. Harkins T.T. Bryant S.V. Randal Voss S. Gardiner D.M. Hunter T. Activation of germline-specific genes is required for limb regeneration in the Mexican axolotl.Dev. Biol. 2012; 370: 42-51Crossref PubMed Scopus (43) Google Scholar), where they are required for efficient regeneration. However, the physiological function of the pathway and its relevance for stem cell maintenance and regenerative capacity have not been explored. Intestinal stem cells (ISCs) of the Drosophila posterior midgut epithelium constitute an experimentally accessible system to address these questions (Ayyaz and Jasper, 2013Ayyaz A. Jasper H. Intestinal inflammation and stem cell homeostasis in aging Drosophila melanogaster.Front. Cell. Infect. Microbiol. 2013; 3: 98Crossref PubMed Scopus (57) Google Scholar). ISCs are the main mitotically competent cell type in the intestinal epithelium of flies, giving rise to an enteroblast (EB) daughter cell that further differentiates into an enterocyte (EC) or an enteroendocrine cell (EE). A complex network of local and systemic signals regulates ISC maintenance and proliferative homeostasis (Ayyaz and Jasper, 2013Ayyaz A. Jasper H. Intestinal inflammation and stem cell homeostasis in aging Drosophila melanogaster.Front. Cell. Infect. Microbiol. 2013; 3: 98Crossref PubMed Scopus (57) Google Scholar). In response to an acute stress signal, such as the infection by a pathogen, ISCs readily increase their proliferative activity to fulfill regenerative demands, and subsequently they shut down the proliferative response to avoid hyperplasia and reinstate epithelial homeostasis (Ayyaz et al., 2015Ayyaz A. Li H. Jasper H. Haemocytes control stem cell activity in the Drosophila intestine.Nat. Cell Biol. 2015; 17: 736-748Crossref PubMed Scopus (98) Google Scholar). Damage to the epithelium induces the expression of cytokines of the Unpaired family (Upd 2 and 3) in ECs, which in turn activate JAK/STAT signaling in ISCs to promote the shift from quiescence to proliferation. JAK/STAT signaling cooperates with epidermal growth factor (EGF) receptor (EGFR) activation to promote proliferation of ISCs, and the return to quiescence is associated with the inhibition of JAK/STAT signaling by negative feedback regulators like SOCS36E (Ayyaz and Jasper, 2013Ayyaz A. Jasper H. Intestinal inflammation and stem cell homeostasis in aging Drosophila melanogaster.Front. Cell. Infect. Microbiol. 2013; 3: 98Crossref PubMed Scopus (57) Google Scholar). The control of proliferative activity is de-regulated in old flies, resulting in dysplasia that is reminiscent of hyper-proliferative phenotypes acquired in young animals in response to chronic stress (Ayyaz and Jasper, 2013Ayyaz A. Jasper H. Intestinal inflammation and stem cell homeostasis in aging Drosophila melanogaster.Front. Cell. Infect. Microbiol. 2013; 3: 98Crossref PubMed Scopus (57) Google Scholar). This is accompanied by widespread mis-differentiation of ISC daughter cells, caused by ectopic activation of Delta/Notch signaling (Biteau et al., 2008Biteau B. Hochmuth C.E. Jasper H. JNK activity in somatic stem cells causes loss of tissue homeostasis in the aging Drosophila gut.Cell Stem Cell. 2008; 3: 442-455Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar). Due to the frequent exposure of ISCs to regenerative pressure, mechanisms that control genomic integrity of ISCs are likely to be critical for long-term functional maintenance of the intestinal epithelium. However, how this is achieved remains unclear. Here we show that the dynamic control of Piwi expression in ISCs is one such mechanism. Piwi is transcriptionally activated in ISCs that are induced to proliferate, and it is required for regenerative capacity and maintenance of ISCs during the regenerative process. Our findings suggest that a decline in the ability of Piwi to maintain heterochromatin contributes to the age-associated deregulation of ISC function and loss of tissue homeostasis. In response to infection with the mild enteropathogen Erwinia carotovora carotovora 15 (Ecc15), ISCs undergo transient activation