Tex19 Paralogs Are New Members of the Pirna Pathway Controlling

RESEARCH ARTICLE Tex19 paralogs are new members of the piRNA pathway controlling retrotransposon suppression Yara Tarabay1,*,§, Mayada Achour1,§, Marius Teletin1,2,TaoYe1, Aurélie Teissandier3, Manuel Mark1,2, Déborah Bourc’his3 and Stéphane Viville1,4,¶,‡

ABSTRACT and pass onto the next generation. TEs account for almost half of the Tex19 genes are mammalian specific and duplicated to give Tex19.1 human and mouse genomes. They are classified into two categories, and Tex19.2 in some species, such as the mouse and rat. It has been class I, which represents the majority of the TE families and class II, ∼ demonstrated that mutant Tex19.1 males display a variable degree of which accounts for 3% of the mammalian genome (Zamudio and ’ infertility whereas they all upregulate MMERVK10C transposons in their Bourc his, 2010; Goodier and Kazazian, 2008). Class I TEs are germ line. In order to study the function of both paralogs in the mouse, believed to be ancient traces of viral infections; they transpose ‘ ’ we generated and studied Tex19 double knockout (Tex19DKO) mutant themselves by using a copy and paste mode involving an RNA mice. Adult Tex19DKO males exhibited a fully penetrant phenotype, intermediate, which is converted into cDNA before its integration. ‘ similar to the most severe phenotype observed in the single Tex19.1KO Class II TEs are DNA transposons that transpose using a cut and ’ mice, with small testes and impaired spermatogenesis, defects in paste mode without any transcription needed (Goodier and Kazazian, ’ meiotic chromosome synapsis, persistence of DNA double-strand 2008; Lander et al., 2001; Zamudio and Bourc his, 2010). breaks during meiosis, lack of post-meiotic germ cells and upregulation Despite an undeniable role in evolution (Furano et al., 2004; ’ of MMERVK10C expression. The phenotypic similarities to mice with Ostertag and Kazazian, 2001; Zamudio and Bourc his, 2010), TEs knockouts in the Piwi family genes prompted us to check and then can be responsible for drastic mutagenic effects, as illustrated by the demonstrate, by immunoprecipitation and GST pulldown followed by growing number of human diseases caused by their transposition mass spectrometry analyses, that TEX19 paralogs interact with PIWI and expression (Cowley and Oakey, 2013). Germ cells have proteins and the TEX19 VPTEL domain directly binds Piwi-interacting developed sophisticated strategies to control TE activities, for RNAs (piRNAs) in adult testes. We therefore identified two new example, at the transcriptional level through DNA methylation and members of the postnatal piRNA pathway. the post-transcriptional level through RNA degradation or by blocking their integration (Gasior et al., 2006; Obbard et al., 2009; KEY WORDS: Meiosis, piRNA pathway, Infertility, Transposable Rollins et al., 2006; Yoder et al., 1997; Zamudio and Bourc’his, elements 2010). For reasons that remain unclear, the control of TEs in the mammalian germ line is gender specific. Indeed, it has been shown INTRODUCTION that the protection of the male germline genome relies on the PIWI In all sexually reproducing species, germ cells are the cell lineage family of Argonaute proteins, which produce and bind small RNAs that is specialized in transferring genetic information from one of ∼24–30 nucleotides long (named Piwi-interacting RNAs, generation to the next (Hajkova, 2011). The genome of germ cells piRNAs) (Ghildiyal and Zamore, 2009). In mice, the PIWI clade needs to be maintained stably and protected in all individuals contains three members, namely MIWI (PIWIL1), MILI (PIWIL2) throughout development. In particular, the genome must be and MIWI2 (PIWIL4) (Kuramochi-Miyagawa et al., 2004), which protected against mobile transposable elements (TEs). These TEs interact with germline-specific members of the TUDOR family can move, amplify themselves and transpose into new genomic loci proteins, such as TDRD1, TDRD5, TDRD6, TDRD7 and TDRD9 prompting a serious threat to the genome. Although most TEs are (Chuma et al., 2003; Hosokawa et al., 2007; Vasileva et al., 2009; inert, some elements have retained activity and become typically Zheng and Wang, 2012). They also interact with other factors such expressed in the germ line where they can transpose into new copies as MOV10L1, MVH (DDX4), MAEL or GASZ (ASZ1) (Costa et al., 2006; Ma et al., 2009; Soper et al., 2008; Zheng et al., 2010; Kotaja et al., 2006; Kuramochi-Miyagawa et al., 2004). Mutation of 1Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de Santéet de Recherche Médicale (INSERM) U964/Centre National de these piRNA pathway members give rise to a similar phenotype Recherche Scientifique (CNRS) UMR 1704/Universitéde Strasbourg, Illkirch consisting of male infertility, due to meiosis defects that can take 67404, France. 2Service de Biologie de la Reproduction, Centre Hospitalier place at different stages of meiosis, and lead to an upregulation of Universitaire, Strasbourg 67000, France. 3Institut Curie, department of Genetics and Developmental Biology, CNRS UMR3215, INSERM U934, 75005 Paris, France. most TEs (Zamudio and Bourc’his, 2010). Despite a large number 4Centre Hospitalier Universitaire, Strasbourg 67000, France. of studies, particularly involving loss-of-function mice, the exact *Present address: Institut de génétique humaine (IGH), 141 rue de la Cardonille, Montpellier 34396, France. ‡Present addresses: Laboratoire de diagnostic mechanism whereby piRNAs control TE expression and the link génetique, UF3472–Infertilité,NouvelHôpital Civil, 1 place de l’Hôpital, 67091 between the loss of TE control and meiosis defects are far from fully Strasbourg cedex and IPPTS, 3, rue Koeberlé, 67000 Strasbourg. understood. Currently, only the process in males is beginning to be §Co-first authors deciphered while that in females remains to be discovered. ¶Author for correspondence ([email protected]) In 2001, Wang et al. described a set of testis-expressed (Tex) genes, including Tex19 (Wang et al., 2001). In a previous study, we Y.T., 0000-0001-5692-8674; M.A., 0000-0003-1044-824X; S.V., 0000-0002- Tex19 5088-3003 showed that is a mammal-specific gene, which is duplicated in the mouse genome into two paralogs, Tex19.1 and Tex19.2.By

