{"paper_id":"136fd724-2af1-47c7-af8b-e2b4f71fff1b","body_text":"Title 1 \nPIWI proximity proteome reveals Set1-mediated piRNA biogenesis for transposon 2 \nsilencing in telomere 3 \n 4 \nAuthors 5 \nWakana Isshiki1, Hiroko Kozuka-Hata2, Masaaki Oyama2, Toshie Kai1,3* , Taichiro 6 \nIki1* 7 \n*Co-corresponding 8 \n 9 \nAffiliations 10 \n1. Graduate School of Frontier Biosciences, The University of Osaka, Osaka, Japan 11 \n2. Medical Proteomics Laboratory, The Institute of Medical Science, The University of 12 \nTokyo, Tokyo, Japan 13 \n3. Graduate School of Biostudies, Kyoto University, Kyoto, Japan 14 \n  15 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nAbstract 16 \nSilencing complexes formed by PIWI -clade Argonaute (Ago) proteins and 17 \nPIWI-interacting RNAs (piRNAs) are essential guardians of genome integrity, 18 \ncontrolling the deleterious activities of transposable elements (TEs) in animal germline. 19 \nHowever, our understanding of PIWI-piRNA-directed TE silencing remains incomplete. 20 \nHere, we systemically characterize the proximity proteome of PIWI members, Piwi, 21 \nAubergine (Aub), and Ago3 in the germline of Drosophila ovaries. Functional screening 22 \nidentifies previously uncharacterized factors involved in TE silencing, including 23 \nH3K4me3 writer  and transcriptional coactivator Set1. Transcriptome analysis reveals 24 \nthat Set1 acts as an  indispensable repressor for TEs , particularly those  forming 25 \ntelomeres. The involvement of Set1 in Piwi pathway is further supported by its critical 26 \nrole in the production of antisense, TE-targeting piRNAs. Notably, catalytic activity of 27 \nSet1 is dispensable for TE silencing . Genome-wide chromatin binding analysis using 28 \nCUT&Tag demonstrates that Set1 preferentially associates with TE sequences and 29 \nlocalizes to subtelomeric piRNA cluster loci , suggesting a role in promoting  piRNA 30 \nprecursor transcription through direct binding . Collectively, these findings uncover a 31 \nnoncanonical function of Set1 in Piwi-mediated TE silencing and telomere control in 32 \ngermline nuclei.  33 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nIntroduction 34 \nTransposable elements (TEs) are mobile DNA sequences constituting a substantial part 35 \nof genomes in most organisms  (Wells & Feschotte, 2020). Although TEs act as drivers 36 \nof genomic evolution and species diversification, the harmful potential as mutagens 37 \nrequires the hosts to establish the control mechanisms. The control is particularly 38 \nimportant in germline cells where activated TEs accumulate damages on the inheritable 39 \ngenomes and impair the production of functional  gametes. In animals, TE control in 40 \ngermline cells relies  on silencing mediated by complexes of PIWI clade Argonaute 41 \n(Ago) family proteins and the sequence-specific guide, PIWI -interacting (pi)RNAs  42 \n(Wang et al, 2023b). 43 \nPIWI-piRNA-directed TE silencing has been studied in  different animals 44 \nincluding the invertebrate model, Drosophila melanogaster. D. melanogaster possesses 45 \nthree PIWI members including Piwi, Aubergine (Aub), and Ago3 , all of which are 46 \nexpressed in germline cells. Piwi differs from the other two  in that it functions in the 47 \nnucleus. Guided by piRNAs, Piwi complexes recognize the nascent transcripts  from 48 \ntarget loci, mediating the (co -)transcriptional silencing and heterochromatin formation 49 \n(Le Thomas et al, 2013; Yu et al, 2015b; Sienski et al, 2015a; Mugat et al, 2020; Ariura et 50 \nal, 2024). In contrast, Aub and Ago3  are cytoplasmic proteins,  localizing to nuage, a 51 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nperinuclear membraneless organelle  formed in germline cells  (Suyama & Kai, 2025; 52 \nKawaguchi et al, 2025). In nuage, Aub and Ago3 mediate the reciprocal cleavage of 53 \ntargets containing sense and antisense TE sequences. 3’ cleavage products generated by 54 \neither of the two proteins are passed to the other (i.e., from Aub to Ago3, or from Ago3 55 \nto Aub), then processed into a piRNA. Newly generated piRNAs direct the next round 56 \nof target cleavage and piRNA production. These cycles are called ping -pong, coupling 57 \nTE degradation and piRNA amplification  in germline cells. Because target cleavage 58 \noccurs between the nucleotides complementary to  10th and 11 th nucleotides of guide 59 \npiRNA, and new piRNA is produced from the 5’ end of cleavage product, germline 60 \npiRNAs exhibit 10-nt overlap signature (ping-pong signature). In addition to ping-pong, 61 \nprecursor fragments incorporated to Aub can be  processed into multiple piRNAs on 62 \nmitochondrial outer membrane. These phasing/trailer piRNAs are mainly loaded onto  63 \nPiwi, translocating the complexes to the nucleus  (Ge et al, 2019). Ping-pong cycles 64 \nprocess diverse transcripts including  intact TE mRNAs and long non -coding RNAs 65 \nderived by noncanonical transcription of heterochromatinized, large intergenic regions  66 \naccumulating truncated TE sequences, defined as piRNA cluster loci (Brennecke et al, 67 \n2007a). 68 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\npiRNA-directed TE silencing is achieved through interactions between PIWI 69 \nproteins and other components . These include nuclear factors involved in 70 \nPiwi-piRNA-directed target heterochromatinization (e.g., Panx, Nxf2, SetDB1/Eggless) 71 \n(Zhao et al, 2019; Murano et al, 2019; Fabry et al, 2019; Batki et al, 2019; Yu et al, 72 \n2015b; Sienski et al , 2015a). In cytoplasm, Tudor domain proteins  (Krimper, Tejas, 73 \nTapas, Qin/Kumo) and RNA helicases (Spn-E, Vas) are enriched in nuage and support 74 \nthe ping-pong cycles mediated by Aub and Ago3  (Webster et al, 2015; Lin et al, 2023; 75 \nHandler et al, 2011; Zhang et al, 2011; Anand & Kai, 2012; Qi et al, 2011; Saito et al, 76 \n2010; Lim & Kai, 2007; Kawaguchi et al , 2025) . Nonetheless, characterizing the 77 \ninteractions between PIWI members and other factors are often challenging because of 78 \ntheir dynamics in piRNA biogenesis and TE silencing. Hence, the repertoire of piRNA 79 \npathway components is heretofore unelucidated, and ou r understanding of 80 \npiRNA-directed TE silencing remains incomplete. 81 \nProximity-dependent biotin labelling (BioID/TurboID) is a powerful tool for 82 \ncharacterizing the physical interactions and proximity relationships  of proteins in vivo 83 \n(Roux et al, 2012; Branon et al, 2018; Choi‐Rhee et al, 2004). These techniques rely on 84 \nthe promiscuous biotin ligase activity exhibited by BirA derived from Escherichia coli. 85 \nTurboID utilizes an efficient and compact BirA variant called mini(m)Turbo established 86 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nthrough directed evolution (Branon et al, 2018), which we previously introduced in male 87 \ngermline cells to study testis -specific factors (Iki et al, 2023; Kai et al, 2025; Iki et al, 88 \n2020). In order to deepen the understanding  of piRNA-directed TE silencing, this study 89 \ncharacterized the proteins  in the physical proximity of individual PIWI proteins in 90 \nfemale germline cells  where TE silencing has been extensively studied . The obtained 91 \nproximity proteome of germline PIWI members not only confirmed reported 92 \ninteractions but also revealed the unrecognized links. Moreover, PIWI proximity 93 \nproteome contained  a list of factors which have  not been characterized in piRNA 94 \npathway thus far. Of those, this study further characterizes Set1, a histone 95 \nmethyltransferase and transcriptional coactivator conserved across eukaryotes. Our data 96 \nhighlight the hitherto unappreciated importance  of Set1 in Piwi-piRNA-directed 97 \nsilencing of TEs forming telomeres in Drosophila genome. 98 \n  99 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nResults 100 \nLabelling of PIWI proximity factors and purification from germline cells 101 \nTo characterize the proximity proteome of PIWI proteins using TurboID, we generated a 102 \nseries of transgene s encoding mTurbo -GFP-fused Piwi, Aub, or Ago3 (Figure 1A). 103 \nThese transgenes were expressed in ovarian germline cells under the control of nos 104 \npromoter activity (UASp/Gal4 binary system  using NGT 40;nos-Gal4 (NN) driver)  105 \n(Rørth, 1998; Grieder et al , 2000) . The biotinylation activities of individual fusion 106 \nproteins were confirmed by detecting the  streptavidin-HRP-dependent signals  (Figure 107 \nS1A). However, pulldown using streptavidin was inefficient  and biotinylated proteins 108 \nlargely remained in the flow through , possibly due to interfer ing factors accumulated 109 \nduring differentiation. To circumvent this technical challenge, we depleted one of the 110 \ndifferentiation factor s, bag of marbles  (bam) (McKearin & Spradling, 1990) , by 111 \ngermline knockdown (GLKD) , and collected germline stem cell  (GSC)-like cells 112 \n(GSCLCs) impaired in  differentiation (Figure 1A, S1BC). Although TurboID in 113 \nGSCLCs showed weaker biotinylation signals in the input, the pulldown efficiency was 114 \ndramatically improved (Figure S1A). 115 \nIn GSCLCs, mTurbo-GFP alone displayed a broad distribution across both the 116 \nnucleus and cytoplasm (Figure 1B). In contrast, mTurbo -GFP-PIWI proteins exhibited 117 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nsubcellular localization patterns consistent with those of their  endogenous counterparts; 118 \nPiwi accumulated in the nucleus, while Aub and Ago3 were enriched in the perinuclear 119 \nnuage. The concordance suggests that N -terminal mTurbo -GFP tagging does not 120 \nsubstantially disrupt the interaction networks  of individual PIWI proteins. Supporting 121 \nthis notion, N-terminal mTurbo-FLAG fusion preserved Aub functions in male germline 122 \n(Iki et al, 2023). Notably, the steady-state levels of fusion proteins were below those of 123 \nendogenous counterparts (Figure S1D) . mTurbo-GFP-Ago3 was the least stable and 124 \nbarely detectable in the input (Figure 1C , anti -GFP), accounting for the weak 125 \nstreptavidin-HRP signals in Ago3 -TurboID condition (Figure 1 C). Nevertheless, the 126 \nstreptavidin pulldown displayed distinct signal patterns, suggesting 127 \nbiotinylation-dependent enrichment of specific factors in each PIWI-TurboID condition. 128 \n 129 \nCharacterization of germline PIWI proximity proteomes 130 \nMass spectrometry analysis and the label-free quantification (LFQ) of streptavidin 131 \npulldown samples identified the proximity factors of individual PIWI proteins (average 132 \nabundance ratios >1 compared to the control GFP-TurboID conditions in biological 133 \nduplicates) (Table S1). These included 37, 33, and 49 factors for Piwi, Aub, and Ago3, 134 \nrespectively (Figure 2A and 2B, S2). Notably, the identified proximity factors contained 135 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nknown piRNA pathway components  (Figure 2A, highlighted in yellow, Table S2), and 136 \nthe significant enrichment was confirmed by Gene Ontology (GO) analysis (Figure 2C). 137 \nThese results suggest that the proteome reflects the physical and functional interactions 138 \nbetween PIWI members and other factors in germline cells. 139 \n 140 \nScreening of  PIWI proximity factors involved in piRNA biogenesis and TE 141 \nsilencing 142 \nMost factors identified in the proximity of PIWI members have not been functionally 143 \ncharacterized in piRNA pathway or TE silencing. Hence, w e investigated the possible 144 \ninvolvement of 54 factors, for which short hairpin-based RNAi lines were available, by 145 \ngermline knockdown (GLKD)  screening using NN driver. Given that impaired piRNA 146 \nbiogenesis can be associated with nuage collapse and mislocalization of its components, 147 \nwe examined the localization of Krimp, a key scaffold of nuage (Lim & Kai, 2007; Patil 148 \n& Kai, 2010). In parallel, we observed the accumulation of Gag protein encoded by a 149 \nnon-LTR retroelement HeT-A, as a readout of TE derepression  (Shpiz et al, 2011). We 150 \nconfirmed that GLKD of  aub leads to the accumulation of HeT-A Gag protein around 151 \nthe oocytes of developing egg chambers (Figure 3A) , and the loss of Krimp from 152 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nperinuclear nuage and the formation of abnormal foci in  the cytoplasm (Figure 3B , 153 \nS3B). 