followed by a return to quiescence when the infection is cleared (Figures 1A and 1B ). To characterize molecular changes that are associated with this transient activation of ISCs, we analyzed transcriptomes of wild-type and STAT-deficient ISCs isolated at various time points after infection by fluorescence-activated cell sorting (FACS; compare Dutta et al., 2015Dutta D. Dobson A.J. Houtz P.L. Gläßer C. Revah J. Korzelius J. Patel P.H. Edgar B.A. Buchon N. Regional cell-specific transcriptome mapping reveals regulatory complexity in the adult Drosophila midgut.Cell Rep. 2015; 12: 346-358Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar and Figures 1A and 1C). STAT was knocked down by the expression of a double-stranded RNA (dsRNA) against STAT using the ISC/EB-specific driver escargot::Gal4 (esg::Gal4, UAS::GFP), combined with ubiquitous expression of temperature-sensitive Gal80 (tub::Gal80ts, TARGET system). RNAi was induced in young (3-day-old) adults for 3 days prior to infection (Figure 1A) to knock down STAT expression (Figure S1A). ISCs were identified in the FACS experiment by their expression of GFP, and they were differentiated from EBs by their smaller size (Figure 1C; note that inhibition of Gal4 in EBs by the expression of Gal80 under the control of a Su(H) promoter [Wang et al., 2014bWang L. Zeng X. Ryoo H.D. Jasper H. Integration of UPRER and oxidative stress signaling in the control of intestinal stem cell proliferation.PLoS Genet. 2014; 10: e1004568Crossref PubMed Scopus (78) Google Scholar] selectively inhibits GFP in the larger cell population). This strategy allows faithful purification of ISCs, as confirmed by the expression of the ISC marker Delta (Dl) and enrichment for progenitor-specific genes (Figures 1D and S1B). This cell population is also depleted for genes expressed in ECs (Figure S1B). Using RNA sequencing (RNA-seq) (Figure S1C), we identified over 655 genes induced in wild-type ISCs at 4 hr after Ecc15 infection. About one-half of those genes required JAK/STAT activity for their induction, and this subset was highly enriched for genes associated with DNA replication and repair (Figures 1E and 1F; Table S1). piwi was found among these genes (Figure 1G), and analysis of independent samples collected in the same conditions, but analyzed by qRT-PCR, confirmed STAT-dependent Piwi induction in ISCs upon regenerative pressure (Figure 1H). Using in situ hybridization (Figure 2A), immunohistochemistry (Figure 2B), a lacZ enhancer trap line (Figure S2A), and a reporter line expressing an N-terminally EGFP-tagged Piwi from the endogenous piwi locus (Figure S2B; Sienski et al., 2012Sienski G. Dönertas D. Brennecke J. Transcriptional silencing of transposons by Piwi and maelstrom and its impact on chromatin state and gene expression.Cell. 2012; 151: 964-980Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar), we found that Piwi was specifically expressed in ISCs and EBs of the posterior midgut, but not in differentiated cell types. In these experiments, ISCs/EBs were detected by GFP expression driven by esg::Gal4 (Figures 2A and 2B) or by co-staining with Dl (Figures S2A and S2B), which co-localized with piwi mRNA or protein signal. We confirmed the induction of Piwi protein after Ecc15 infection (Figures 2B and S2A–S2D) and the dependence on STAT signaling (Figure 2B). Piwi protein detected using immunohistochemistry (Figure 2B) or with the GFP signal of the tagged Piwi protein (Figure S2B) further revealed that Piwi localizes both to the cytoplasm and to the nucleus. Piwi expression was also induced, in a STAT-dependent manner, in ISCs/EBs of flies exposed to other damaging insults, such as bleomycin (Figure S2E), and in conditions where proliferative pressure is imposed by the overexpression of constitutively active Ras (Rasv12; Figure S2F). Furthermore, activation of STAT signaling, through overexpression of a constitutively active form of the JAK kinase hopscotch (hopTumL; Hanratty and Dearolf, 1993Hanratty W.P. Dearolf C.R. The Drosophila Tumorous-lethal hematopoietic oncogene is a dominant mutation in the hopscotch locus.Mol. Gen. Genet. 