Received 17 March 2016; Accepted 27 February 2017 multiple sequence alignment of mammalian TEX19 proteins, two Journal of Cell Science

1463 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 1463-1474 doi:10.1242/jcs.188763 highly conserved domains, named MCP and VPTEL, respectively, western blotting of testes extracts from double wild-type (DWT), were identified. However, neither of them has any homology with Tex19.1KO, Tex19.2KO or Tex19DKO samples (Fig. 1C and data known proteins, preventing functional prediction (Kuntz et al., not shown). 2008). Both genes are co-expressed in the ectoderm, then in Interbreeding of Tex19 double heterozygote (DHZ) animals primordial germ cells (PGCs). Later on, they are co-expressed from resulted in a statistically significant deviation of the frequency of embryonic day (E)13.5 until adulthood in testes. However, only double homozygous animals from the expected Mendelian 1:2:1 Tex19.1 transcripts are present in developing and adult ovaries as ratio [207 (29.3%) Tex19DWT mice, 440 (62.3%) Tex19 double well as in the placenta (Celebi et al., 2012). Tex19.1-knockout heterozygote (DHZ) mice and 59 (8.3%) Tex19DKO mice, P<0.05, (Tex19.1KO) mice show a variable degree of male infertility due to Student’s t-test]. Yang et al., 2010 previously described higher level impaired meiosis associated with an upregulation of MMERVK10C of lethality in females among Tex19.1KO animals, an observation retrotransposon expression (Öllinger et al., 2008; Tarabay et al., which was not shared by other independent studies including ours 2013). More recently, it was shown that Tex19.1 plays an important (Öllinger et al., 2008; Tarabay et al., 2013; Yang et al., 2010). role in placenta function, as Tex19.1KO mouse embryos exhibit Nonetheless, we found that the number of Tex19DKO females intra-uterine growth retardation and have small placentas due to surviving for 2 weeks or more was significantly reduced when reduced number of spongiotrophoblast, glycogen trophoblast and compared to Tex19DKO males (32.2% of animals were females and sinusoidal trophoblast giant cells (Reichmann et al., 2013; Tarabay 67.8% males, P<0.05), suggesting a gender-specific lethality. et al., 2013). Tex19.2KO mice are fertile presenting only a subtle Surviving Tex19DKO mice did not present gross somatic phenotype with discrete seminiferous tubule degeneration in adult abnormalities. Females displayed normal fertility, but males were male testes (Y.T., M.T. and S.V. unpublished observations). The co- sterile. expression of Tex19.1 and Tex19.2 in PGCs, gonocytes and Tex19DKO males displayed a constant and severe reduction in spermatocytes suggests that these two genes could play redundant testis size (Fig. 2A). The weight of Tex19DKO adult testes (from functions during spermatogenesis. 19 mg up to 35 mg, mean 24.4 mg±10.5 mg per testis) was By generating a double knockout of both Tex19.1 and Tex19.2 threefold less than that of WT testes (from 109 mg up to 135 mg, genes (Tex19DKO), we demonstrate here that TEX19 paralogs mean 113.5 mg±5.52 mg per testis) (Fig. 2B). In contrast to WT exhibit redundant functions, which are essential for male fertility. seminiferous tubules (Fig. 2C), Tex19DKO testes histology showed We show that the Tex19DKO phenotype is fully penetrant, degenerate seminiferous epithelium with tubules containing only mimicking the most severe phenotype observed in single early spermatogenic cells, but a complete lack of post-meiotic germ Tex19.1KO mice. All adult DKO males are infertile and display cells, suggesting a meiotic arrest (Fig. 2D); consequently, the impaired spermatogenesis: meiosis is blocked at the pachytene stage epididymis was free of spermatozoa (Fig. S1A,B). Adult – during which chromosome synapsis is not correctly formed or Tex19DKO seminiferous tubules exhibited a drastically increased incomplete – and this leads to testis degeneration. Furthermore, level of apoptosis when compared to WT (Fig. S1C). This MMERVK10C retrotransposon expression is upregulated during phenotype is comparable to the most severe phenotype observed meiosis in Tex19DKO testes. Through GST pulldown and in the single Tex19.1KO (Tarabay et al., 2013). Thus, Tex19 immunoprecipitation (IP) experiments, we found that TEX19 paralogs are required for male meiosis and are essential for male paralogs interact with PIWI proteins and their accessory partners; fertility. moreover, they have the ability to bind RNAs of ∼30 nucleotides, a To further investigate the meiotic defect of Tex19DKO mutants, diagnostic size for piRNAs, but are not required for their biogenesis. we tested the assembly of the synaptonemal complex by co- Detailed analysis further showed that TEX19 directly interacts with immunostaining of the central and lateral elements with antibodies piRNAs via its VPTEL domain. Taken together, our study provides against SYCP1 and SYCP3, respectively. In contrast to WT novel evidence that TEX19 proteins are new members of the PIWI/ spermatocytes where all autosomes were fully synapsed at piRNA pathway acting during post-natal spermatogenesis. pachytene (Fig. 2E), synapsis failed to occur properly in Tex19DKO, as evidenced by the complete or partial absence of RESULTS the SYCP1 signal (Fig. 2F). Furthermore, as progression of Tex19 paralogs play an essential role in male fertility homologous recombination and chromosome synapsis are Tex19.1KO mice show a variable level of male infertility: one third interdependent, we checked whether homologous recombination of the KO male mice are almost indistinguishable from wild-type was also affected by studying DNA double-strand break (DSB) (WT) mice, one third display a mild phenotype and the last third a distribution, by immunostaining against phosphorylated histone severe phenotype with a complete meiosis arrest at pachytene γH2AX. γH2AX staining is normally present throughout chromatin (Öllinger et al., 2008; Tarabay et al., 2013). Independently of the during the zygotene stage. As synapsis proceeds, the DSBs are severity of the phenotype, a twofold upregulation of MMERVK10C resolved, resulting in γH2AX disappearance from autosomes, but elements was consistently observed among Tex19.1KO animals not from the sex chromosomes. In WT pachytene cells, synapsis (Öllinger et al., 2008; Tarabay et al., 2013). In contrast, Tex19.2KO was complete and only the sex chromosomes were stained for mice are fertile and their testes are undistinguishable from controls. γH2AX (Fig. 2G). However, the incompletely synapsed pachytene However, some seminiferous tubules show epithelium chromosomes in Tex19DKO nuclear spreads exhibited strong vacuolization (Y.T., M.T. and S.V. unpublished observations). diffuse γH2AX staining, suggesting that the disruption of Tex19 Considering the overlapping profile of Tex19.1 and Tex19.2 paralogs caused meiotic arrest at the pachytene stage because of the expression (Celebi et al., 2012), we wondered whether there was persistence of DSBs (Fig. 2H). Alternatively, γH2AX retention in any functional redundancy between the two genes in testes. We spermatocytes from Tex19DKO may be a consequence of asynapsis. therefore generated conditional double KO mice for Tex19.1 and In order to date the developmental onset of the Tex19DKO Tex19.2 genes, which exist as tandem duplication (Fig. 1A). phenotype, we analyzed testis sections at post-natal day (P)10, P16 The KO of both alleles (called Tex19DKO) was confirmed by and P20 and at 16 weeks of age (Fig. 3). At P10, Tex19DKO