154 \nGLKD screening identified several factors  including Hrb27C and Set1 , the 155 \nproximity factor of Aub and Piwi, respectively  (Table S3). Both factors are conserved 156 \nacross eukaryotes , and their molecular and physiological functions have been 157 \ncharacterized. Hrb27C (also known as Hrp48) is a n hnRNP family member required in 158 \nGSC for the maintenance (Yan et al, 2014; Ables et al, 2016; Finger et al, 2023). Set1 is 159 \nan enzyme acting inside COMPASS complex responsible for di- and tri-methylation of 160 \nhistone H3 at lysine 4 (H3K4me2/3), a modification associated with transcription start 161 \nsites (TSSs) for RNA polymerase (pol)II activation (Ardehali et al, 2011; Mohan et al, 162 \n2011; Wang et al , 2023a) . We observed distinct phenotypes  in hrb27C-GLKD and 163 \nset1-GLKD. In hrb27C-GLKD, abnormal Krimp foci appeared in the cytoplasm, similar 164 \nto aub-GLKD (Figure 3B). However, HeT-A Gag protein was under detectable level 165 \n(Figure 3A). In set1-GLKD, though abnormal Krimper foci formation was not clear, 166 \nHeT-A Gag protein accumulated around the oocyte. In both hrb27c- and set1-GLKD 167 \nconditions, perinuclear Krimp signals could be weaker but still  observed (Figure 3B). 168 \nConsistently, Aub and Piwi did not show drastic changes in their localization (Figure 3C, 169 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nS3C). Of note, RT -qPCR confirmed target downregulation in individual GLKD 170 \nconditions (Figure S3D). 171 \n 172 \nset1 and hrb27C functionally interact with piwi and aub, respectively, to control 173 \nTEs  174 \nTo investigate the global effect of  Hrb27C or Set1 depletion in the  germline on TE  175 \nexpression, we compared the transcriptome from hrb27C- or set1-GLKD ovaries with 176 \nthat from gfp-GLKD control ovaries (Figure 4 A, S4 A). In parallel, aub- and 177 \npiwi-GLKD ovary transcriptomes were generated as references for TE derepression.  178 \nOur analysis included  germline stem cell -like cell (GSCLC) condition in which PIWI 179 \nproximity proteome was characterized (Figure 4B, S4B).  First, comparison of  180 \ngfp-GLKD transcriptomes between non-GSCLC and GSCLC contexts revealed that TE 181 \ntranscript levels are generally elevated in GSCLCs (Figure S4C). This baseline increase 182 \nmay account for the relatively modest TE derepression observed for  aub- or 183 \npiwi-GLKD in GSCLC setting (Figure 4AB). 184 \nHrb27C was identified as the  proximity factor of Aub  (Figure 2). H owever, 185 \nunlike aub-GLKD, hrb27C-GLKD did not result in  clear TE derepression in 186 \nnon-GSCLC ovaries (Figure 4A, S4A) . In contrast , TE derepression was evident in 187 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nGSCLC ovaries (Figure 4B, S4B) , and the patterns were positively correlated between 188 \nhrb27C- and aub-GLKD conditions (r = 0.57, Figure S4D). These suggest a functional 189 \nlink between Hrb27C and Aub in GSCs. 190 \nFor Set1 identified as the proximity factor of Piwi (Figure 2), its GLKD caused 191 \nremarkable derepression of a subset of  TEs in non-GSCLC ovaries (Figure 4A, S4A). 192 \nThese set1-sensitive TEs included HeT-A, consistent with its Gag protein accumulation 193 \n(Figure 3) , as well as  TART family members , which together with  HeT-A maintain 194 \ntelomeres as HTT array (Villasante et al, 2007). TAHRE, another HTT component, was 195 \nnot analyzed due to the lack of annotated insertions. In addition to  HTT, several LTR 196 \nretrotransposons including HMS-Beagle, Max-element, diver, 3S18, and gypsy12 197 \nexhibited robust derepression (Figure 4A) . This result  was corroborated by analyses 198 \nbased on mapping reads to consensus sequences of individual TE families (Figure 4C). 199 \nOverall, the derepression profile of set1-GLKD showed a strong positive correlation  200 \nwith that of piwi-GLKD (r = 0. 73), but not with aub-GLKD (r = 0.02, Figure 4 C). 201 \nConsistent with this, re-analysis of  publicly available dataset (GSE103582) indicated 202 \nthat HTT members are more sensitive to the loss of piwi than that of aub (Figure 4D, 203 \nS4E) (Teixeira et al , 2017a). Moreover, similar to set1-GLKD, HTT members were 204 \nstrongly derepressed upon loss of  Panoramix (Panx), an essential cofactor of Piwi 205 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\n(GSE71374) (Sienski et al, 2015b; Yu et al, 2015a). In contrast to the pronounced effect 206 \nobserved in germline, knockdown of set1 in ovarian follicle cells using traffic jam-Gal4 207 \ndid not derepress  zam and gypsy, which are silenced by piwi in this somatic lineage 208 \n(Figure S4F). Taken together, these results suggest that Set1 function is associated with 209 \nPiwi-mediated TE silencing in germline cells. 210 \n 211 \nCatalytic activity-independent role of Set1 in TE silencing 212 \nThe C-terminal SET domain of Set1 is responsible for H3K4me3 modification (Mohan 213 \net al , 2011; Ardehali et al , 2011; Wilson et al , 2002) . To investigate  whether this 214 \nmethyltransferase activity is required for TE silencing , we performed rescue 215 \nexperiments by expressing RNAi-resistant transgene encoding GFP -tagged wild -type 216 \nSet1 (Set1 WT) or a catalytically inactive variant (Set1E1613K) in germline cells depleted 217 \nof endogenous Set1 (set1-GLKD) (Figure 5A) (Vidaurre et al, 2024; Hallson et al, 2012). 218 \nBoth GFP-Set1WT and GFP -Set1E1613K proteins accumulated in the germline  nuclei 219 \n(Figure 5B). Weaker GFP signals observed for E1613K variant suggest reduced protein 220 \nstability.  221 \nTranscriptome analysis revealed that HTT and other TE families  were fully 222 \nre-repressed by GFP -Set1WT introduced in the  set1-GLKD background, indicating the 223 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\ndepletion of set1 is causal for their derepression (Figure 5C). Remarkably, although less 224 \neffective than GFP-Set1WT, expression of GFP-Set1E1613K led to substantial 225 \nre-repression of TEs. This effect was most pronounced for HTT, with TART-A1 as an 226 \nexception (Figure 5C). Together, these results suggest that  Set1 plays a noncanonical, 227 \ncatalytic activity-independent role in TE silencing. 228 \nTo further support the above notion , we analyzed H3K4me3 signals in 229 \nset1-GLKD and the transgene-rescue conditions. Ovary immunotaining showed that 230 \nH3K4me3 signals were depleted from germline nuclei in set1-GLKD (Figure 5 B). 231 \nGermline H3K4me3 signals were fully recovered by GFP -Set1WT, while no  recovery 232 \nwas observed with GFP-Set1E1613K. We further employed CUT&Tag to genome-widely 233 \ncharacterize H3K4me3 signals. H3K4me3 peaks were identified around  the 234 \ntranscription start site (TSS) of genes including ago3 and aub (Figure 5D). Consistent 235 \nwith the immunostaining data, H3K4me3 signals on ago3 and aub were markedly 236 \nreduced in set1-GLKD ovaries, and  fully restored by GFP-Set1WT, but not by 237 \nGFP-Set1E1613K (Figure 5D). These results confirm the catalytic inactivity of  E1613K 238 \nvariant and further support the catalytic activity -independent role in TE silencing . 239 \nNotably, despite these changes in H3K4me3 signals,  steady-state transcript levels of 240 \npiRNA pathway components were only modestly affected in set1-GLKD ovaries 241 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\n(Figure S5 A). Moreover, RT -qPCR analysis showed that depletion of COMPASS 242 \nsubunits led to milder derepression of HeT-A compared with that of set1 (Figure S5B). 243 \nTaken together, these results argue against the notion that Set1 functions solely as an 244 \nH3K4me3 writer and  represses TEs simply by  promoting the expression  of piRNA 245 \npathway components. 246 \n 247 \nLoss of TE-targeting piRNAs in set1-depleted ovaries  248 \nTE derepression pattern suggests the functional link between Set1 and  Piwi (Figure 4, 249 \nS4). Moreover, consistent with the ir close proximity (Figure 2), Set1 and Piwi interact 250 \nphysically, as Piwi co-precipitated with GFP-Set1WT and GFP -Set1E1613K expressed in 251 \ngermline cells (Figure 6A). Notably, the interaction appears transient or weak, given the 252 \ndependency on crosslinking (Figure S6A) . These physical and functional links 253 \nprompted us to examine  whether piRNA expression is affected by set1 depletion. 254 \nDeep-sequencing of ovarian small RNAs showed that overall abundance of piRNAs  255 \n(23~29-nt fragments and those derived from TE sequences)  was largely unchanged in 256 \nset1-GLKD (Figure 6B). However, analysis at the level of individual TE  families 257 \nrevealed a striking loss of piRNAs mapping to  telomeric HTT members (Figure 6C). 258 \nNotably, antisense piRNAs, which directly target cognate TE transcripts, were reduced 259 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nmore severely  than sense piRNAs.  A prominent example  is HeT-A, for which  sense 260 \npiRNAs were largely unaffected whereas antisense species were nearly lost (Figure 261 \n6CD). Supporting the link to Piwi pathway, re -analysis of published small RNA -seq 262 \ndata (GSE71374) revealed a similar  antisense-biased reduction of HTT-mapping 263 \npiRNAs in ovaries lacking Panx, a key cofactor of Piwi (Figure 6E)  (Yu et al, 2015a; 264 \nSienski et al, 2015b). An antisense-biased reduction of piRNAs was also observed for 265 \nother Set1-controlled TEs, including 3S18, gypsy12, Max-element, diver, and 266 \nHMS-Beagle (Figure 6C). Despite this loss, the ping-pong signature ( 10-nt overlap 267 \nfrequency) of piRNAs  was preserved (Figure S 6). This is  consistent with the 268 \nmaintenance of nuage structure in set1-GLKD ovaries  (Figure 3B) . Together, t hese 269 \nfindings indicate that  Set1 is required to express  TE-targeting functional piRNAs, 270 \nsupporting its crucial role in Piwi-mediated TE silencing. 271 \n 272 \nSet1 binds TE sequences and localizes to subtelomeric piRNA cluster loci 273 \nHow does Set1 contribute to the expression of antisense piRNAs? To address this  274 \nquestion, we analyzed the chromatin binding by GFP-Set1 expressed in set1-depleted 275 \ngermline cells, using CUT&Tag with anti-GFP antibodies. In parallel with GFP-Set1WT, 276 \nGFP-Set1E1613K was included in the analysis , given the functionality  in TE silencing 277 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\n(Figure 5) . Comparison between GFP -Set1 and GFP control conditions identified 278 \nSet1-binding peaks ( p<0.01, Figure S7A ). Notably, genes and TEs associated with  279 \npeaks from Set1WT or Set1 E1613K showed limited  overlap (Figure 7A) . However, this 280 \ndoes not indicate their targets are distinct; rather, it reflects differential affinity between 281 \nSet1WT and Set1 E1613K to shared targets (r=0.85, Figure 7B). Consistent with its role as 282 \nH3K4me3 writer, targets identified from Set1WT peaks were predominantly endogenous 283 \ngenes (541 of 548, Figure 7A). These bindnig peaks appeared around transcription start 284 \nsite ( TSS) and coincided with set1-dependent H3K4me3 signals, as exemplified with 285 \naub (Figure 7C). In contrast, targets identified from Set1E1613K peaks were enriched with 286 \nTEs (Figure 7A, 84 of 306) , including  HeT-A, TART, diver, HMS-Beagle, and 287 \nMax-element, regulated by Set1 (Figure 4A, 5C). At the level of consensus sequences , 288 \ntwo major peaks were typically observed: one spanning the 5’UTR-ORF1 region and 289 \nanother within the 3’UTR (Figure 7 D, S7CD). Collectively, these results revealed the 290 \naffinity of Set1 to TE sequences. 