1993; 238: 33-37Crossref PubMed Scopus (121) Google Scholar), was sufficient to induce Piwi expression in progenitor cells (Figure S2F). Conversely, blocking EGFR signaling prevented Ecc15-dependent Piwi induction (Figure S2F). Thus, Piwi is induced in ISCs/EBs of the Drosophila midgut under proliferative pressure, independently of the type of regenerative stimulus. To test if Piwi activity is required to regulate or maintain ISC function during regeneration, we analyzed the effects of Piwi knockdown (Figure 3A). Expression of an RNAi against Piwi for 3 days in ISCs/EBs of young adult flies was sufficient to significantly reduce its expression in the midgut (Figures S2G and S2H). Loss of Piwi impaired regenerative capacity of the gut (Figure 3A), leading to reduced density of both ECs and progenitor cells in infected midguts containing Piwi-deficient progenitors. This was likely associated with the inability to maintain ISCs rather than with defects in ISC activation, as the number of Dl+ cells was significantly reduced in midguts containing Piwi-deficient ISCs, while mitotic activity (determined using phosphorylated Histone H3 [pH3]) was similar to wild-type guts at earlier time points (Figures 3A and S3A). Similarly, piwi2 homozygous flies had significantly lower numbers of Dl+ ISCs already 48 hr after Ecc15 infection, and a large number of these ISCs also expressed the EC marker PDM1, indicating mis-differentiation (Figure S3B). The requirement of Piwi for ISC function under regenerative pressure was further confirmed by lineage-tracing ISCs homozygous for the piwi3 loss-of-function allele (Lin and Spradling, 1997Lin H. Spradling A.C. A novel group of pumilio mutations affects the asymmetric division of germline stem cells in the Drosophila ovary.Development. 1997; 124: 2463-2476Crossref PubMed Google Scholar), using mosaic analysis with a repressible cell marker (MARCM) during regeneration. Consistent with the previous results, piwi3 mutant ISCs generated smaller MARCM clones than wild-type controls (Figure 3B). piwi-deficient MARCM clones were generally composed of 2–3 cells rather than single ISCs, indicating that, before arresting, ISCs underwent a few rounds of division after the induction of piwi3 homozygosity. Survival after enteropathogen infection is an indicator of effective regeneration, as the inability to restore epithelial integrity can result in an animal’s death. Thus, we monitored survival following infection with the strong enteropathogen Pseudomonas entomophila (P.e.). Flies with Piwi-deficient ISCs and EBs died faster after infection with P.e. (Figures 3C and 3D), consistent with an inability to efficiently regenerate the intestinal epithelium (Figure 3C). The role of Piwi in maintaining ISC function during proliferative pressure was not limited to infection conditions, as loss of Piwi limited the growth of ISC tumors generated by Notch deficiency (Figure S3C). Our results suggest that, even in these conditions, Piwi-deficient ISCs cannot sustain a high rate of proliferative activity. We asked if Piwi was also required for long-term maintenance of ISCs and midgut homeostasis. We knocked down Piwi in ISCs of young adults and analyzed the effects 7 and 14 days later. While Piwi-deficient intestines did not exhibit major defects at 7 days after Piwi knockdown (data not shown), loss of epithelial homeostasis became apparent at 14 days after Piwi depletion, as evidenced by a reduction in the density of ECs (Figure 3E, large polyploid nuclei stained with DAPI) and progenitor cells (Figure 3E). These effects of Piwi loss are region specific, with most of ISC/EB loss occurring in the R4bc morphological subdomain (Buchon et al., 2013Buchon N. Osman D. David F.P. Fang H.Y. Boquete J.P. Deplancke B. Lemaitre B. Morphological and molecular characterization of adult midgut compartmentalization in Drosophila.Cell Rep. 2013; 3: 1725-1738Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar) of the midgut (Figure 3E). To determine the cell type