PCR (Fig. 1B) and verifying the absence of the encoded proteins by sections were histologically indistinguishable from WT and Journal of Cell Science

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Fig. 1. Genetic targeting of Tex19 paralogs in mice. (A) The loci of the Tex19 paralogs are shown before and after homologous recombination upon removal of the neomycin (Neo)-encoding gene by crossing the mice with the Flp-recombinase strain. LoxP sites surrounding Tex19.1, Lox5171 sites surrounding Tex19.2 and PacI restriction sites (PacI RS) are depicted. E, exon. The KO allele is selected after a crossing with a mouse line expressing the Cre-recombinase. (B) Examples of PCR genotyping; one example of each genotype for Tex19.1KO, Tex19.2KO and Tex19DKO is shown. WT alleles are indicated by +/+, whereas mutant alleles are indicated by +/– and −/− for homozygous and heterozygous, respectively. (C) Western blot using the 6Tex:4D2 antibody recognizing both paralogs on adult testes extracts from WT, Tex19.1KO and Tex19DKO mice showing the absence of TEX19 proteins in the Tex19DKO. β-Tubulin is used as a loading control. M, molecular size marker.

Tex19DHZ testes (Fig. 3A–C). Tex19DKO showed vacuolization be responsible for the meiotic defects seen in Tex19.1KO mice and seminiferous epithelium degeneration as soon as P16 (Fig. 3D–F). (Öllinger et al., 2008; Tarabay et al., 2013). In Tex19.DKO mice, we Thus, the Tex19DKO testes phenotype became histologically observed a twofold and fourfold upregulation of MMERVK10C visible between P10 and P16, corresponding to the timing when expression at P10 and P16, respectively; no upregulation was the first spermatocytes reach the pachytene stage during the first post- observed at other TEs (LINEs and IAP) (Fig. S2), confirming the natal wave of spermatogenesis. This confirmed a spermatogenesis specific impact of Tex19 deficiency in the control of arrest occurring between the zygotene and the early pachytene MMERVK10C. stages of meiosis prophase I. Finally, increased TE expression has been proposed to be TEX19 associate with PIWI proteins and pachytene piRNAs responsible for impaired chromosome synapsis and meiotic defects To gain insight into TEX19 function, we sought to further identify during spermatogenesis in Dnmt3L, Miwi2 and Mili mutant mice its protein partners through GST pulldown and IP experiments. (Aravin et al., 2007; Bestor and Bourc’his, 2004; Carmell et al., For the GST pulldown experiment, GST alone, GST–TEX19.1 or 2007; Kuramochi-Miyagawa et al., 2004; Zamudio et al., 2015). GST–Tex19.2 was incubated with total testicular protein extract of

Overexpression of MMERVK10C retrotransposons could similarly adult mice (Fig. S3); complexes were then analyzed by mass Journal of Cell Science