291 \nWe mention that  H3K4me3 signals on TEs behaved differently from those at 292 \ngene TSS: they increased upon set1 depletion and decreased upon expression of  both 293 \nSet1WT and Set1 E1613K, indicating a lack of  correlation with Set1’s catalytic  activity 294 \n(Figure 7D, S7CD). Instead, TE-associated H3K4me3 signals were positively correlated 295 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nwith the transcript abundance (Figure 5) , suggesting that these marks are  deposited 296 \neither by residual Set1 in the set1-depleted germline cells or through Set1-independent 297 \nmechanisms (Ardehali et al, 2011).  298 \n3’UTRs of HTT members possess promoter and TSS for bidirectional 299 \ntransctiption (Figure 7D)  (Danilevskaya & Arkhipova, 1997; Radion et al , 2017; 300 \nMaxwell et al, 2006). Intriguingly, HTT 3’UTRs interacting with Set1 are maintained as 301 \ntruncated insertions in subtelomeric piRNA cluster loci. HeT-A{}6278 in cluster 3 is one 302 \nof those 3’UTR fragments.  Alignment to the consensus sequence suggests that 303 \nHeT-A{}6278 maintains promoter and antisense TSS but not sense TSS (Figure S7E). 304 \npiRNA hot spot starting from th is 3’UTR fragment implies the involvement in piRNA 305 \nprecursor transcription  (Figure 7E). Set1 showed affinity to other sites on cluster 3 , 306 \nwhich is contrastive to non-telomeric clusters lacking, if not any,  Set1-binding signals 307 \n(Figure S7F). Insertions from set1-sensitive LTR retrotransposons are rare within major 308 \npiRNA clusters. Max{}2206 is one of exceptions found in 42AB cluster, but it did not 309 \nexhibit Set1 binding (Figure S7F). Hence, Set1 binding to cluster loci  is biased toward 310 \nsubtelomeric regions, whose piRNA  production is  extremely set1-dependent (Figure 311 \n7E). Set1 binding to LTR retrotransposons may instead reflect its affinity for potentially 312 \nmobile copies located outside piRNA clusters (Wang et al, 2018). Collectively, t hese 313 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nresults support a model in which  Set1 binding to TE  sequences promotes precursor 314 \ntranscription for piRNA biogenesis in germline cells (Figure 7F).  315 \n  316 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nDiscussion 317 \nTo deepen the understanding of piRNA -directed TE silencing, this study explored the 318 \nproximity factors of individual PIWI  proteins expressed in female germline cells. The 319 \nproteome provided additional clues supporting the known interactions, and, more 320 \nimportantly, revealed otherwise unrecognized  links between factors in piRNA 321 \nbiogenesis. First, we point out that Zucchini (Zuc), an endonuclease on mitochondrial 322 \nouter membrane, was identified in the proximity of both Aub and Piwi . These three 323 \nproteins are together involved in phased piRNA production  (Ge et al, 2019), but their 324 \nphysical links have not been reported in ovaries. It should be noted, a study conducting 325 \nTurboID using Zuc as bait did not find Aub or Piwi in its proximity (Nguyen et al, 2023). 326 \nThis might be due to the difference of drivers (matalfa -Gal4 vs nos -Gal4), stages of 327 \ngermline cells (differentiating cells vs stem -cell like cells), or bait proteins (Zuc vs 328 \nPIWI proteins) . In addition to Zuc, our data also showed that Nxf3 and  Bootlegger 329 \n(Boot) mediating the export of  piRNA precursors are in the proximity of  Ago3, but not 330 \nof Aub (ElMaghraby et al, 2019; Kneuss et al, 2019). The selective proximity supports  331 \nthe model whereby precursor transcripts delivered by Nxf3 and Boot from nucleus to 332 \nnuage in cytoplasm are targeted by Ago3-piRNA complexes (Wang et al, 2023b). 333 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nHrb27C emerged as a functionally relevant partner of Aub (Figure 2-4). As a 334 \nmember of hnRNP family, Hrb27C regulates the transport, localization, and translation 335 \nof interacting mRNAs  during oogenesis (Yano et al, 2004; Goodrich et al, 2004; Huynh 336 \net al, 2004). Both Hrb27C and Aub are intrinsic factors required for GSC maintenance 337 \n(Ma et al, 2017; Rojas‐Ríos et al, 2017; Rojas-Ríos et al, 2024; Finger et al, 2023). In this 338 \nstudy, we provided evidence supporting the involvement of Hrb27C in Aub -mediated 339 \nTE silencing in GSCLCs  (GSC-like cells induced by depletion of differentiation factor, 340 \nbam) (Figure 4). The cooperation between Hrb27C and Aub in GSCs warrants further 341 \ndetailed investigation. 342 \nH3K4 methyltransferase Set1 was identified and characterized as a key factor 343 \nin Piwi-mediated TE silencing  in female germline  (Figure 2-7). We initially as sumed 344 \nthat, despite the  proximity to Piwi, TE silencing by Set1 could be attributable to  its 345 \ngeneral coactivator function (Ardehali et al, 2011; Mohan et al, 2011). Set1 acting as 346 \nH3K4me3 writer inside COMPASS potentially underlies the expression of germline 347 \ngenes including piRNA pathway components. However, our results argue against this 348 \nnotion but instead shed light on its specialized role in Piwi pathway. First, set1 and piwi 349 \nbut not aub showed similar TE derepression pattern upon depletion from germline cells 350 \n(Figure 4) . Second, set1 depletion caused severe loss of piRNAs derived from HTT, 351 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nwhich we found ocurring in  panx-deficient ovaries disrupting Piwi pathway (Figure 6). 352 \nMoreover, w e demonstrated that Set1 can control TEs , HTT members in particular,  353 \nwithout its catalytic activity (Figure 5). Last, CUT&Tag revealed the affinity of Set1 to 354 \nTE sequences, which is associated with piRNA production (Figure 7). 355 \nHow Set1 recognizes TE sequences has remained elusive. Set1 interacts with 356 \nphosphorylated C -terminal domain ( CTD) of RNA polII in elongation phase, which 357 \nallows targeted recruitment and H3K4me3 deposition on actively transcribed genes (Ng 358 \net al, 2003). In o ur CUT&Tag analysis, H3K4me3 signal s within TE sequences were 359 \nlargely overlap ped with Set1 -binding regions. Although our results did not support a 360 \nrole for Set1 in depositing these TE-associated H3K4me3 (Figure 7D), RNA polII on 361 \ntranscriptionally active TEs could contribute to  Set1 recruitment. However, HeT-A 362 \nshowed only mild derepression by downregulation of a COMPASS component, Wdr82 363 \nthat is the orthologue of yeast Swd2 linking Set1 and RNA polII (Figure S5A) (Bae et al, 364 \n2020). Hence, RNA polII-dependent recruitment of Set1 as part of COMPASS may be 365 \ninsufficient, and additional mechanisms may contribute. The e levated TE binding 366 \nobserved for the catalytically inactive Set1 variant, compared with the wild-type protein, 367 \nsupport a possible alternative mode of recruitment  (Figure 7B). Given the physical and 368 \nfunction links, involvement of Piwi machinery in recruiting Set1 to TEs is a plausible 369 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\npossibility. Our proximity proteome did not identify SetDB1/Eggless, the H3K9me3 370 \nwriter responsible for heterochromatin formation at Piwi-piRNA target loci (Rangan et 371 \nal, 2011; Osumi et al , 2019; Akkouche et al , 2017) . This absence suggests that our 372 \ndataset may be  biased toward a specific stage at which  Piwi cooperates with Set1 . 373 \nFurther characterization of additional factors  identified by our  Piwi proxiome should 374 \nprovide deeper insight into the mode of actions of Set1 in TE silencing. 375 \nMost piRNAs are derived from defined genomic regions called piRNA c luster 376 \nloci (Brennecke et al , 2007b) . The heterochromati c structure of these loci  requires 377 \nnoncanonical RNA polII recruitment for piRNA precursor transcription, mediated by 378 \nH3K9me3 reader Rhino and the cofactors  (Klattenhoff et al, 2009; Mohn et al, 2014; 379 \nAndersen et al , 2017) . Recent studies uncovered cluster-specific molecular 380 \nunderpinnings for piRNA precursor transcription.  These include DNA-binding protein, 381 \nKipferl, and enhancer of zeste, E(z), the writer of H3K27me3 , each required at distinct 382 \ncluster loci  (Akkouche et al, 2025; Baumgartner et al, 2022). In gonadal soma, Traffic 383 \njam (Tj), a fly orthologue of large Maf transcription factors , activates soma-restricted 384 \ncluster, flamenco (Rivera et al, 2025; Alizada et al, 2025). Subtelomeric piRNA cluster 385 \nloci involve Set1 (this study) and NSL complex (Iyer et al , 2023). Together, these 386 \nfindings indicate that precursor transcription and piRNA production are governed by  387 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\ndiverse chromatin modifiers and transcriptional regulators . Such diversity may be 388 \nadvantageous, enabling spaciotemporal control of the piRNA repert oire to support 389 \nmultitasks of a common set of PIWI proteins. By localizing to subtelomeric cluster loci, 390 \nSet1 would exert its role in telomere  control with Piwi . Characterization of  telomere 391 \nphenotypes in set1-depleted germline cells will be an important direction for  future 392 \nstudies. Beyond piRNA clusters, Set1 also exhibited affinity for several TE families 393 \nincluding HMS-Beagle and Max-element, both of which maintains mobility in female 394 \ngermline and transmits new copies to next generations through oocyte targeting (Yang et 395 \nal, 2023; Wang et al , 2018). The role of Set1 in silencing of such active TEs also 396 \nrepresent a future avenue.   397 \nFly Set1 can silence TEs forming telomeres regardless of its methyltransferase 398 \nactivity (Figure 5). This finding is reminiscent of molecular features reported for yeast 399 \nSet1, which was originally identified as a transcriptional repressor in the telomere  and 400 \nsilent mating loci (Nislow et al, 1997). Notably, its repressive functions on genes and 401 \nTEs are often associated with H3K4 methylation-independent mechanisms (Jezek et al, 402 \n2023; Lee et al, 2018; Lorenz et al, 2012). However, t he m echanisms underlying TE 403 \nsilencing are different between the two organisms, as yeast lacks PIWI clade proteins 404 \nand does not produce  piRNAs. Nonetheless, Set1-dependent antisense non-coding 405 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nRNAs have been characterized as repressors of retrotransposons in yeast (Berretta et al, 406 \n2008). The role of  Set1 in generating antisense , silencing-competent transcripts for 407 \ngenome defense may be conserved across broader contexts. 408 \n409 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nMaterials and Methods 410 \nFly stocks and culture 411 \nAll stocks and crosses were raised at 25 ˚C on a molasses/yeast medium [5% (w/v) dry 412 \nyeast, 5% (w/v) corn flower, 2% (w/v) rice bran, 10% (w/v) glucose, 0.7% (w/v) agar, 413 \n0.2% (v/v) propionic acid, and 0.05% (v/v) p -hydroxy butyl benzoic acid]. Fly stocks 414 \nused in this study are listed in Table S4. 415 \n 416 \nPlasmid construction and generation of transgenic flies 417 \nAll the primers used for plasmid construction are listed in Table S 5. To generate 418 \nUASp-mTurbo-GFP, UASp -mTurbo-GFP-Piwi, UASp -mTurbo-GFP-Aub and 419 \nUASp-mTurbo-GFP-Ago3, mTurbo fragment was amplified by PCR using 420 \nattB_miniTurbo_Fw_general and miniTurbo_linker_Rv_general as primers and 421 \n3xHA-miniTurboNLS_pCDNA3 (addgene #107172) as template. GFP fragment was 422 \namplified by PCR using 2 -1vLinker>GFP5_F and Ti876 -GFP-G5-R as primers.  Piwi, 423 \nAub or Ago3 fragment was amplified by PCR using primer pairs of Linker_to_Piwi and 424 \nPiwi_to_Vector, Ti893-G5-Aub-CDS-F and Ti894 -Aub-UASp-R1, or Linker_to_Ago3 425 \nand Ago3 _to_Vector, respectively, with cDNA from yw ovaries as template . PCR 426 \nfragments were introduced into the XbaI site in pUASp-K10-attB vector (Koch et al, 427 \n2009) using In -Fusion HD Cloning (Takara). UASp-mTurbo-GFP and  428 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nUASp-mTurbo-GFP-Ago3 were integrated to attP40 , while UASp -mTurbo-GFP-Piwi 429 \nand UASp-mTurbo-GFP-Aub were integrated to attP2, and attP-3B sites, respectively. 