specificity of this effect, we used ISC-specific and EB-specific drivers to knock down Piwi. ISC-specific knockdown of Piwi resulted in a significant loss of ISCs (Figure S3D), while EB-specific knockdown of Piwi had no effect on ISC or EB numbers (Figure S3E). Altogether, these data suggested that Piwi acts in an ISC-autonomous fashion to maintain ISC function and that, accordingly, loss of Piwi is accompanied by a progressive loss of ISC function in the posterior midgut. This observation was reproduced in piwi2 mutant flies (Figure S3F) and by lineage-tracing piwi mutant ISCs: clones derived from piwi3 homozygous ISCs were significantly smaller than clones derived from wild-type ISCs (Figures S3G–S3I). This phenotype was region specific (Figure S3H; also after Ecc15 infection, Figure S3J) and recapitulated the loss of ISCs observed after Piwi knockdown, as there was a significantly higher proportion of clones derived from piwi3 homozygous ISCs that were completely depleted of Dl+ ISCs (Figure S3I). Piwi proteins have both cytoplasmic and nuclear functions, having been implicated in the maintenance of the chromatin state in the nucleus (Rozhkov et al., 2013Rozhkov N.V. Hammell M. Hannon G.J. Multiple roles for Piwi in silencing Drosophila transposons.Genes Dev. 2013; 27: 400-412Crossref PubMed Scopus (189) Google Scholar). To assess if Piwi nuclear function is critical for ISC maintenance, we lineage-traced ISCs that were homozygous for a piwi allele in which the 26 N-terminal amino acids containing the nuclear localization signal are absent (piwiNT). This mutant Piwi lacks the ability to translocate to the nucleus, thus preventing its ability to regulate chromatin structure, while retaining its cytoplasmic function (Klenov et al., 2011Klenov M.S. Sokolova O.A. Yakushev E.Y. Stolyarenko A.D. Mikhaleva E.A. Lavrov S.A. Gvozdev V.A. Separation of stem cell maintenance and transposon silencing functions of Piwi protein.Proc. Natl. Acad. Sci. USA. 2011; 108: 18760-18765Crossref PubMed Scopus (121) Google Scholar). piwiNT MARCM clones phenocopied the piwi3 loss-of-function mutant, suggesting that the nuclear function of Piwi is required for the maintenance of ISC function (Figure S3K). The silencing of transposons in the Drosophila germline relies in part on the formation and maintenance of heterochromatic regions by Piwi family proteins, through interaction with heterochromatin-forming pathways (Sienski et al., 2012Sienski G. Dönertas D. Brennecke J. Transcriptional silencing of transposons by Piwi and maelstrom and its impact on chromatin state and gene expression.Cell. 2012; 151: 964-980Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar). To test this function of nuclear Piwi in ISCs, we assessed the extent of heterochromatinization globally using a Position-Effect Variegation (PEV) reporter line (Lu et al., 1996Lu B.Y. Bishop C.P. Eissenberg J.C. Developmental timing and tissue specificity of heterochromatin-mediated silencing.EMBO J. 1996; 15: 1323-1332Crossref PubMed Scopus (74) Google Scholar). The In(3L)BL1 line contains a heat shock-inducible HS-lacZ gene insertion juxtaposed to pericentric heterochromatin, resulting in variegated expression of lacZ. Thus, lacZ expression can be evaluated at the level of single cells as an indicator of heterochromatin status (Figure 4A). piwi3 heterozygous mutants showed an increase in the number of lacZ-expressing progenitor cells in the posterior midgut at 20 days of age (Figure 4B) and in conditions of acute regenerative pressure caused by infection with Ecc15 (Figure S4A), associated with a significant increase in LacZ transcript abundance (Figures 4B and S4A). This indicates that a significant loss of constitutive heterochromatin occurred in those animals and that the nuclear function of Piwi that is required for ISC maintenance may involve the regulation of heterochromatin. Supporting this view, loss of Piwi was associated with de-repression of TE expression and with TE mobilization. Knockdown of Piwi, but not of Aub or AGO3, was accompanied by an accumulation of several TE transcripts (Figure 4C). The requirement of Piwi for TE repression was further confirmed in the piwi3 heterozygous background, where a significant induction of Gypsy could be detected, in homeostasis and after Ecc15 infection (Figure S4B). Furthermore, the number of integration events in piwi3 heterozygous flies, assessed using a Gypsy-TRAP reporter system (Li et al., 2013Li W. Prazak L. Chatterjee N. Grüninger S. Krug L. Theodorou D. Dubnau J. Activation of transposable elements during aging and neuronal decline in Drosophila.Nat. Neurosci. 