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Fig. 2. Tex19DKO testicular defects. (A) Testes from Tex19DKO (DKO) mice are smaller than testes from double wild-type (DWT) littermates (8 weeks old). (B) Adult testes weight in mg. Testes weight (24.5 mg for DKO and 113.3 mg for DWT) is significantly reduced in Tex19DKO mice. Data are shown as mean±s.d. *P<0.01 (Student’s t-test). (C,D) Histological sections through the testes of adult mice stained with hematoxylin and eosin. (E–H) Double immunostaining of spread nuclei using antibodies against SYCP1 and either SYCP3 or γH2AX, as indicated. In E,F, the synaptonemal complexes (SC) appear yellow from overlapping of SYCP3 (green) and SYCP1 (red) signals. In both DWT and Tex19DKO spermatocytes at the zygotene stage, short fragments of SC begin to form. In DWT spermatocytes at the pachytene stage, all 19 bivalents are fully synapsed. In ∼20% of the Tex19DKO spermatocytes at the pachytene stage, only a few chromosomes are fully (arrowheads) or partially (arrow) synapsed, while the majority are unsynapsed (thin green strands). (I,K) DWT and Tex19DKO spermatocytes at the zygotene stage express γH2AX (green signal) throughout the nucleus. (J) In DWT spermatocytes at the pachytene stage, γH2AX expression becomes restricted to the (almost) unsynapsed XY body. (L) In ∼80% of the Tex19DKO spermatocytes at the pachytene stage, γH2AX remains widely distributed throughout the nucleus, even in regions that are apparently synapsed as assessed from the presence of thick SYCP3-positive strands (in red). Scale bars: 100 µm (D), 10 µm (in L). spectrometry (MS). In GST–TEX19.1 and GST–TEX19.2 samples, antibody, we further immunopurified TEX19.1 complexes in we detected bands corresponding to MAEL, MIWI, MILI, TDRD6, adult testes and identified individual polypeptides by MS. Among RANBP9 and MVH, while these bands were absent in the the identified proteins, we found MAEL, MIWI, MILI and MVH, GST-only sample (Fig. 4B). These interactions were further which were already identified by GST pulldown, but also TDRD8, confirmed by western blotting after co-immunoprecipitation TDRD6, EDC4, PABPC2, PSMC3, RANBP9, ANGEL1, SRPK1, experiments. Using a specific anti-TEX19.1 monoclonal DDX20 and DDX46. We confirmed the presence of MAEL, MIWI, Journal of Cell Science

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Fig. 3. Testes histology in DWT, DHZ and DKO animals. In DWT (A,D,G,J) and DHZ (B,E,H,K) testis histology reveals tissues are normal. The seminiferous tubules contain Sertoli cells, and germline cells are found at all studied ages. (L) Tex19DKO testes exhibit a severe phenotype with a diminished tubule diameter, vacuolization of the seminiferous epithelium and a reduction of the germ cell number at 16 weeks (16w). (C) An abnormal testicular phenotype starts to be visible at 16 days of age (P16) (F) and increases at 20 days of age (P20) (I,L) but is absent at 10 days of age (p10) (C). Thus, the histological phenotype starts to be visible between P10 and P16. Scale bar: 100 μm.

MILI, MVH, EDC4, TDRD6 and RANBP9 in TEX19.1 labeling and gel electrophoresis (Fig. 5). As expected, MILI immunopurified complexes isolated from cytoplasmic adult testes associated with ∼26–30 nucleotide (nt) small RNAs. TEX19.1 extracts by western blotting (Fig. 4C,D). These interactions were purification also revealed an interaction with small RNAs ranging in further validated by the reciprocal detection of TEX19.1 in size between 26 and 30 nt, migrating similarly to MILI-associated immunopurified MAEL, MIWI, MILI, MVH, EDC4 and piRNAs. Using the 6Tex:4D2 antibody, which recognizes both RANBP9 complexes (Fig. 4D). Taken together these results TEX19 paralogs, on an adult Tex19.1KO testicular extract from a suggest that TEX19.1 and TEX19.2 interact with proteins of the normal size testis from a 12-week-old male, we showed that TEX19.2 post-natal PIWI/piRNA pathway, namely core components such as also interacted with small RNAs of the same size range (Fig. 5). All MIWI and MILI, and accessory proteins such as EDC4, TDRD6, bands disappeared upon RNase treatment, confirming that these RANBP9 and MVH. entities were RNA. Furthermore, no such band was detected in the The identification of TEX19 paralogs as partners of the PIWI ascitic fluid control sample or in the sample precipitated with a proteins along with the phenotypic similarities observed between specific anti-TEX19.1 monoclonal antibody in Tex19.1KO testicular Tex19KO mice and piRNA mutant mice prompted us to check extract, confirming the specificity of the interaction between whether TEX19s could bind small RNAs, and in particular piRNAs. TEX19.1 and small RNAs. Thus, both TEX19 paralogs interact For this purpose, we immunoprecipitated TEX19.1, TEX19.2 and with small RNAs in a size range compatible with piRNAs. MILI (as a positive control) in adult mouse (aged 12 weeks) testes To further identify the small RNA species associated with TEX19 32 extracts and assessed for the presence of small RNAs by P5′ end- proteins, we generated small RNA-seq libraries by Illumina Journal of Cell Science

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Fig. 4. GST pulldown and IP of TEX19 paralogs. (A) Western blot showing GST– TEX19.1 and GST–TEX19.2. (B) Western blot analysis of the GST pulldown revealing that MAEL, MIWI, MILI, MVH, TDRD6 and RANBP9 are present in the analyzed pulldown samples. (C) Reciprocal IP experiment confirming the association of MAEL, MILI, MIWI, MVH, RANBP9, EDC4 with TEX19.1 by western blotting. (D) Co-IP of TEX19.1 by anti- TEX19.1 (7Tex: 1F11) and the detection by western blotting of either MAEL, MILI, MIWI, MVH, TDRD6, RANBP9 or EDC4. An immunoblott with the corresponding IP antibody (IBIPAB) is also shown. Ascitic fluid from an unimmunized animal (AFC) is used as a negative control.