430 \n 431 \nProximity-dependent biotin pulldown in ovarian germline cells 432 \nAfter eclosion, female s expressing mini(m)Turbo -GFP-PIWI proteins were reared at 433 \n25˚C for 2 -3 ( non-GSCLC ovaries) or 5 -7 days (GSCLC ovaries) in the modified 434 \nmolasses/yeast medium supplemented with 100 μM biotin (Nacalai). For individual 435 \nconditions, ~ 300 ovaries  from 150 progenies were collected . Ovaries were 436 \nhomogenized in 150 μL of PI -lysis buffer [50 mM t ris-HCl (pH 7.5), 500 mM NaCl, 2 437 \nmM EDTA, 2 mM dithiothreitol (DTT), 0.4% (w/v) SDS, and cOmplete Protease 438 \nInhibitor Cocktail Tablet (Roche)] , using Bioruptor (Diagenode) for 30s (power H) for 439 \nsix times with 30s intervals. Triton X -100 was then added to sa mples at a final 440 \nconcentration of 2% (v/v), and homogenization was further performed for 30s for three 441 \ntimes with 30s intervals. After centrifugation (20,000 g, 10 min, 4 ˚C), the supernatant 442 \nwas diluted with equal amount of 50 mM Tris -HCl (pH 7.5) buffer and incubated with 443 \npre-equilibrated 15-μL slurry volume of Dynabeads MyOne Streptavidin C1 (Thermo 444 \nFisher Scientific), overnight at 4 ˚C with gentle rotation. Next day, beads were washed 445 \ntwice with W1 buffer [50 mM tris -HCl (pH 7.5), 250 mM NaCl, 0.2% (w/v)  SDS, 1 446 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nmM EDTA, and 1 mM DTT], twice with W2 buffer [50 mM H EPES-KOH (pH 7.4), 447 \n500 mM NaCl, 0.1% (w/v) deoxycholate (Nacalai), 1% (v/v) Triton X -100, and 1 mM 448 \nEDTA], twice with W3 buffer [10 mM tris -HCl (pH 8.0), 250 mM LiCl, 0.5% (w/v) 449 \ndeoxycholate, 0.5% (v/v) NP-40 (Nacalai), and 1 mM EDTA], and four times with W4 450 \nbuffer [50 mM tris -HCl (pH 7.5) and 50 mM NaCl].  The purified proteins bound to 451 \nbeads were stored at -80˚C until analysis. 452 \n 453 \nImmunoblotting and biotinylated protein detection 454 \nProtein s amples wer e denatured at 95°C for 5 min in 2× protein loading buffer [4% 455 \n(w/v) SDS, 200 mM DTT, 0.1% (v/v) bromophenol blue (BPB), and 20% (v/v) 456 \nglycerol] saturated with biotin, resolved by SDS –polyacrylamide gel electrophoresis 457 \n(SDS-PAGE) and transferred to 0.2 -μm polyvinylidene difluoride membrane (Wako) 458 \nusing the semi-dry system (Trans-blot Turbo, Bio-Rad). The membrane was blocked in 459 \n4% (w/v) skim milk (Nacalai) in 1× phosphate -buffered saline (PBS) supplemented 460 \nwith 0.1% (v/v) Tween 20 and further incubated with  primary antibodies: rabbit 461 \nanti-GFP (1:1000, Clonetech), mouse anti -Piwi (1:100)  (Saito et al, 2006), guinea pig 462 \nanti-Aub (1:1000)  and rat anti -Ago3 (1:200 ) (Lim et al, 2022), mouse anti -Tubulin 463 \n(1:3000, Santa Cruz) . Secondary antibodies were anti-guinea pig 464 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nimmunoglobulins-HRP (1:1000; Dako), anti -rabbit immunoglobulin G (IgG) –HRP 465 \n(1:3000; Bio-Rad), anti-mouse IgG-HRP (1:3000; Bio -Rad), and anti-rat IgG (1:1000; 466 \nDAKO). HRP-conjugated Streptavidin (Clonetech) was used for detecting biotinylated  467 \nProteins. The chemiluminescent signals generated with Chemi-Lumi One (Nacalai) 468 \nwere detected by Chemidoc MP Imaging system (Bio-Rad). The images were processed 469 \nwith Fiji. 470 \n 471 \nProteomic Sample Preparation and Label -Free Quantification using Orbitrap 472 \nEclipse Tribrid Mass Spectrometer 473 \nBiotinylated proteins bound to magnetic beads were reduced with 1 mM dithiothreitol 474 \n(DTT) and alkylated with 5.5 mM iodoacetamide. Proteins were digested overnight at 475 \n37°C with Trypsin Gold, Mass Spectrometry Grade (Promega, Madison, WI). The 476 \nresulting peptides were captured and desalted using ZipTip C18 (Millipore, Billerica, 477 \nMA). Shotgun proteomic analysis was performed using an Orbitrap Eclipse Tribrid 478 \nmass spectrometer equipped with a FAIMS Pro interface (Thermo Fisher Sci entific, 479 \nWaltham, MA), which was coupled to a Vanquish Neo UHPLC system (Thermo Fisher 480 \nScientific, Waltham, MA). Peptides were separated on a reversed -phase column using a 481 \nlinear gradient of 2 –24% acetonitrile in 0.1% formic acid at a flow rate of 300 nL/m in. 482 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nFull MS scans were acquired in the Orbitrap at a resolution of 120,000, followed by 483 \nMS/MS scans in the ion trap using higher -energy collisional dissociation (HCD) with a 484 \nnormalized collision energy of 35% and a maximum injection time of 10 ms.  Label-free 485 \nquantification (LFQ) was performed using Proteome Discoverer version 2.5 (Thermo 486 \nFisher Scientific, Waltham, MA) with the Sequest HT search engine. MS/MS spectra 487 \nwere searched against the UniProt Drosophila melanogaster reference proteome 488 \n(UP000000803). Protein identifications were filtered at a false discovery rate (FDR) of 489 \n<1%. Peptide intensities were extracted using the Minora Feature Detector node and 490 \nused for protein quantification. GO analysis was conducted using MetaScape. (Zhou et 491 \nal, 2019) 492 \n 493 \nCroslinking and immunoprecipitation 494 \nOvaries were dissected from 50 females expressing GFP, GFP -Set1WT, or 495 \nGFP-Set1E1613K in phosphate -buffered saline (PBS) and  immediately fixed with 0.1% 496 \n(w/v) paraformaldehyde (Electron Microscopy Sciences) for 10 min at room 497 \ntemperature (RT). Fixation was quenched with 125 mM glycine. Samples were then 498 \nsnap-frozen in liquid nitrogen and stored at -80°C until use . Ovary s amples were 499 \nhomogenized with a pestle in lysis buffer [20 mM Tris -HCl (pH 7.5), 135 mM NaCl , 500 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\n1.5 mM MgCl 2, 10% (v/v) glycerol, 0.2% (v/v) TritonX-100] supplemented with 501 \ncOmplete Protease Inhibitor Cocktail (Roche). Pre-equilibrated a nti-GFP 502 \nantibody-conjugated magnetic beads (MBL, D153-11) were added to ovarian lysate and 503 \nincubated for 1h at 4°C. After three times of washing with lysis buffer, bead-bound 504 \nproteins were extracted by boiling in SDS-PAGE loading buffer [50 mM Tris–HCl (pH 505 \n6.8), 2% (w/v) SDS, 100 mM 1,4-dithiothreitol (DTT), 10% (v/v) glycerol, 0.05%(w/v) 506 \nbromophenol blue]. 507 \n 508 \nHistochemistry and image acquisition 509 \nOvaries were dissected from adult females in 1x PBS buffer supplemented with 0.4% 510 \n(w/v) bovine serum albumin (BSA; Wako) and fixed in 5.3% (v/v) paraformaldehyde 511 \n(Nacalai) in 0.67x PBS buffer for 10 min. To observe DNA, ovaries were incubated 512 \nwith 1 μM 40,6-diamidino-2-phenylindole (DAPI) in PBX buffer [1x PBS containing 513 \n0.2% (v/v) Triton X -100]. For immunostaining, fixed ovaries were washed with PBX 514 \nand incubated with Image -iT™ FX Signal Enhancer (Invitrogen) for 30 min and PBX 515 \ncontaining 2% (w/v) BSA for 30 min for blocking. The primary antibody incubation 516 \nwas performed overnight at 4 ˚C, and ovaries were washed with PBX at RT for 1h. The 517 \nsecondary antibody incubation was then performed at RT for 2 h, and then ovaries were 518 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nwashed with PBX at RT for 1 h. The antibodies used for immunostaining were rabbit 519 \nanti-HeT-A Gag (1:2000),(Lin et al, 2023) guinea pig anti-Krimp (1:2000),(Lim & Kai, 520 \n2007) mouse anti-Piwi (1:50, Santa Cruz, sc-390946), guinea pig anti-Aub (1:500),(Lim 521 \net al, 2022) mouse anti-GFP (1:50, Invitrogen, A11120) . Secondary antibodies were as 522 \nfollows: Alexa Fluor 488 - and 555 -conjugated goat antibodies at 1:500 (Molecular 523 \nProbes) and CF®633 goat antibodies at 1:500 (Biotium) . Antibodies were  diluted in 524 \n0.4% (w/v) BSA containing PBX as the working solution. Images were taken by ZEISS 525 \nLSM 900 using C-Apochromat 40x/1.20 W Korr  objective lens and processed with 526 \nZEISS ZEN 3.0 and Fiji. 527 \n 528 \nReverse transcription and qPCR analysis 529 \nTotal RNA was extracted from ovaries  using TRIzol™ LS (Invitrogen) following the 530 \nmanufacturer’s instructions. Using DNase I (NEB) –treated RNA, cDNA was 531 \nsynthesized with 2.5 μM oligo(dT) adaptor using SuperS cript III reverse transcriptase 532 \n(Thermo Fisher Scientific). Quantitative reverse transcription PCR (qPCR) reaction was 533 \nperformed using SYBR™ Green qPCR Master Mix (Thermo Fisher Scientific) and 534 \ngene-specific primers (Table S5) in QuantStudio 5 Real-Time PCR system (ABI). 535 \n 536 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nTranscriptome analysis 537 \nTotal RNA was extracted from 100 ovaries  (non-GSCLC ovaries or GSCLC ovaries) 538 \nusing TRIzol™ LS (Invitrogen). To obtain GSCLC ovaries expressing shRNA for aub, 539 \nhrb27C, set1 or gfp, shRNA lines were combined ( bam shRNA; aub shRNA, bam 540 \nshRNA; hrb27C shRNA, bam shRNA; set1 shRNA, bam shRNA; gfp shRNA) and 541 \ncrossed with NN. DNase I-treated RNA was sent to Rhelixa (Japan) for library 542 \nconstruction and deep -sequencing. Polyadenylated RNA selection was performed with 543 \nNEBNext Poly(A) mRNA Magnetic Isolation Module (NEB). Libraries were 544 \nconstructed with the NEBNext Ultra II Directional RNA Library Prep Kit (NEB) and 545 \nsequenced by using NovaSeq 6000 (Illumina).  Trimming was performed by Cutadapt 546 \n(v1.18, -j 12 -a AGATCGGAAGAGCA CACGTCTGAACTCCAGTCA -A 547 \nAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT, -m 20).  Trimmed paired-end 548 \nreads were mapped to the genome (BDGP6.46, dm6)  using STAR ( v2.7.10b, 549 \n--outFilterMultimapNmax 100 --outSAMmultNmax 1 --outMultimapperOrder Random). 550 \nRead counting was performed with featureCounts (-M -p --countReadPairs -t exon -g 551 \ngene_id) using Drosophila_melanogaster.BDGP6.46.112.gtf (ensembl.org) . Using the 552 \nsum of  RPK ( read per kilobase) for individual genes and TEs, TPM ( transcript per 553 \nmillion) was calculated. Trimmed paired -end reads were  also mapped to consensus 554 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nsequences of individual TE families  (transposon_sequence_set.embl.v.9.41, flybase ) 555 \nwith STAR using the same options . Mappers on individual TE families were counted  556 \nusing pileup.sh (BBMap). TPM were calculated using the sum of RPK for genes and 557 \nTEs obtained in genome mapping . Published small RNA data ( GSE103582 and 558 \nGSE71374) were processed in the same way. 559 \n 560 \nSmall RNA sequencing and analysis  561 \nBiological duplicate dataset for Set1 -present condition was provided by gfp-GLKD 562 \novaries (control RNAi condition), and set1-GLKD ovaries expressing GFP -Set1WT 563 \n(rescue condition). Dataset for Set1 -depleted condition was provided by set1-GLKD 564 \novaries, and th e siblings of rescue conditions (CyO). T otal RNA was extracted using 565 \nTRIzol™ LS (Invitrogen) from 60 to 100 ovaries of ~3 days old adult females. After the 566 \naddition of chloroform and centrifugation (12,000g 15min, 4˚C), short (< ~200 nt) RNA 567 \nin the aqueous  phase was purified using RNA Clean & Concentrator 5 (R1015 Zymo ). 