2013; 16: 529-531Crossref PubMed Scopus (209) Google Scholar), was higher than in wild-type controls (Figures 4D and 4E). The Gypsy-TRAP reporter consists of a GAL80 (Gal4 inhibitor) transgene expressed under the control of a tubulin promoter separated by a gypsy target site (Tub::OvoSite::GAL80), so that insertions of gypsy prevent GAL80 expression. Combined with esg::Gal4, UAS::GFP, this system allows the detection of de novo gypsy integration events in ISCs and EBs (Figure 4D). To further characterize the consequences of Piwi loss in ISCs, we analyzed the transcriptome of Piwi-deficient ISCs (Table S1) isolated by FACS. Gene ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway classification analysis of genes upregulated after Piwi knockdown indicated enrichment for components involved in proteasome-mediated degradation, DNA damage response (DDR) pathways, and apoptosis (Figures S4C–S4E). Interestingly, we found several subunits of the proteasome complex overexpressed, in agreement with previously reported effects of piwi loss in the germline (Le Thomas et al., 2013Le Thomas A. Rogers A.K. Webster A. Marinov G.K. Liao S.E. Perkins E.M. Hur J.K. Aravin A.A. Tóth K.F. Piwi induces piRNA-guided transcriptional silencing and establishment of a repressive chromatin state.Genes Dev. 2013; 27: 390-399Crossref PubMed Scopus (330) Google Scholar). The upregulated DDR components and molecules associated with apoptosis suggested an increase in DNA damage, which may result in checkpoint activation and the triggering of apoptotic pathways. Using qRT-PCR in an independent set of samples of ISCs isolated by FACS, we confirmed the upregulation of several of the genes listed above, as well as of TEs in Piwi-deficient ISCs (Figures S4F and S4G). As predicted by the transcriptome analysis, Piwi deficiency led to an increase in ISC apoptosis. Apoptotic cells were detected using either an antibody against the cleaved form of the Drosophila effector caspase, also known as death caspase 1 (cDcp1), or terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) (Sarkissian et al., 2014Sarkissian T. Timmons A. Arya R. Abdelwahid E. White K. Detecting apoptosis in Drosophila tissues and cells.Methods. 2014; 68: 89-96Crossref PubMed Scopus (53) Google Scholar). We observed a significant increase in the percentage of cDcp1+/Dl+ ISCs expressing an RNAi against Piwi during Ecc15-induced regeneration and in 5-day-old piwi2 homozygous flies (Figures S4H and S4I). Similarly, we observed a significant increase of TUNEL+ ISCs/EBs after piwi knockdown (Figure S4J). In addition, we used a GFP-based caspase activity reporter (Apoliner; Bardet et al., 2008Bardet P.L. Kolahgar G. Mynett A. Miguel-Aliaga I. Briscoe J. Meier P. Vincent J.P. A fluorescent reporter of caspase activity for live imaging.Proc. Natl. Acad. Sci. USA. 2008; 105: 13901-13905Crossref PubMed Scopus (122) Google Scholar) comprising two fluorophores, mRFP and EGFP, linked by a specific caspase-sensitive site. piwi knockdow" @default.
• W2756245919 created "2017-09-25" @default.
• W2756245919 creator A5006886237 @default.
• W2756245919 creator A5034084898 @default.
• W2756245919 creator A5043842215 @default.
• W2756245919 creator A5063179526 @default.
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• W2756245919 date "2017-09-01" @default.
• W2756245919 modified "2023-10-10" @default.
• W2756245919 title "Piwi Is Required to Limit Exhaustion of Aging Somatic Stem Cells" @default.
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