sequencing after TEX19 and TEX19.1 IP from adult WT testes. To Because the three libraries were very similar in size and content, infer the TEX19.2-bound fraction, we analyzed TEX19 complexes but the TEX19.1 sample had a better quality, we focused the rest of in Tex19.1KO testes. Size distribution and genomic content were our analysis on the latter. Out of 1.6 million reads, we noticed a then compared to available datasets from MILI and MIWI-bound strong preference (94.42%) for a uridine at position one (U1 bias), in piRNAs from adult testes (Robine et al., 2009). MIWI was formerly agreement with the biogenesis mode of piRNAs (Aravin et al., shown to preferentially associate with 29–30 nt piRNA species, 2006; Frank et al., 2010; Kawaoka et al., 2011) (Table S1, Fig. S4). while the main piRNA size in MILI complexes is shorter, at ∼26– The genomic distribution appeared identical to both MILI and 27 nt. Interestingly, TEX19-, TEX19.1- and TEX19.2-associated MIWI IPs (Fig. 6B): the majority (70%) of the TEX19.1-derived small RNAs were found to segregate in size with MIWI-bound reads originated from intergenic regions, while less than 20% of the piRNAs, with a diagnostic 30 nt size (Fig. 6A). TEX19.2 showed a reads mapped to repeated elements. This is in line with the known stronger association to 22–23 nt long RNAs, which is a diagnostic genomic distribution of pachytene piRNA species, which are size for microRNAs; however, as small RNA libraries are expressed produced in adult testes from a discrete number of unique and as relative rather than absolute values, this may reflect a weaker intergenic piRNA clusters. Accordingly, 90% TEX19.1-associated affinity of TEX19.2 towards piRNAs but a genuine preferential reads uniquely mapped onto the genome (Fig. 6C), and, moreover, association with miRNAs cannot be excluded. we found the large majority of TEX19.1-associated small RNAs to Journal of Cell Science

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(Table S2) and it has a U at position one (denoted U1). We found that the piR-117061 was ‘shifted’ up upon electrophoresis in the presence of GST–TEX19.1, GST–TEX19.2, GST–VPTEL-Cter (amino acids 125–351) or GST–VPTEL of TEX19.1, but not in the presence of GST alone or GST–MCP (amino acids 1–124) or GST– Cter (amino acids 163–351) of TEX19.1. This result stresses the specificity of the interaction between piR-117061 and TEX19.2 and TEX19.1 via the VPTEL domain (Fig. 7A). The specificity of this interaction was further confirmed by showing that a modified form of piR-117061, in which we replaced U1 and A10 by a C nucleotide was unable to bind to TEX19.1, its VPTEL domain or TEX19.2 (Fig. 7B) and was not able to replace piR-117061 in a competition experiment (Fig. 7C). By adding an increased concentration of the non-radiolabeled piR-117061 to a fixed concentration of GST– VPTEL (5 µM pmole) and radiolabeled piR-117061, we showed that the non-radiolabeled piR-117061 completely displaced the radiolabeled piR-117061 when the concentration was 100-fold higher (Fig. 7D). Finally, we showed that the quantity of piR- 117061 ‘shifted’ up was proportional to the quantity of the TEX19.1 VPTEL domain (Fig. 7D).

DISCUSSION Tex19 Fig. 5. Interaction of TEX19.1 and TEX19.2 with ∼30 nt RNAs. MILI, genes are specific to mammals and can be found as a tandem TEX19.1 or TEX19.2 were immunoprecipitated from 2-month-old mouse testis duplication in the mouse and rat genomes. In the mouse, Tex19.1 protein extract by specific antibodies and bound RNAs were then 32P-end- and Tex19.2 expression is restricted to the germ line and the placenta labeled and separated on a 15% denaturing urea-polyacrylamide gel. (Celebi et al., 2012). Analyses of Tex19.1KO mice have previously Immunoprecipited RNAs are predominantly ∼30 nt long. The size and mobility highlighted a variable phenotype. In the severest form, Tex19.1- of the oligoribonucleotide markers is indicated on the right. The asterisk deficient males were infertile and presented an arrest at the indicates RNase treatment. Ctrl-, negative control (ascitic fluid from an unimmunized animal). pachytene stage of meiosis in association with a failure to silence MMERVK10C elements (Öllinger et al., 2008; Tarabay et al., 2013). Tex19.2KO males are fertile; however, a subtle phenotype of be derived from the same piRNA loci clusters previously defined as testes degeneration could be observed in adult mice (Y.T., M.T. and regulated by MILI and MIWI (Fig. 6D) (Beyret et al., 2012). We S.V. unpublished observations). The present study describes the nonetheless examined the content of the repeated fraction of the functional consequences of deleting both TEX19 paralogs and the TEX19.1 library and found that ∼50% were derived from long involvement of these proteins in the piRNA pathway. Surviving terminal repeat (LTR) retrotransposons, among which ∼1% Tex19DKO male and female mice were perfectly healthy, but males belonged to the MMERVK10C class (Fig. 6B). This means that exhibited a fully penetrant phenotype similar to the severest among all 25–30 nt-bound RNAs, only 0.1% derived from Tex19.1KO phenotype (Öllinger et al., 2008; Tarabay et al., MMERVK10C elements. Finally, to address the role of TEX19.1 2013). Indeed, Tex19DKO males have highly degenerated testes, in the biogenesis of piRNAs, we sequenced whole testes small RNA with a lack of germ cells beyond the pachytene stage, which is libraries in three adult (12 weeks of age) Tex19.1KO animals linked to defects in chromosome synapsis during meiosis. showing normal size testis: the size distribution appeared similar to Compared to Tex19.1KO testes (Öllinger et al., 2008; Tarabay WT testes, and, in particular, there was no relative redistribution et al., 2013), we showed, here, a stronger upregulation of towards 22–23 nt microRNA species, as an indication of impaired MMERVK10C retrotransposon in the double knockout piRNA production (Fig. 6E). Enrichment in 30 nt small RNAs was (Tex19DKO) testes of twofold at P10 and fourfold at P16 even higher in the Tex19.1KO background compared to WT: this compared to WT counterparts. This fully penetrant phenotype likely reflects different cellular composition, and in particular an confirms the hypothesis of a functional redundancy between overrepresentation of pachytene cells in Tex19.1KO testes, due to Tex19.1 and Tex19.2. Moreover, this phenotype bears striking the meiotic arrest. Taken together, these results suggest that TEX19 resemblance to the Dnmt3L, Miwi2 and Mili mutant phenotypes proteins sample the same set of primary, unique piRNA species as (Aravin et al., 2007; Bestor and Bourc’his, 2004; Carmell et al., MILI and MIWI, at the time of their production at the pachytene 2007; Kuramochi-Miyagawa et al., 2004). One explanation was stage in the adult testis. However, they are not required for their proposed by Zamudio et al., who suggested that improper TE synthesis, and likely act as simple cargo. silencing could alter the meiotic process through local remodeling of the chromatin landscape (Zamudio et al., 2015). TEX19 paralogs interact directly with piRNAs The essential role of TEX19 may extend beyond the context of In order to assess a direct interaction between TEX19.1, TEX19.2 spermatogenesis as we report here a severe lethality of Tex19DKO and piRNAs, we performed electrophoretic mobility shift assays animals, with only 8.3%, instead of the expected 25%, mutant (EMSA) using GST–TEX19.1 and GST–TEX19.2 proteins as well animals surviving at over 2 weeks of age. Moreover, this lethality as different domains of TEX19.1 fused to GST (Figs S5 and S6) was sexually dysmorphic as among the survivors, only one third against the piR-117061 piRNA. We choose this piRNA because it were females. We did not observe such lethality in our single has a very significant absolute count (2361 and 2028) in two Tex19.1KO mouse models (Tarabay et al., 2013), but Yang et al. biological replicates prepared using anti-TEX19.1 antibody (2010) had reported such female-specific lethality in their Journal of Cell Science