568 \nTo deplete 2S ribosomal RNA (rRNA) , 2 pmol of complementary oligo DNA 569 \n(5’-AGTCTTACAACCCTCAACCATATGTAGTCCAAGCAGCACT-3’) was added 570 \nper 1 μg of RNA. The mixture was heated (95 ˚C, 2min), gradually cooled down to form 571 \nDNA/RNA hybrid, and then treated with RNase H (NEB) at 37 ˚C for 30min. RNase H 572 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nwas inactivated by heating at 65 ˚C for 20  min. The 2S rRNA -depleted RNA mixture 573 \nwas loaded onto 8 M urea -polyacrylamide gel (12%) and size-separated in parallel with 574 \nvisible RNA ladder (Dynamarker DM253, BioDynamics Laboratory Inc.) in 0.5x 575 \ntris-borate EDTA buffer. Gel within the range from 20 to 30nt was excised and passed 576 \nthrough a small hole opened by needle (23Gx1, TERUMO) at the bottom of 0.5  mL 577 \nDNA LoBind tubes (Eppendorf) by centrifugation (15,000g). The fine particles were 578 \nrecovered in 2.0  mL DNA LoBind tubes, and RNAs were eluted overnight at 4 ˚C with 579 \ngentle rotation in the presence of 300 mM NaOAc (pH 5.2). After removing the 580 \nparticles using cell ulose 0.22  µm membrane filter Spin -X (Costar, 8160), RNA was 581 \nprecipitated in the presence of 80% (v/v) ethanol and glycogen (40 μg/mL) (Nacalai) for 582 \novernight at -20˚C. After centrifugation (20,000g, 20min, 4˚C), RNA pellet rinsed twice 583 \nwith 80% (v/v) ethanol was resuspended in RNase-free water. Small RNA libraries were 584 \nprepared using NEBNext® Multiplex Small RNA Library Prep Set for Illumina (NEB, 585 \nE7300S) following manufacture’s procedure. After 15 cycles of PCR amplification, the 586 \nlibraries were purifi ed using MagMAX™ Pure Bind Beads (Applied Biosystems), 587 \nsize-separated in 3% (w/v) low melting agarose (HydraGene) in the presence of SYBR 588 \ngold (Thermo Fisher). ~150 -bp library fragments containing 20~30 -bp inserts were 589 \npurified using QIAquick gel extractio n kit (Qiagen). Libraries were sequenced by 590 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nRIMD NGS core facility ( The University of Osaka ) using the NovaSeqX Plus platform 591 \n(Illumina). Adaptor sequences (AGATCGGAAGAGC) were removed by Trim Galore 592 \n(v0.6.2, --length 15) with quality filter cut off (Phred ≥20). Adaptor-trimmed reads were 593 \nmapped to rRNA, tRNA, snoRNA, and snRNA sequences using bowtie (v1.3.1), and 594 \nthe unmappers were collected (samtools view -f 4). From the unmappers, 18~29 -nt 595 \nreads were selected as small RNAs using seqkit (v2.4.0 ) and  mapped to miRNA 596 \nprecursors using bowtie ( -v 0) to collect “miRNA” populations. The size -selected 597 \n(23~29-nt) unmappers were considered as “piRNA”. These piRNA fragments were 598 \nmapped to TE consensus sequences (transposon_sequence_set.embl.v.9.41, flybase ) 599 \nusing bowtie allowing up to 3 mismatches and taking one from multi mappers (-v 3 -M 600 \n1 --best --strata). Fw and Rv mappers were counted for individual TEs using pileup.sh. 601 \nRead counts were normalized using the sum of 18~29 -nt reads and  reads per million 602 \n(RPM) was obtained. For visualization, Fw and Rv mappers were separated using 603 \nsplitsam.sh. Bedgraph was generated using bedtools (v2.26.0, genomecov -bga -split), 604 \nvalues normalized using scale factors given by the sum of 18~29-nt reads. Mean values 605 \nof biological replicates were obtained using bedtools unionbedg. Track view was 606 \ngenerated in IGV (v2.16.0). piRNA overlap scores (z) were measured using signature.py 607 \n(Antoniewski, 2014) . piRNA fragments were also mapped to Drosophila genome 608 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\n(BDGP6.46, dm6) using bowtie without allowing mismatch (-v 0 -M 1 --best --strata). 609 \nBedgraph generation, normalization, averaging, and visualization  follow the processes 610 \nin the analysis using TE consensus sequences. Published small RNA data (GSE71374) 611 \nwere processed in the same way. 612 \n 613 \nCUT&Tag sequencing and analysis 614 \nWe referred (Anderson et al, 2023) for performing whole ovary C UT&Tag. Twenty 615 \novaries from 10 adult females at ~ 3 days old were dissected in PBS buffer 616 \nsupplemented with cOmplete Protease Inhibitor (Roche). Ovaries were permeabilized in 617 \nPBX buffer (PBX containing 0.2%(v/v) Triton-X100) for 30 min at RT. After removing 618 \nPBX, ovaries were washed once with Wash+ buffer ( 20mM HEPES [pH 7.4], 150  mM 619 \nNaCl, 0.5  mM spermidine[Nacalai], 2  mM EDTA, 1%[w/v] BSA, cOmplete Protease 620 \nInhibitor) and incubated overnight at 4 ˚C with primary antibodies diluted by 1:50 with 621 \nWash+ buffer. Anti-H3K4me3 (#61979, mouse, Active Motif), or anti-GFP (#598, rabbit, 622 \nMBL) antibodies were used . Next day, ovaries were washed three times with Wash+ 623 \nbuffer, and incubated with secondary antibodies diluted by 1:50 with Wash+ buffer.  624 \nAnti-mouse IgG (#52885L, Cell Signalling), or anti -rabbit IgG (#35401S, Cell 625 \nSignalling) were used. After removing the secondary antibody solution, ovaries were 626 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nwashed three times with 300Wash+ buffer (20 mM HEPES [pH 7.4], 300 mM NaCl, 0.5 627 \nmM spermidine, cOmplete Protease Inhibitor ) and incubated for 1h at RT with loaded 628 \npAG-Tn5 ( #79561S, Cell Signalling) diluted by 1:25 with 300Wash+ buffer. After 629 \nremoving the Tn5 solution, ovaries were washed three times with 300Wash+ buffer. 630 \nTagmentation was performed by incubating ovaries in 300Wash+ buffer supplemented 631 \nwith 10 mM MgCl2 for 1h at 37˚C. After removing the supernatant, ovaries were treated 632 \nwith collagenase (2  mg/mL, Sigma C9407) in HEPESCA buffer (50  mM HEPES [pH 633 \n7.4], 360 µM CaCl 2) for 1h at 37 ˚C. Then, the reaction mixture was added with SDS, 634 \nEDTA, and proteinase K (Nacalai) (final concentration 0.2%[w/v], 16  mM, and 0.3  635 \nmg/mL, respectively), and further incubated for 1h at 58 ˚C. After the reaction, DNA 636 \nwas purified by using Quick -DNA Microprep kit (D30 20, ZYMO). Using the purified 637 \nDNA as template, libraries were synthesized by PCR (72 ˚C 5min, 98 ˚C 30s, 14 cycles 638 \nof [98 ˚C 10s, 63 ˚C 15s], 65 ˚C 5min) using Ultra II Q5 Master Mix (NEB) and 639 \ndual-indexed primers (Table S 5). Generated libraries were purified using x1.3 volume 640 \nof AmpureXP (beckman). The concentration of libraries was measured using Qubit 641 \ndsDNA HS assay kit (Thermo). Libraries were sequenced with NovaSeqX (Illumina) 642 \nand 150+ 150bp paired -end reads were obtaine d (H3K4me3 libraries sequenced by 643 \nRIMD in The University of Osaka , GFP libraries sequenced by Rhelixa ). Adaptor 644 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\ntrimming was performed by Cutadapt ( -a 645 \nCTGTCTCTTATACACATCTCCGAGCCCACGAGAC -A 646 \nCTGTCTCTTATACACATCTGACGCTGCCGACGA, -m 20). Trimmed reads were 647 \nmapped to D. melanogaster genome (BDGP6.46, dm6) using Bowtie 2 -p 12 --no-unal 648 \n--end-to-end --very-sensitive --no-mixed --no-discordant -q --phred33 -I 10 -X 700 ). 649 \nRead counts were normalized using deeptools (bamCoverage --normalizeUsing CPM 650 \n-of bigwig --binSize=1). Average bigwig was generated from biological duplicate 651 \nbigwig data using wiggletools and ucsc-wigtobigwig (wigToBigWig). Track view was 652 \ngenerated in IGV (v2.16.0). Counting reads on gene  TSS±500bp (H3K4me3) or exon 653 \n(GFP) was performed using featureCounts (v2. 1.1, -M -p --countReadPairs -O) with 654 \nDrosophila_melanogaster.BDGP6.46.112.gtf (Ensembl). RPM (read per million) was 655 \ncalculated using the number of aligned reads in Bowtie2 mapping . Trimmed reads were 656 \nalso mapped to TE consensus sequences  (transposon_sequence_set.embl.v.9.41, 657 \nflybase). Bigwig was generated using deeptools (bamCoverage), normalizing the values 658 \nwith scale factors given by the sum of  aligned reads on the genome . Mean values of 659 \nbiological replicates were obtained using wiggletools and wigToBigWig 660 \n(ucsc-wigtobigwig). Track view was generated in IGV (v2.16.0). 661 \n  662 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nAcknowledgement 663 \nWe thank Dr. Prof. Xin Chen for providing fly stocks.  We thank Chisato Yanagisawa, 664 \nMinako Moriguchi for their helps with maintenance of fly lines. We are also grateful to 665 \nBloomington Drosophila Stock Center for providing fly stocks. We also thank all 666 \nmembers in our lab for their insightful discussion and suggestions.  This study is 667 \nsupported by JSPS Grant -in-Aid for Scientific Research C (22K06081) for TI, Life 668 \nScience Foundation of Japan (J231503018) for TI, Takeda Foundation (J241503007) for 669 \nTI, Daiichi Sankyo Foundation of Life Science (J241503010) for TI, Naito Foundation 670 \n(J241503011) for TI, JSPS Grant -in-Aid for Scientific Research B (21H02401)  for TK, 671 \nJSPS Grant -in-Aid for Transformative Research Areas A (21H05275) for TK,  672 \nGrant-in-Aid for JSPS Fellows (23KJ1521)  for WI, and Grant from Open and 673 \nTransdisciplinary Research Initiatives (OTRI) RNA Frontier Science Division for TI.  674 \nThe founders had no role in study design, data collection and analysis, decision to 675 \npublish, or preparation of the manuscript. 676 \n 677 \nAuthor contributions 678 \nConceptualization: TI and TK 679 \nMethodology: TI, TK, and WI 680 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nInvestigation: TI, WI, HK-H and MO 681 \nSupervisions: TI and TK 682 \nWriting-original draft: WI and TI 683 \nWriting-review and editing: TI, WI and TK 684 \n 685 \nCompeting interest statement 686 \nThe authors declare no competing interests. 687 \n 688 \nData availability 689 \nNewly generated transcriptome data are available with BioProject accession ID: 690 \nPRJNA1345876; Drosophila ovary polyA transcriptome sequencing, PRJNA1345540; 691 \nDrosophila ovary small RNA sequencing, PRJNA1345531; Drosophila ovary 692 \nCUT&Tag sequencing 693 \n  694 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nFigure legends 695 \nFigure 1. PIWI proximity biotin labelling in germline stem cell-like cells (GSCLCs) 696 \n(A) Experimental design for PIWI proximity biotin labelling in female germline cells . 697 \nmTurbo-GFP-Piwi, -Aub, or -Ago3 is expressed in germline cells using UASp/Gal4 698 \nsystem with NGT40; nos-Gal4 (NN) drive r. mTurbo-GFP serves as negative contr ol. 699 \nDepletion of bam using short hairpin (sh)RNA enables TurboID in GSCLC s. (B) 700 \nSubcellular localization of mTurbo-GFP fusion proteins in GSCLC ovary. Upper panels; 701 \nfluorescent signals  from GFP (green). Lower panels; immunostaining signals from  702 \nendogenous Piwi, Aub, or Ago3 (green). DAPI (blue) for nuclei. Scale bar = 50 μm or 5 703 \nμm in the insets. (C) Blotting images for p roteins extracted from GSCLC ovaries 704 \nexpressing mTurbo fusion proteins (Input), and the streptavidin-bound fraction  705 \n(Pulldown). Upper panels; immunoblotting with anti-GFP antibody. Lower panels; 706 \nblotting with streptavidin-HRP. White and black arrowheads indicate mTurbo -GFP and 707 \nmTurbo-GFP-PIWI proteins, respectively. 708 \n 709 \nFigure 2. Proximity proteome of PIWI proteins in germline cells 710 \n(A) Network diagram summarizing proximity factors of individual PIWI proteins (Piwi, 711 \nAub, and Ago3) identified by mass spectrometry ( nanoLC-MS/MS) followed by  712 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nlabel-free quantification. Yellow nodes: factors known to be involved in piRNA pathway. 713 \nSize of c ircle reflects abundance ratio (mTurbo -GFP-PIWI/mTurbo-GFP) based on  714 \nbiological duplicate TurboID data. (B) Venn diagram show s the number of proximity 715 \nproteins shared by or unique to individual PIWI members. (C) GO enrichment analys is 716 \nof PIWI proximity factors. Hierarchical clustering and heatmap show significantly 717 \nenriched GO terms. Color intensity represents the significance of enrichment ( -log10 718 \np-value). 719 \n 720 \nFigure 3. Germline knockdown (GLKD) screening of PIWI proximity factors 721 \n(A) Immunofluorescence signals from HeT-A Gag protein (top panels) in egg chambers 722 \nof control (y w) or GLKD of indicated genes. DNA stained with DAPI (bottom panels). 723 \nArrowheads; Gag proteins accumulating around oocyte. Scale bars  = 20 μm. (B) 724 \nImmunofluorescence signals of Krimp (green) in the egg chambers of control ( y w) or 725 \nof indicated GLKD conditions . DNA stained with DAPI  (magenta). Fluorescence 726 \nsignals along yellow arro ws are plotted in the bottom panels (signals normalized by 727 \nsetting 1 for the highest value of DAPI). Scale bars = 10 μm. (C) Immunofluorescence 728 \nsignals of Aub or Piwi (green) in the egg chambers of indicated conditions. 