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Fig. 6. piRNA populations isolated from TEX19 paralogs and MILI complexes were cloned, sequenced and annotated. (A) Size distribution of small RNA-seq samples immunoprecipitated with TEX19 and TEX19.1 from WT adult testes, and with TEX19 from Tex19.1KO mice. These samples exhibit a peak at ∼29–30 nt, which is reminiscent of MIWI-associated piRNAs; in contrast, MILI- associated piRNAs are ∼26–27 nt. (B) Genomic annotation of TEX19.1-associated reads compared to MILI and MIWI-associated reads. In agreement with piRNA size, the analysis was restricted to reads of 25–32 nt. (C) The vast majority of TEX19.1 reads have unique hits on the genome, which is similar to MILI and MIWI- associated small RNAs. (D) Venn diagram distribution of the piRNA cluster defined by the piRNA population associated with TEX19.1 compared to that of MILI and MIWI. (E) Size distribution of whole adult testes small RNA-seq libraries obtained from one WT and three Tex19.1KO animals. The 22–23 nt peak corresponds to microRNAs, whereas the piRNAs are clustered in two peaks, at 26–27 nt and at 29–30 nt. Journal of Cell Science

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Fig. 7. Tex19 paralogs interact directly with ∼26-30 nt piRNAs. (A,B) EMSAs were performed in the presence of 5 µM of GST alone, or GST-tagged full-length TEX19.1, GST-tagged TEX19.1 domains MCP (amino acids 1–124), VPTEL-Cter (amino acids 125–351), VPTEL (amino acids 125–163; VPTEL) or Cter (amino acids 164–351) or GST-tagged TEX19.2. All proteins were incubated with 125 femtomoles (8000 cpm) of radiolabeled piR-117061 or modified piR-117061. (C) An increased concentration of the modified non-radiolabeled piR-117061(2C) was used with a fixed concentration (5 µM) of the TEX19.1 VPTEL domain and a fixed quantity of radiolabeled piR-117061 (8000 cpm). (D) An increased concentration of the non-radiolabeled piR-117061 was used with a fixed concentration of the VPTEL domain of TEX19.1 (5 µM) and a fixed quantity of radiolabeled piR-117061 (8000 cpm).

Tex19.1KO mice and suggested that the variable severity among et al., 2013), but also suggests a functional redundancy between different studies could be linked to strain specificities (Yang et al., Tex19.1 and Tex19.2 during embryo development. The exact reason 2010; Kwon et al., 2003). However, our studies on Tex19.1KO or for the preferential female lethality of the Tex19DKO mutation Tex19DKO mice were conducted in the same genetic background remains unclear, but may be linked to X chromosome-specific (C57Bl/6). A strong bias in sexual lethality was also observed in effects. C57Bl/6 mice by Reichmann et al., but only when females were The main findings provided by our work relate to the lactating and nursing a pre-existing litter during pregnancy identification of the protein partners of TEX19 in adult testes. (Reichmann et al., 2013). However, none of our studies were Through GST pulldown and IP experiments, we indeed showed for conducted under such lactating conditions. Consequently, this the first time that TEX19.1 exists in a complex with members of the difference between Tex19.1KO and Tex19DKO phenotypes is quite PIWI and/or piRNA pathway: MAEL, MIWI, MILI and MVH. We surprising since Tex19.2 is not expressed in the placenta, neither in also confirmed a significant level of interaction between TEX19.1 WT nor in Tex19.1KO animals. This early postnatal lethality could and UBR2 by IP, as has been previously reported (Yang et al., be indicative of defects in placenta (Reichmann et al., 2013; Tarabay 2010). Most importantly, we found that both TEX19 paralogs bind Journal of Cell Science