729 \n 730 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nFigure 4. Germline knockdown (GLKD) effect on ovary transcriptome  731 \n(A-B) Transcriptome analysis and comparison in non-GSCLC ovaries (A) or in GSCLC 732 \novaries (B). Transcript levels (TPM) of genes and TE insertions in set1-, hrb27C-, aub-, 733 \nor piwi-GLKD condition were compared with those in gfp-GLKD control. Dots in the 734 \nvolcano plots; genes (gray) and TEs (gray with red outline) included in the differential 735 \nexpression analysis (EdgeR). ( C) Scatter plot c omparing the effect of GLKD on the 736 \ntranscript levels  of i ndividual TE families  (consensus sequence mapping). Left : 737 \nset1-GLKD compared with aub-GLKD. Right: set1-GLKD compared with piwi-GLKD. 738 \nFold change from control gfp-GLKD (TPM/TPM) were shown. r = pearson correlation 739 \ncoefficient. (D) Transcript levels (TPM)  for HeT-A and TAHRE in different 740 \ntranscriptome datasets. GSE103582 including aub and piwi knockouts, and GSE71374 741 \nincluding panx knockout were analyzed together(Yu et al, 2015a; Teixeira et al, 2017b). 742 \nError bars indicate ±standard deviation (SD) of biological duplicate data. 743 \n 744 \nFigure 5. Effect of set1 transgene expression on H3K4me3 modification and TE 745 \nexpression in set1-GLKD ovaries 746 \n(A) Schemetic of GFP-Set1 transgenes (Vidaurre et al, 2024). Nucleotide substitution to 747 \nreplace 1613 th glutamate ( E) of Set1 polypeptide to lysine ( K) abolishes catalytic 748 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nactivity. Nonsense substitutions render resistance to  siRNA targeting. Both placed 749 \ndownstream of UASp and expressed by germline driver NN. (B) GFP fluorescence (top) 750 \nand immunofluorescence staining for H3K4me3 and Vas (bottom) in egg chambers. Vas 751 \nmarks germline cells. Scale bar = 10μm. (C) Transcript levels (TPM) of TE  families in 752 \ngfp-GLKD, set1-GLKD, and transgene expression  conditions ( GFP-Set1WT or 753 \nGFP-Set1E1613K expressed in set1-GLKD). Error bars  indicate ±SD from biological 754 \nduplicates. (D) CUT&Tag analysis of H3K4me3 in ovaries of indicated conditions. 755 \nLeft: track view at the ago3 and aub gene loci. Bar graph on the right shows H3K4me3 756 \nsignals (mean CPM) around TSS (±500bp) of ago3, aub and piwi . Error bars indicate 757 \n±SD from biological duplicates. 758 \n 759 \nFigure 6. Co-precipitation of Piwi with Set1, and e ffect of set1-GLKD on piRNA 760 \naccumulation in ovaries  761 \n(A) Immunoprecipitation of GFP or GFP -Set1 proteins from ovaries using anti -GFP 762 \nantibody (GFP-IP) after crosslinking treatment.  Tubulin serves as loading control.  (B) 763 \nProportion (%) of miRNA and piRNA in ovarian small RNAs. miRNA: reads mapping 764 \nto miRNA precursor s. piRNA: 23~29-nt reads  (excluding rRNA, tRNA, snoRNA, 765 \nsnRNA, and miRNA mappers), or 23~29-nt reads mapping to TE consensus sequences. 766 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nControl: mean values given by single replicate of  gfp-GLKD and GFP-Set1WT-rescue 767 \nconditions. set1-GLKD: mean values given by  biological duplicate of set1-GLKD 768 \ncondition. Error bars indicate ±SD of biological duplicates. (C) Fold changes (FC) of 769 \npiRNA levels (RPM) in set1-GLKD compared to control condition. piRNA : 23~29-nt 770 \nmappers on individual TE families . TEs having few mappers ( RPM <5) are excluded. 771 \nSense or antisense piRNA : mappers having TE sense strand or the reverse 772 \ncomplementary sequences.  (D) Track view for 23~29-nt mappers on consensus 773 \nsequences of telomeric TE families. (E) Fold change (FC) of the levels (RPM) of 774 \npiRNA mapping to  telomeric TE families . Small RNA libraries for panx KO and the 775 \nheterozygous control conditions (GSE71374) are included (Yu et al, 2015a). 776 \n 777 \nFigure 7. Genome-wide Set1-binding analysis, and a model for Set1 function in TE 778 \nsilencing 779 \n(A) Venn diagram for genes and TEs  having GFP-Set1WT- or GFP-Set1E1613K-binding 780 \npeaks. Peak calling by MACS2 (p<0.01).  (B) Scatter plot comparing fold differences of 781 \nCUT&Tag signals  (GFP-Set1E1613K - GFP GFP -Set1WT - GFP) on genes and TEs 782 \nidentified by peak calling . r =  pearson correlation coefficient.  (C, D) Track view for 783 \nCUT&Tag signals of GFP proteins (green) and H3K4me3 (black). aub (C) and HeT-A 784 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\n(D) represent genes or TEs, respectively. TSS: transcription starting site. (E) Track view 785 \nfor CUT&Tag signals of GFP proteins (green) and piRNA levels on subtelomeric 786 \npiRNA cluster 3. piRNAs having genomic plus strand sequence (Blue) or  minus strand 787 \nsequences (Red) are separately shown . (F) A model for Set1 -mediated production of 788 \nantisense-biased piRNAs on subtelomeric cluster loci and on individual TE insertions  789 \noutside of cluster loci. Subtelomere regions maintain fragments of 3’UTR derived from 790 \nHTT, which are bound by Set1 to initiate  antisense transcription. Catalytic activity of 791 \nSet1 is dispensable. Similar mechanism is applied for antisense piRNA production from 792 \npotentially mobile TE insertions outside of piRNA cluster loci . Antisense piRNAs are 793 \nloaded onto Piwi to reinforce transcriptional silencing. 794 \n 795 \nFigure S1. Optimization of germline PIWI proximity labelling and purification 796 \n(A) Comparison of biotinylation efficiency and biotinylated protein purification 797 \nbetween non-GSCLC and GSCLC ovaries. Co omassie brilliant blue (CBB) staining 798 \nserving as protein loading control. Ovaries expressing mTurbo -GFP or 799 \nmTurbo-GFP-Aub were analyzed with negative control (without mTurbo protein  800 \nexpression). Asterisks : non-specific signals.  (B) Schematic illustration f or germline 801 \ndifferentiation and the function of Bam. Left; the germarium in wild -type, non-GSCLC 802 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\novaries. A germline stem cell (GSC, blue) divides to produce a daughter cell (red) which 803 \nexpresses Bam and starts the differentiation. Two-to-three GSCs are normally 804 \nmaintained in a germarium. Right: by bam-GLKD, daughter cells cannot differentiate 805 \nand thus accumulate as GSCLCs. (C) A pair of wild -type ovaries (y w, left) and that of 806 \nbam-GLKD ovaries (right). Scale bar  = 1 mm. (D) Immunoblotting images com paring 807 \nthe expression level of mTurbo -GFP-tagged Piwi or Aub with that of endogenous Piwi 808 \nor Aub in GSCLC ovaries. 809 \n 810 \nFigure S2. Quality assessment of PIWI proximity proteome data 811 \nV olcano plots for the proximity factors of Piwi, Aub, or Ago3 in individual biological 812 \nreplicates. Red dots highlight the factors showing significant enrichment (abundance 813 \nratio > 1, adjusted p < 0.05). Statistics relies on background-based t-test.  814 \n 815 \nFigure S3. Germline knockdown (GLKD) efficiency and the effect on protein 816 \nsubcellular localization 817 \n (A-C) Plotting the fluorescence signals from egg chambers of indicated genotypes, 818 \nrelated to Figure 3B. (A) Krimp (green) and DAPI (magenta) , (B) Aub (green) and  819 \nDAPI (magenta), (C) Piwi (green) and DAPI (magenta) . (D) Transcript levels of aub, 820 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nhrb27C and set1 in individual GLKD conditions (ΔΔCt method using rp49 as reference). 821 \nError bars  indicate standard deviation (SD) from three biological  replicates. p values 822 \nfrom unpaired Student's t-test. 823 \n 824 \nFigure S4. GLKD effect on ovary transcriptome, and somatic KD effect on TEs 825 \n(A-B) Transcriptome analysis and comparison in non-GSCLC ovaries (A) or in GSCLC 826 \novaries (B). Transcript levels (TPM) of genes and TE insertions in set1-, hrb27C-, aub-, 827 \nor piwi-GLKD conditions were compared with those in the control gfp-GLKD. Dots in 828 \nthe MA plots; genes (gray) and TEs (gray with red outline) included in the differential 829 \nexpression analysis (EdgeR). (C) Transcript levels (TPM) of i ndividual TE families in 830 \ngfp-GLKD (consensus sequence mapping) compared between non-GSCLC and GSCLC 831 \novaries. (D) Scatter plot c omparing the effect of  GLKD on the transcript levels  of 832 \nindividual TE families  (consensus sequence mapping). Left : hrb27C-GLKD compared 833 \nwith aub-GLKD. Right : hrb27C-GLKD compared with piwi-GLKD. F old change s 834 \n(TPM/TPM) from control gfp-GLKD are shown. r = pearson correlation coefficient. (E) 835 \nTranscript levels (TPM)  for TART-A1, TART-B, and TART-C, in different transcriptome 836 \ndatasets. GSE103582  including aub and piwi knockout conditions, and GSE71374 837 \nincluding panx knockout condition were analyzed. Error bars  indicate ± SD of 838 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nbiological duplicates. (F) RT-qPCR measurement of TE transcript levels in ovaries upon 839 \nsomatic KD of gfp, piwi or set1 using tj-Gal4 driver. ΔΔCt method using rp49 as 840 \nreference. Error bars indicate ±SD from three biological replicates. 841 \n 842 \nFigure S5. Effect of COMPASS subunit GLKD on HeT-A expression, and effect of 843 \nset1-GLKD on the expression of piRNA pathway factors 844 \n(A) RT-qPCR measurement of HeT-A transcript levels in COMPASS subunit GLKD 845 \novaries. ΔΔCt method using rp49 as a reference. Error bars indicate ±SD from three 846 \nbiological replicates. p value from unpaired Student's t -test is indicated. Asterisk; p < 847 \n0.05. (B) Bar graph shows the transcript levels (TPM) of piRNA pathway components 848 \nin gfp-GLKD and set1-GLKD ovaries. Error bar s indicate ± SD of biological 849 \nduplicates. 850 \n 851 \nFigure S6. Immunoprecipitation of  GFP-Set1 without crosslinking, and e ffect of 852 \nset1-GLKD on ping-pong signature of piRNAs 853 \n(A) Immunoprecipitation of GFP or GFP -Set1 proteins from ovaries using anti -GFP 854 \nantibody (GFP -IP) without crosslinking treatment. Tubulin serves as loading control.  855 \n(B) Ping-pong signature analysis on piRNAs mapping to telomeric TE families . Z10  856 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nscores (reflecting 10-nt overlap frequenc y within piRNA groups) were highlighted . 857 \nError bars indicate ±SD of biological duplicates. 858 \n 859 \nFigure S7. Genome-wide Set1-binding analysis 860 \n(A) Venn diagram shows overlap of genes and TEs between replicate 1 and 2 of 861 \nGFP-Set1WT CUT&Tag (peak calling using GFP control) . (B) Venn diagram shows 862 \noverlap of genes and TEs between replicate 1 and 2 of GFP -Set1E1613K CUT&Tag (peak 863 \ncalling using GFP control) . (C) Track view for CUT&Tag signals of GFP proteins 864 \n(green) and H3K4me3 (black) on TAHRE (left) and TART-C (right). (D) Track view for 865 \nCUT&Tag signals of GFP proteins (green) and H3K4me3 (black) on HMS-Beagle (left) 866 \nand Max-element (right). (E) Alignment of HeT-A{}6278 to HeT-A consensus sequence. 867 \n(F) Track view for CUT&Tag signals of GFP proteins (green) and piRNA levels on 868 \n42AB locus. Blue and Red indicate piRNAs containing genomic plus and minus strand 869 \nsequences, respectively. 870 \n  871 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nReferences 872 \nAbles ET, Hwang GH, Finger DS, Hinnant TD & Drummond-Barbosa D (2016) A Genetic Mosaic 873 \nScreen Reveals Ecdysone-Responsive Genes Regulating Drosophila Oogenesis. G3 874 \nGenes|Genomes|Genetics 6: 2629–2642 875 \nAkkouche A, Kneuss E, Bornelöv S, Renaud Y, Eastwood EL, van Lopik J, Gueguen N, Jiang 876 \nM, Creixell P, Maupetit-Mehouas S, et al (2025) Binding of heterochromatin 877 \nprotein Rhino to a subset of piRNA clusters depends on a combination of two histone 878 \nmarks. Nat Struct Mol Biol 32: 1517–1527 879 \nAkkouche A, Mugat B, Barckmann B, Varela-Chavez C, Li B, Raffel R, Pélisson A & 880 \nChambeyron S (2017) Piwi Is Required during Drosophila Embryogenesis to License 881 \nDual-Strand piRNA Clusters for Transposon Repression in Adult Ovaries. Mol Cell 882 \n66: 411-419.e4 883 \nAlizada A, Martins A, Mouniée N, Rodriguez Suarez J V., Bertin B, Gueguen N, Mirouse 884 \nV, Papameletiou A-M, Rivera AJ, Lau NC, et al (2025) The transcription factor 885 \nTraffic jam orchestrates the somatic piRNA pathway in Drosophila ovaries. Cell 886 \nRep 44: 115453 887 \nAnand A & Kai T (2012) The tudor domain protein Kumo is required to assemble the nuage 888 \nand to generate germline piRNAs in Drosophila. EMBO J 31: 870–882 889 \nAndersen PR, Tirian L, Vunjak M & Brennecke J (2017) A heterochromatin-dependent 890 \ntranscription machinery drives piRNA expression. Nature 549: 54–59 891 \nAnderson JT, Henikoff S & Ahmad K (2023) Chromosome-specific maturation of the 892 \nepigenome in the Drosophila male germline. Elife 12 893 \nAntoniewski C (2014) Computing siRNA and piRNA Overlap Signatures. In Methods in 894 \nMolecular Biology pp 135–146. Humana Press Inc. 895 \nArdehali MB, Mei A, Zobeck KL, Caron M, Lis JT & Kusch T (2011) Drosophila Set1 is 896 \nthe major histone H3 lysine 4 trimethyltransferase with role in transcription. 897 \nEMBO J 30: 2817–2828 898 \nAriura M, Solberg T, Ishizu H, Takahashi H, Carninci P, Siomi H & Iwasaki YW (2024) 899 \nDrosophila Piwi distinguishes transposons from mRNAs by piRNA complementarity 900 \nand abundance. Cell Rep 43: 115020 901 \nBae HJ, Dubarry M, Jeon J, Soares LM, Dargemont C, Kim J, Geli V & Buratowski S (2020) 902 \nThe Set1 N-terminal domain and Swd2 interact with RNA polymerase II CTD to recruit 903 \nCOMPASS. Nat Commun 11: 2181 904 \nBatki J, Schnabl J, Wang J, Handler D, Andreev VI, Stieger CE, Novatchkova M, 905 \nLampersberger L, Kauneckaite K, Xie W, et al (2019) The nascent RNA binding 906 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\ncomplex SFiNX licenses piRNA-guided heterochromatin formation. Nat Struct Mol 907 \nBiol 26: 720–731 908 \nBaumgartner L, Handler D, Platzer SW, Yu C, Duchek P & Brennecke J (2022) The Drosophila 909 \nZAD zinc finger protein Kipferl guides Rhino to piRNA clusters. Elife 11 910 \nBerretta J, Pinskaya M & Morillon A (2008) A cryptic unstable transcript mediates 911 \ntranscriptional trans -silencing of the Ty1 retrotransposon in S. cerevisiae. 912 \nGenes Dev 22: 615–626 913 \nBranon TC, Bosch JA, Sanchez AD, Udeshi ND, Svinkina T, Carr SA, Feldman JL, Perrimon 914 \nN & Ting AY (2018) Efficient proximity labeling in living cells and organisms 915 \nwith TurboID. Nat Biotechnol 36: 880–887 916 \nBrennecke J, Aravin AA, Stark A, Dus M, Kellis M, Sachidanandam R & Hannon GJ (2007a) 917 \nDiscrete Small RNA-Generating Loci as Master Regulators of Transposon Activity 918 \nin Drosophila. Cell 128: 1089–1103 919 \nBrennecke J, Aravin AA, Stark A, Dus M, Kellis M, Sachidanandam R & Hannon GJ (2007b) 920 \nDiscrete Small RNA-Generating Loci as Master Regulators of Transposon Activity 921 \nin Drosophila. Cell 128: 1089–1103 922 \nChoi‐Rhee E, Schulman H & Cronan JE (2004) Promiscuous protein biotinylation by 923 \nEscherichia coli biotin protein ligase. Protein Science 13: 3043–3050 924 \nDanilevskaya ON & Arkhipova IR (1997) Promoting in Tandem: The Promoter for Telomere 925 \nTransposon HeT-A and Implications for the Evolution of Retroviral LTRs 926 \nElMaghraby MF, Andersen PR, Pühringer F, Hohmann U, Meixner K, Lendl T, Tirian L & 927 \nBrennecke J (2019) A Heterochromatin-Specific RNA Export Pathway Facilitates 928 \npiRNA Production. Cell 178: 964-979.e20 929 \nFabry MH, Ciabrelli F, Munafò M, Eastwood EL, Kneuss E, Falciatori I, Falconio FA, 930 \nHannon GJ & Czech B (2019) piRNA-guided co-transcriptional silencing coopts 931 \nnuclear export factors. Elife 8 932 \nFinger DS, Williams AE, Holt V V & Ables ET (2023) Novel roles for RNA binding proteins 933 \nsquid , hephaesteus , and Hrb27C in Drosophila oogenesis. Developmental Dynamics 934 \n252: 415–428 935 \nGe DT, Wang W, Tipping C, Gainetdinov I, Weng Z & Zamore PD (2019) The RNA-Binding 936 \nATPase, Armitage, Couples piRNA Amplification in Nuage to Phased piRNA Production 937 \non Mitochondria. Mol Cell 74: 982-995.e6 938 \nGoodrich JS, Clouse KN & Schüpbach T (2004) Hrb27C, Sqd and Otu cooperatively regulate 939 \ngurken RNA localization and mediate nurse cell chromosome dispersion in 940 \nDrosophila oogenesis. Development 131: 1949–1958 941 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nGrieder NC, Cuevas M de & Spradling AC (2000) The fusome organizes the microtubule 942 \nnetwork during oocyte differentiation in Drosophila. Development 127: 4253–4264 943 \nHallson G, Hollebakken RE, Li T, Syrzycka M, Kim I, Cotsworth S, Fitzpatrick KA, 944 \nSinclair DAR & Honda BM (2012) dSet1 Is the Main H3K4 Di- and 945 \nTri-Methyltransferase Throughout Drosophila Development. Genetics 190: 91–100 946 \nHandler D, Olivieri D, Novatchkova M, Gruber FS, Meixner K, Mechtler K, Stark A, 947 \nSachidanandam R & Brennecke J (2011) A systematic analysis of Drosophila TUDOR 948 \ndomain-containing proteins identifies Vreteno and the Tdrd12 family as essential 949 \nprimary piRNA pathway factors. EMBO J 30: 3977–3993 950 \nHuynh J-R, Munro TP, Smith-Litière K, Lepesant J-A & Johnston DS (2004) The Drosophila 951 \nhnRNPA/B Homolog, Hrp48, Is Specifically Required for a Distinct Step in osk mRNA 952 \nLocalization. Dev Cell 6: 625–635 953 \nIki T, Kawaguchi S & Kai T (2023) miRNA/siRNA-directed pathway to produce noncoding 954 \npiRNAs from endogenous protein-coding regions ensures Drosophila 955 \nspermatogenesis. Sci Adv 9 956 \nIki T, Takami M & Kai T (2020) Modulation of Ago2 Loading by Cyclophilin 40 Endows 957 \na Unique Repertoire of Functional miRNAs during Sperm Maturation in Drosophila. 958 \nCell Rep 33: 108380 959 \nIyer SS, Sun Y, Seyfferth J, Manjunath V, Samata M, Alexiadis A, Kulkarni T, Gutierrez 960 \nN, Georgiev P, Shvedunova M, et al (2023) The NSL complex is required for piRNA 961 \nproduction from telomeric clusters. Life Sci Alliance 6: e202302194 962 \nJezek M, Sun W, Negesse MY, Smith ZM, Orosz A & Green EM (2023) Set1 regulates telomere 963 \nfunction via H3K4 methylation–dependent and -independent pathways and calibrates 964 \nthe abundance of telomere maintenance factors. Mol Biol Cell 34 965 \nKai T, Zheng J & Iki T (2025) Paralog‐Dependent Specialization of <scp>Paf1C</scp> 966 \nSubunit, Ctr9, for Sex Chromosome Gene Regulation and Male Germline 967 \nDifferentiation in Drosophila. Genes to Cells 30: e70040 968 \nKawaguchi S, Isshiki W & Kai T (2025) Factories without walls: The molecular 969 \narchitecture and functions of non-membrane organelles in small RNA-guided genome 970 \nprotection. Biochimica et Biophysica Acta (BBA) - General Subjects 1869: 130811 971 \nKlattenhoff C, Xi H, Li C, Lee S, Xu J, Khurana JS, Zhang F, Schultz N, Koppetsch 972 \nBS, Nowosielska A, et al (2009) The Drosophila HP1 Homolog Rhino Is Required for 973 \nTransposon Silencing and piRNA Production by Dual-Strand Clusters. Cell 138: 974 \n1137–1149 975 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nKneuss E, Munafò M, Eastwood EL, Deumer U-S, Preall JB, Hannon GJ & Czech B (2019) 976 \nSpecialization of the Drosophila nuclear export family protein Nxf3 for piRNA 977 \nprecursor export. Genes Dev 33: 1208–1220 978 \nKoch R, Ledermann R, Urwyler O, Heller M & Suter B (2009) Systematic Functional 979 \nAnalysis of Bicaudal-D Serine Phosphorylation and Intragenic Suppression of a 980 \nFemale Sterile Allele of BicD. PLoS One 4: e4552 981 \nLee KY, Chen Z, Jiang R & Meneghini MD (2018) H3K4 Methylation Dependent and 982 \nIndependent Chromatin Regulation by JHD2 and SET1 in Budding Yeast. G3 983 \nGenes|Genomes|Genetics 8: 1829–1839 984 \nLim AK & Kai T (2007) Unique germ-line organelle, nuage, functions to repress selfish 985 \ngenetic elements in Drosophila melanogaster. Proceedings of the National Academy 986 \nof Sciences 104: 6714–6719 987 \nLim L-X, Isshiki W, Iki T, Kawaguchi S & Kai T (2022) The Tudor Domain-Containing 988 \nProtein, Kotsubu (CG9925), Localizes to the Nuage and Functions in piRNA 989 \nBiogenesis in D. melanogaster. Front Mol Biosci 9: 1–15 990 \nLin Y, Suyama R, Kawaguchi S, Iki T & Kai T (2023) Tejas functions as a core component 991 \nin nuage assembly and precursor processing in Drosophila piRNA biogenesis. 992 \nJournal of Cell Biology 222 993 \nLorenz DR, Mikheyeva I V., Johansen P, Meyer L, Berg A, Grewal SIS & Cam HP (2012) 994 \nCENP-B Cooperates with Set1 in Bidirectional Transcriptional Silencing and 995 \nGenome Organization of Retrotransposons. Mol Cell Biol 32: 4215–4225 996 \nMa X, Zhu X, Han Y, Story B, Do T, Song X, Wang S, Zhang Y, Blanchette M, Gogol M, 997 \net al (2017) Aubergine Controls Germline Stem Cell Self-Renewal and Progeny 998 \nDifferentiation via Distinct Mechanisms. Dev Cell 41: 157-169.e5 999 \nMaxwell PH, Belote JM & Levis RW (2006) Identification of multiple transcription 1000 \ninitiation, polyadenylation, and splice sites in the Drosophila melanogaster 1001 \nTART family of telomeric retrotransposons. Nucleic Acids Res 34: 5498–5507 1002 \nMcKearin DM & Spradling AC (1990) bag-of-marbles: a Drosophila gene required to 1003 \ninitiate both male and female gametogenesis. Genes Dev 4: 2242–2251 1004 \nMohan M, Herz H-M, Smith ER, Zhang Y, Jackson J, Washburn MP, Florens L, Eissenberg 1005 \nJC & Shilatifard A (2011) The COMPASS Family of H3K4 Methylases in Drosophila. 1006 \nMol Cell Biol 31: 4310–4318 1007 \nMohn F, Sienski G, Handler D & Brennecke J (2014) The Rhino-Deadlock-Cutoff complex 1008 \nlicenses noncanonical transcription of dual-strand piRNA clusters in Drosophila. 1009 \nCell 157: 1364–1379 1010 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nMugat B, Nicot S, Varela-Chavez C, Jourdan C, Sato K, Basyuk E, Juge F, Siomi MC, 1011 \nPélisson A & Chambeyron S (2020) The Mi-2 nucleosome remodeler and the Rpd3 1012 \nhistone deacetylase are involved in piRNA-guided heterochromatin formation. Nat 1013 \nCommun 11: 2818 1014 \nMurano K, Iwasaki YW, Ishizu H, Mashiko A, Shibuya A, Kondo S, Adachi S, Suzuki S, 1015 \nSaito K, Natsume T, et al (2019) Nuclear RNA export factor variant initiates 1016 \npiRNA‐guided co‐transcriptional silencing. EMBO J 38 1017 \nNg HH, Robert F, Young RA & Struhl K (2003) Targeted Recruitment of Set1 Histone 1018 \nMethylase by Elongating Pol II Provides a Localized Mark and Memory of Recent 1019 \nTranscriptional Activity. Mol Cell 11: 709–719 1020 \nNguyen TTM, Munkhzul C, Kim J, Kyoung Y, Vianney M, Shin S, Ju S, Pham-Bui H-A, Kim 1021 \nJ, Kim J-S, et al (2023) In vivo profiling of the Zucchini proximal proteome in 1022 \nthe Drosophila ovary. Development 150 1023 \nNislow C, Ray E & Pillus L (1997) SET1 , A Yeast Member of the Trithorax Family, 1024 \nFunctions in Transcriptional Silencing and Diverse Cellular Processes. Mol Biol 1025 \nCell 8: 2421–2436 1026 \nOsumi K, Sato K, Murano K, Siomi H & Siomi MC (2019) Essential roles of Windei and 1027 \nnuclear monoubiquitination of Eggless/ <scp>SETDB</scp> 1 in transposon 1028 \nsilencing. EMBO Rep 20 1029 \nPatil VS & Kai T (2010) Repression of Retroelements in Drosophila Germline via piRNA 1030 \nPathway by the Tudor Domain Protein Tejas. Current Biology 20: 724–730 1031 \nQi H, Watanabe T, Ku H-Y, Liu N, Zhong M & Lin H (2011) The Yb Body, a Major Site 1032 \nfor Piwi-associated RNA Biogenesis and a Gateway for Piwi Expression and 1033 \nTransport to the Nucleus in Somatic Cells. Journal of Biological Chemistry 286: 1034 \n3789–3797 1035 \nRadion E, Ryazansky S, Akulenko N, Rozovsky Y, Kwon D, Morgunova V, Olovnikov I & 1036 \nKalmykova A (2017) Telomeric Retrotransposon HeT-A Contains a Bidirectional 1037 \nPromoter that Initiates Divergent Transcription of piRNA Precursors in 1038 \nDrosophila Germline. J Mol Biol 429: 3280–3289 1039 \nRangan P, Malone CD, Navarro C, Newbold SP, Hayes PS, Sachidanandam R, Hannon GJ & 1040 \nLehmann R (2011) piRNA Production Requires Heterochromatin Formation in 1041 \nDrosophila. Current Biology 21: 1373–1379 1042 \nRivera AJ, Lee J-HR, Gupta S, Yang L, Goel RK, Zaia J & Lau NC (2025) Traffic Jam 1043 \nactivates the Flamenco piRNA cluster locus and the Piwi pathway to ensure 1044 \ntransposon silencing and Drosophila fertility. Cell Rep 44: 115354 1045 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nRojas-Ríos P, Chartier A, Enjolras C, Cremaschi J, Garret C, Boughlita A, Ramat A 1046 \n& Simonelig M (2024) piRNAs are regulators of metabolic reprogramming in stem 1047 \ncells. Nat Commun 15: 8405 1048 \nRojas‐Ríos P, Chartier A, Pierson S & Simonelig M (2017) Aubergine and pi 1049 \n<scp>RNA</scp> s promote germline stem cell self‐renewal by repressing the 1050 \nproto‐oncogene