1471 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 1463-1474 doi:10.1242/jcs.188763 small RNAs that have all the characteristics of piRNAs as they are 4.4 kb and a 3.5 kb fragment corresponding to the 5′ and the 3′ arms, ∼30 nt in size and there is a strong preference for a uridine as the first respectively. The MCI vector contains a Lox5171 site as well as a F3- nucleotide (Frank et al., 2010; Kawaoka et al., 2011; Brennecke surrounded hygromycin resistance cassette. A unique PacI restriction site was inserted upstream of the 5′ loxP site. The linearized construct was et al., 2007). Finally, this interaction seems to be direct and to occur Tex19.1 through the well-conserved VPTEL domain of TEX19 proteins. We electroporated in one fully valid KO clone. After hygromycin selection, targeted clones were identified by PCR using external primers and can therefore conclude that TEX19 paralogs are new members of the further confirmed by Southern blotting with 5′ and 3′ external probes. family of proteins that bind piRNAs and that the VPTEL domain Restriction digestion with PacI followed by Southern blotting allowed could be a new small RNA-binding motif. While this study reveals selection of a clone showing targeting of both constructs on the same allele. the piRNA-binding function of the VPTEL domain, we cannot This clone was injected into C57BL/6J blastocysts, and male chimeras tested speculate about the function of the MCP domain, which lacks for germline transmission. The NeoR and HygroR cassettes were deleted by homology with any known proteins. PIWI proteins have, in addition breeding the F1 mice with Flp mice (Rodríguez et al., 2000). Excision of to their ability to bind piRNAs, an RNA-guided nuclease function both Tex19.1 and Tex19.2 inter-Lox fragments was performed by breeding linked to the catalytic triad DDH localized in the PIWI domain and the mice with a Cre deleter line (Dupe et al., 1997). The absence of Tex19.1 Tex19.2 which is required for piRNA biogenesis (Martinez and Tuschl, and mRNA and protein was tested by RT-PCR, using 2004). No such triad can be found in TEX19 proteins. Accordingly, oligonucleotides described previously (Tarabay et al., 2013), and by – Tex19 western blotting, respectively. the production of small RNAs of 25 30 nt is not altered in - All animal experimental procedures were performed according to the deficient testes. This suggests that while TEX19 proteins bind to European authority guidelines. piRNAs, they are not involved in the catalysis of piRNA synthesis. Finally, it is now well established that TEX19 paralogs are Antibody production, western blotting, and preparation of required for the control of MMERVK10C transposons in testes cytoplasmic and nuclear fractions (Öllinger et al., 2008; Tarabay et al., 2013). Despite a consistent Injecting the entire protein or the peptide DLGPEDAEWTQALPWRC into effect of Tex19 deficiency on these elements, we found that mice allowed the generation of anti-TEX19 monoclonal antibodies. The MMERVK10C-derived reads only represent 0.1% of all TEX19- specificity of both antibodies was tested as described elsewhere (Tarabay bound small RNAs of 25–30 nt. In contrast, TEX19 mostly binds et al., 2013). The 7Tex:1F11 antibody is TEX19.1 specific, whereas the unique, non-transposon-derived small RNAs in adult testes, which 6Tex:4D2 antibody detects both TEX19.1 and TEX19.2. Cytoplasmic and are similar to the MILI and MIWI-associated piRNAs. This is not nuclear fractions were prepared as described at http://openwetware.org/wiki/ surprising, as the post-natal PIWI machinery is responsible for Cytoplasm_and_nuclear_protein_extraction producing piRNAs derived from discrete intergenic regions as male RT-PCR and real-time quantitative RT-PCR germ cells enter the pachytene stage (Aravin et al., 2006; Girard RNA was prepared using the RNeasy mini or micro kit (Qiagen) following et al., 2006), although the function of these mammalian post-natal the manufacturer’s instructions. After DNase I digestion (Roche, piRNAs has not been fully determined (Goh et al., 2015). This poor Mississauga, ON, Canada), 1 µg RNA was reverse-transcribed by random enrichment of TEX19-bound small RNAs in MMERVK10C priming using Superscript II (Invitrogen/Life Technologies, Burlington, fragments raises the question as to whether the reactivation of ON, Canada). The quantitative PCR was performed by using SYBR® green MMERVK10C in Tex19.1KO and Tex19DKO mice reflects a direct JumpStartTM Taq ReadyMixTM (Sigma-Aldrich, Oakville, ON, Canada) role of TEX19 proteins in silencing these elements – likely via post- and LightCycler 480 (Roche, Mississauga, ON, Canada). The efficiency transcriptional mechanisms – or an indirect effect of the Tex19 and specificity of each primer pair was checked using a cDNA standard mutation. Further work should be dedicated to resolving this curve. All samples were normalized to β-actin (Actb) and Rrm2 expression question. (Tarabay et al., 2013). In conclusion, our analysis of the double-mutant mice for the two TEX19 paralogs undoubtedly reveals the crucial role of these Histology Testes were collected and fixed in Bouin’s fluid for 48 h and then embedded proteins for male fertility in mammals, likely via their association in paraffin. Adult male testis were dissected at 16 weeks of age. For with the PIWI/piRNA pathway. histological analyses, 5-µm-thick sections were stained with hematoxylin and eosin. All slides were examined using a DMLA microscope (Leica) with MATERIALS AND METHODS 10×, 20×, 40× and 100× objectives with apertures of 0.3, 0.5, 0.7 and 1.3, Generation of Tex19 double knockout mice respectively. Images were taken with a digital camera (CoolSnap; The Tex19 double mutant mouse line was established at the MCI (Mouse Photometrix) using CoolSnap v.1.2 software and then processed with Clinical Institute, Institut Clinique de la Souris, Illkirch, France; http://www- Photoshop CS2 v.9.0.2 (Adobe). mci.u-strasbg.fr). For Tex19.1, the targeting vector (Fig. 1A) was constructed by sequentially subcloning three PCR fragments (from Immunostaining of meiotic chromosome spreads 129S2/SvPas ES cell genomic DNA) into an MCI proprietary vector, Meiotic chromosome spreads were prepared as described previously (Mark namely, one 2.2 kb fragment corresponding to inter-loxP fragment et al., 2008). encompassing exons 2 and 3, and a 4.5 kb and a 3.1 kb fragment corresponding to the 5′ and 3′ arms, respectively. The MCI vector TEX19 protein production and purification contains a LoxP site as well as a floxed and flipped neomycin resistance Tex19.1 cDNA or its domains were cloned, through RT-PCR starting from a cassette. A unique PacI restriction site was inserted downstream to the 3′ testis cDNA library, into pCR®4-TOPO (Invitrogen, Life Technologies, loxP site. The linearized construct was electroporated in 129S2/SvPas Burlington, ON, Canada), then into pENTR 1A (Life Technologies) then by mouse embryonic stem (ES) cells. After G418 selection, targeted clones LR recombinase into pHGGWA or pGGWA. The GST-tagged recombinant were identified by PCR using external primers and further confirmed by proteins TEX19, MCP, VPTEL-Cter, VPTEL, Cter and protein GST alone Southern blotting with 5′ and 3′ external probes. were individually expressed in E. coli BL21 cells. The induction by 0.1 mM For Tex19.2, the targeting vector was constructed by sequentially of IPTG was performed at a cell density of A600=0.8 at 20°C overnight and a subcloning three PCR fragments (from 129S2/SvPas ES cell genomic portion of the proteins examined by SDS-PAGE (10% gels). Then, DNA) into an MCI proprietary vector, namely, a 2.0 kb fragment sonication was performed for 3 s ON and 3 s OFF for 5 min at amplitude