Cbl. EMBO J 36: 3194–3211 1051 \nRørth P (1998) Gal4 in the Drosophila female germline. Mech Dev 78: 113–118 1052 \nRoux KJ, Kim DI, Raida M & Burke B (2012) A promiscuous biotin ligase fusion protein 1053 \nidentifies proximal and interacting proteins in mammalian cells. Journal of Cell 1054 \nBiology 196: 801–810 1055 \nSaito K, Ishizu H, Komai M, Kotani H, Kawamura Y, Nishida KM, Siomi H & Siomi MC (2010) 1056 \nRoles for the Yb body components Armitage and Yb in primary piRNA biogenesis in 1057 \nDrosophila. Genes Dev 24: 2493–2498 1058 \nSaito K, Nishida KM, Mori T, Kawamura Y, Miyoshi K, Nagami T, Siomi H & Siomi MC (2006) 1059 \nSpecific association of Piwi with rasiRNAs derived from retrotransposon and 1060 \nheterochromatic regions in the Drosophila genome. Genes Dev 20: 2214–2222 1061 \nShpiz S, Olovnikov I, Sergeeva A, Lavrov S, Abramov Y, Savitsky M & Kalmykova A (2011) 1062 \nMechanism of the piRNA-mediated silencing of Drosophila telomeric 1063 \nretrotransposons. Nucleic Acids Res 39: 8703–8711 1064 \nSienski G, Batki J, Senti K-A, Dönertas D, Tirian L, Meixner K & Brennecke J (2015a) 1065 \nSilencio/CG9754 connects the Piwi–piRNA complex to the cellular heterochromatin 1066 \nmachinery. Genes Dev 29: 2258–2271 1067 \nSienski G, Batki J, Senti KA, Dönertas D, Tirian L, Meixner K & Brennecke J (2015b) 1068 \nSilencio/CG9754 connects the piwi-piRNA complex to the cellular heterochromatin 1069 \nmachinery. Genes Dev 29: 2258–2271 1070 \nSuyama R & Kai T (2025) pi <scp>RNA</scp> processing within non‐membrane structures 1071 \nis governed by constituent proteins and their functional motifs. FEBS J 292: 2715–1072 \n2736 1073 \nTeixeira FK, Okuniewska M, Malone CD, Coux R-X, Rio DC & Lehmann R (2017a) 1074 \npiRNA-mediated regulation of transposon alternative splicing in the soma and germ 1075 \nline. Nature 552: 268–272 1076 \nTeixeira FK, Okuniewska M, Malone CD, Coux RX, Rio DC & Lehmann R (2017b) 1077 \nPiRNA-mediated regulation of transposon alternative splicing in the soma and germ 1078 \nline. Nature 552: 268–272 1079 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nLe Thomas A, Rogers AK, Webster A, Marinov GK, Liao SE, Perkins EM, Hur JK, Aravin 1080 \nAA & Tóth KF (2013) Piwi induces piRNA-guided transcriptional silencing and 1081 \nestablishment of a repressive chromatin state. Genes Dev 27: 390–399 1082 \nVidaurre V, Song A, Li T, Ku WL, Zhao K, Qian J & Chen X (2024) The Drosophila histone 1083 \nmethyltransferase SET1 coordinates multiple signaling pathways in regulating 1084 \nmale germline stem cell maintenance and differentiation. Development 151 1085 \nVillasante A, Abad JP, Planelló R, Méndez-Lago M, Celniker SE & de Pablos B (2007) 1086 \nDrosophila telomeric retrotransposons derived from an ancestral element that was 1087 \nrecruited to replace telomerase. Genome Res 17: 1909–1918 1088 \nWang H, Fan Z, Shliaha P V., Miele M, Hendrickson RC, Jiang X & Helin K (2023a) H3K4me3 1089 \nregulates RNA polymerase II promoter-proximal pause-release. Nature 615: 339–1090 \n348 1091 \nWang L, Dou K, Moon S, Tan FJ & Zhang ZZ (2018) Hijacking Oogenesis Enables Massive 1092 \nPropagation of LINE and Retroviral Transposons. Cell 174: 1082-1094.e12 1093 \nWang X, Ramat A, Simonelig M & Liu M-F (2023b) Emerging roles and functional mechanisms 1094 \nof PIWI-interacting RNAs. Nat Rev Mol Cell Biol 24: 123–141 1095 \nWebster A, Li S, Hur JK, Wachsmuth M, Bois JS, Perkins EM, Patel DJ & Aravin AA (2015) 1096 \nAub and Ago3 Are Recruited to Nuage through Two Mechanisms to Form a Ping-Pong 1097 \nComplex Assembled by Krimper. Mol Cell 59: 564–575 1098 \nWells JN & Feschotte C (2020) A Field Guide to Eukaryotic Transposable Elements. Annu 1099 \nRev Genet 54: 539–561 1100 \nWilson JR, Jing C, Walker PA, Martin SR, Howell SA, Blackburn GM, Gamblin SJ & Xiao 1101 \nB (2002) Crystal Structure and Functional Analysis of the Histone 1102 \nMethyltransferase SET7/9. Cell 111: 105–115 1103 \nYan D, Neumüller RA, Buckner M, Ayers K, Li H, Hu Y, Yang-Zhou D, Pan L, Wang X, Kelley 1104 \nC, et al (2014) A regulatory network of Drosophila germline stem cell self-renewal. 1105 \nDev Cell 28: 459–473 1106 \nYang F, Su W, Chung OW, Tracy L, Wang L, Ramsden DA & Zhang ZZ (2023) Retrotransposons 1107 \nhijack alt-EJ for DNA replication and eccDNA biogenesis. Nature 620: 218–225 1108 \nYano T, de Quinto SL, Matsui Y, Shevchenko A, Shevchenko A & Ephrussi A (2004) Hrp48, 1109 \na Drosophila hnRNPA/B Homolog, Binds and Regulates Translation of oskar mRNA. 1110 \nDev Cell 6: 637–648 1111 \nYu Y, Gu J, Jin Y, Luo Y, Preall JB, Ma J, Czech B & Hannon GJ (2015a) Panoramix enforces 1112 \npiRNA-dependent cotranscriptional silencing. Science (1979) 350: 339–342 1113 \nYu Y, Gu J, Jin Y, Luo Y, Preall JB, Ma J, Czech B & Hannon GJ (2015b) Panoramix enforces 1114 \npiRNA-dependent cotranscriptional silencing. Science (1979) 350: 339–342 1115 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nZhang Z, Xu J, Koppetsch BS, Wang J, Tipping C, Ma S, Weng Z, Theurkauf WE & Zamore 1116 \nPD (2011) Heterotypic piRNA Ping-Pong Requires Qin, a Protein with Both E3 Ligase 1117 \nand Tudor Domains. Mol Cell 44: 572–584 1118 \nZhao K, Cheng S, Miao N, Xu P, Lu X, Zhang Y, Wang M, Ouyang X, Yuan X, Liu W, et 1119 \nal (2019) A Pandas complex adapted for piRNA-guided transcriptional silencing 1120 \nand heterochromatin formation. Nat Cell Biol 21: 1261–1272 1121 \nZhou Y, Zhou B, Pache L, Chang M, Khodabakhshi AH, Tanaseichuk O, Benner C & Chanda 1122 \nSK (2019) Metascape provides a biologist-oriented resource for the analysis of 1123 \nsystems-level datasets. Nat Commun 10: 1523 1124 \n  1125 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nFigure 1\nA\nmTurbo GFP PIWI\nUASp\n(Piwi, Aub or Ago3)\nGal4\nB\nGSCLC ovaries\nmTurbo-GFP-PiwimTurbo-GFP\n mTurbo-GFP-Aub mTurbo-GFP-Ago3\nGFP\nDAPI\nGSCLC ovaries (bam-GLKD) expressing:\nanti-Piwi\nDAPI\nanti-Aub\nDAPI\nanti-Ago3\nDAPIbam shRNAUASp\nGal4\n♀\nGal4\nnos promoter\nInput\nStreptavidin\npulldown\n180\n140\n100\n75\n60\nanti-GFP\n180\n140\n100\n75\n60\n45\n35\nStreptavidin-HRP\nC\n(kDa)\nmTurbo-GFP\nmTurbo-GFP-Piwi\nmTurbo-GFP-Aub\nmTurbo-GFP-Ago3\nmTurbo-GFP\nmTurbo-GFP-Piwi\nmTurbo-GFP-Aub\nmTurbo-GFP-Ago3\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nFigure 2\nA B\nC\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\n0 5\n0\n0. 5\n1\n0 5\nFigure 3\naub-GLKDy w hrb27C-GLKD set1-GLKD\nKrimp HeT-A Gag\nA\nB aub-GLKDy w hrb27C-GLKD set1-GLKD\n0 5 0 5\n0\n1\n2\n0 5 0 5 0 5\nNormalized \nsignal\nDistance (µm)\n0 5\nDAPIAubDAPI\nNormalized \nsignal\nDAPI\nNormalized \nsignal\nPiwiDAPI\nDistance (µm) Distance (µm) Distance (µm)\n0\n1\n0 5 10 0 5 10 0 5 100 5 10\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nGSE71374\ngfp-GLKD\nset1-GLKD\naub-GLKD\npiwi-GLKD\npanx het\npanx KO\n0\n10 00\n20 00\n30 00\n0\n20 0\n40 0\n60 0\nB\n80\n40\n0\n120\n160\n200\nlog2FC  (hrb27C-GLKD / gfp-GLKD) log2FC  (aub-GLKD / gfp-GLKD)log2FC  (set1-GLKD / gfp-GLKD)\nHeT-A\nHMS-Beagle\nTART-B\nHeT-A\ngypsy12\nlog2FC  (hrb27C-GLKD / gfp-GLKD)\nFigure 4\nA\nC\nNon-GSCLC ovary (Genes and individual TE insertions)\nD\nGSCLC ovary (Genes and individual TE insertions)\n-log\n10\nFDR\n-log\n10\nFDR\nlog2FC  (set1-GLKD / gfp-GLKD)\nTranscript level (TPM)\nr = -0.02\nlog2FC  (aub-GLKD / gfp-GLKD)\nlog\n2FC  (\nset1\n-GL\nKD\n/gfp\n-GLKD\n)\nTEs included in \nEdgeR output\nGenes and TEs \nincluded in EdgeR output\nTAHRE\nNon-GSCLC ovary (Individual TE families)\nlog2FC  (piwi-GLKD / gfp-GLKD)\nlog2FC  (piwi-GLKD / gfp-GLKD)\nlog2FC  (aub-GLKD / gfp-GLKD)\n80\n40\n0\n100\n120\n140\n60\n20\n-10 -5 0 5 10 15 -10 -5 0 5 10 15 -10 -5 0 5 10 15\nTEs included in \nEdgeR output\nGenes and TEs \nincluded in EdgeR output\n-10 -5 0 5 10 15 -10 -5 0 5 10 15 -10 -5 0 5 10 15\n-10 -5 0 5 10 15\n-10 -5 0 5 10 15\nlog2FC  (piwi-GLKD / gfp-GLKD)\nmdg3\nDM88\ncopia\nCirce\nmdg3HMS-Beagle\nflea\nMax-element\ndiver\nflea\nHMS-Beagle\ndiver\n4\n0\n-4\n6\n8\n10\n2\n-2\nHeT-A\ngypsy12\nTAHRE\nTART-A1\nTART-B\nTART-C\nMax-element\n40-4 6 8 102-2\ndiver\nHMS-Beagle\nflea\nmdg3\n4\n0\n-4\n6\n8\n10\n2\n-2\n40-4 6 8 102-2\nr = 0.73\nHeT-A\nTAHRE\ngypsy12\nTART-B\nTART-A1\nTART-C\nMax-element HMS-\nBeagle\ndiver\nHeT-A\nHeT-A\nmdg3\ninvader3\nHMS-Beagle\nTART-A\nMax-element\ndiver\ngypsy12\nTART-C\n3S18\n3S18\n3S18\nTART-B\nTART-A\nrover\naub\nhet\naub\nKO\npiwihet\npiwiKO\nThis study GSE103582\n3S18 3S18\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nFigure 5\nA\nB\nGFPGFP-Set1WT SET\nRNAi-resistant nonsense substitutions\nGFPGFP-Set1E1613K SET\nE1613K\nTranscript level (mean TPM)\nGLKD\nGFP\nset1\nset1\nset1\nGFP-set1WT\nGFP-set1E1613K\nTransgene400\n300\n200\n100\n0\n400\n300\n200\n100\n0\n3000\n2000\n1000\n0\nHeT-A TAHRE TART-A1 TART-B TART-C diver HMS-\nBeagle\n3S18 Max-\nelement\nTelomeric TE families\nC\nD\n[0-50]\n[0-50]\n[0-50]\n[0-50]\nGLKD\nGFP\nset1\nset1\nset1\nGFP-set1WT\nGFP-set1E1613K\nTransgene ago3\nago3 aub piwi\n200\n160\n120\n80\n40\n0\nH3K4me3 signal (mean CPM)\naub\n[0-15]\n[0-15]\n[0-15]\n[0-15]\nTSS\nRRM RRM\nGFPH3K4me3Vas\nset1-GLKDgfp-GLKD GFP-Set1WT in set1-GLKD GFP-Set1E1613K in set1-GLKD\nH3K4me3 (CUT&Tag) TSS\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nHMS-Beagle (0.51)\ndiver (0.59)\n0 60 120\nFigure 6\nD\nlog2FC 23-29nt mapper (set1-GLKD / control)\nC\nB\nControl\nset1\nGLKD\n6,063 bp\nHeT-A\n10,463bp\nTAHRE\n13,424bp\nTART-A1\n0\n1\n2\n-1\n-2\n-3\n0 60 120\nIndividual TE families ordered by FC value\nProportion (%) \nControl\nset1-GLKD\n60\n50\n40\n30\n20\n10\n0\n0 60 120\nSense Antisense Sense+Antisense\nTART-C (0.16)\nTART-A1 (0.24)\nTART-B (0.35)\nTAHRE (0.49)\nHeT-A (1.10)\nTART-C (0.18)\nTART-A1 (0.19)\nTAHRE (0.20)\nHeT-A (0.23)\nTART-B (0.28)\nTART-C (0.16) \nTART-A1 (0.20)\nTART-B (0.29)\nTAHRE (0.31)\nHeT-A (0.51)\nMax-element (1.36)\ndiver (1.60)\nHMS-Beagle (1.86)\ngypsy12 (0.72)\ngypsy12, 3S18 (0.52)\nMax-element (0.71)\ngypsy12 (0.66)\nMax-element (0.84)\nHMS-Beagle (0.99)\ndiver (1.01)\nA\nE set1-GLKD / Control\nSense Antisense\nlog2FC 23-29nt mapper\nSense Antisense\nHeT-A\nTAHRE\nTART-A1\nTART-B\nTART-C\n1\n0\n-1\n-2\n-3\n1\n0\n-1\n-2\n-3\nHeT-A\nTAHRE\nTART-A1\nTART-B\nTART-C\nHeT-A\nTAHRE\nTART-A1\nTART-B\nTART-C\nHeT-A\nTAHRE\nTART-A1\nTART-B\nTART-C\n3S18 (2.04)\n3S18 (1.13)\nCPM\n[0-250]\n[0-250]\nCPM\n[0-250]\n[0-250]\n10,176bp\nTART-B\nSense\nAntisense\nSense\nAntisense\npanx KO / Het (GSE71374)\n11,124bp\nTART-C\n[0-250]\n[0-250]\n[0-250]\n[0-250]\n[0-50]\n[0-50]\n[0-50]\n[0-50]\n[0-50]\n[0-50]\n[0-50]\n[0-50]\n[0-50]\n[0-50]\n[0-50]\n[0-50]\nWT EKGFP GFP-Set1\nPiw i\nGFP-Set1\nGFP\nTubulin\nInput\nWT EKGFP GFP-Set1\nGFP-IP (Crosslinking)\n245\n35\n100\n60\npiRNA\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint \n\nSubtelomeric \nHTT cluster\nset1-GLKD\nFigure 7\nA B\nTE 84\n(HeT-A, TART, diver,\nHMS-Beagle, Max, etc)\nGene 222\nTE 7\nGene 541\n(aub, etc)\nTE 1\nGene 91\nPeak call (p<0.01)\nGFP-Set1 (WT or E1613K) / GFP control\n-4\n-2\n0\n2\n4\n6\n8\n10\n12\n-4 -2 0 2 4 6 8 10 12\nlog2 fold difference (GFPSet1WT - GFP)\nTEs with peaks\nGenes and TEs with peaks\nC D\nGFP\nGFP-Set1-WT in set1-GLKD\nH3K4me3\ngfp-GLKD [0-15]\n[0-15]\n[0-15]\n[0-15]\n[0-50]\n[0-50]\n[0-50]\nORF1\nTSSHeT-A (consensus)\nTSS aub\n[0-500]\n[0-500]\n[0-500]\n[0-500]\n[0-50]\n[0-50]\n[0-50]\n5’ 3’\nGFP-Set1-EK in set1-GLKD\nGFP-Set1-WT in set1-GLKD\nGFP-Set1-EK in set1-GLKD\nGFP\nE\nCUT&Tag\nCluster 3 (chr4:1,267,100 -1,348,250)\nTE\nGene track\nGFP\nGFP-Set1-WT in set1-GLKD\n[0-15]\n[0-15]\n[0-15]\nGFP-Set1-EK in set1-GLKD\nGFP(CUT&Tag )\nControl\n[0-10] plus st rand (antisense)\n[0-10] minus strand (sense)\n[0-10] plus st rand (antisense)\n[0-10] minus strand (sense)\nset1-GLKD\npiRNA\n6083\nF\nPrecursors\npiRNA (Sense < Antisense)\n3’UTR HTT\nPiwi\nTE\nIndividual\nTE insertions\nTelomere\nPrecursor\nAntisense piRNA\nPiwi\nSet1\nlog2 fold difference (GFPSet1E1613K - GFP) r = 0.85\nSet1Set1 Set1 Set1 Set1\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted March 30, 2026. ; https://doi.org/10.64898/2026.03.30.715253doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}