corresponding to inter-lox P fragment encompassing exons 2 and 3, and a 33%. Cleared lysates were prepared in lysis buffer (50 mM Tris-HCl pH 9, Journal of Cell Science

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400 mM NaCl, 2 mM DTT, Triton 1% and protease inhibiting cocktail) and were then annotated by using RepeatMasker and refGene databases, and the centrifuged for 1 h at 20,000 rpm. The soluble fusion proteins were purified positional read coverage was weighted by mapping site numbers. by a batch procedure, incubating the lysate with 500 µl of glutathione– The piRNA cluster analysis was performed as described previously Sepharose (Amersham Biosciences) for 1 h at 4°C with gentle shaking. (Beyret et al., 2012). A cluster was defined as a group of piRNAs where the Then the glutathione–Sepharose was collected by centrifugation at 1000 reads were less than 1500 bp away from each other. Clusters with at least rpm for 5 min at 4°C and washed five times with lysis buffer. After boiling, 1000 normalized read counts were reported. The UCSC genome tracks were the bound proteins were analyzed by SDS-PAGE (10% gels) followed generated by using the HOMER software (v4.3) using unique mapped reads. by western blotting using anti-TEX19 monoclonal antibodies 7Tex:1F11 (1:1000) or 6Tex:4D2 (1:1000). Acknowledgement For GST pulldown assays, total testicular extracts of adult mice (aged We would like to thank Robert Drillien for his critical reading of the manuscript. We 12 weeks) were added to recombinant protein fixed to glutathione– are grateful to the Institute of Genetics and Molecular and Cellular Biology (IGBMC) Sepharose beads for 2 h at 4°C. After five washes, proteins were platforms. separated on 10% SDS-PAGE gels and subsequently stained with Bio- Safe Coomassie (Bio-Rad, Hercules, CA). The gel was then sent to Taplin Competing interests The authors declare no competing or financial interests. Biological Mass Spectrometry Facility to be analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS; Taplin, Boston, Author contributions MA). Purified recombinant proteins were used in EMSA assays as described Y.T., M.A., M.T., T.Y., A.T., M.M., D.B., S.V.: Data analysis and interpretation, below. manuscript writing, final approval of manuscript.

Co-IP Funding Antibodies were chemically cross-linked to protein-G–Sepharose beads by The study was funded by Agence Nationale de la Recherche (ANR-11-BSV2-002 – using dimethyl pimelimidate (DMP; Sigma-Aldrich, D-8388) and used to TranspoFertil) and Agence de la Biomédecine – AMP, diagnostic prénatal et purify TEX19.1, TEX19.2, MAEL, MIWI, MILI and MVH complexes diagnostic génétique. This work was supported by the French Centre National de la from cytoplasmic fractions of adult mouse testes (aged 12 weeks). 30 mg of Recherche Scientifique (CNRS), Institut National de la Santéet de la Recherche ́ ̀ ’ ́ clarified DWT testes cytoplasmic extracts were incubated with 300 µl Medicale (INSERM), the Ministere de l Enseignement Superieur, de la Recherche ’ protein-G–Sepharose beads coupled to antibodies as follows: anti-TEX19.1 Scientifique et des Technologies de l Information et de la Communication, and the Universitéde Strasbourg (University Hospital of Strasbourg). (7Tex:1F11, 100 µl), anti-TEX19.1/TEX19.2 (6Tex:4D2, 10 ml of hybridoma supernatant), anti-MAEL (18 µg, Abcam, Toronto, ON, Supplementary information Canada), anti-MIWI (20 µg, Aviva System Biology, San Diego, CA), Supplementary information available online at anti-MILI (40 µg, Abcam), anti-MVH (12 µg, Abcam), anti-TDRD66 http://jcs.biologists.org/lookup/doi/10.1242/jcs.188763.supplemental (50 µg, Millipore/Upstate, Etobicode, ON, Canada), anti-EDC4 (12 µg, Abcam), anti-RANBP9 (8 µg, Proteintech, Rosemonte, IL, USA) or an References ascitic fluid control (negative control, 100 µl) in co-IP buffer (50 mM Tris- Aravin, A., Gaidatzis, D., Pfeffer, S., Lagos-Quintana, M., Landgraf, P., Iovino, N., HCl, pH 8, 150 mM NaCl, 1 mM EDTA, complete protease inhibitor) Morris, P., Brownstein, M. J., Kuramochi-Miyagawa, S., Nakano, T. et al. (2006). overnight at 4°C. After five washes with co-IP buffer, the retained proteins A novel class of small RNAs bind to MILI protein in mouse testes. Nature 442, were denatured by using Laemmli buffer with 5% β-mercaptoethanol heated 203-207. Aravin, A. A., Hannon, G. J. and Brennecke, J. (2007). The Piwi-piRNA pathway to 100°C, then separated by SDS-PAGE as described above. If required, gels provides an adaptive defense in the transposon arms race. Science 318, 761-764. were stained with Coomassie Blue and sent to Taplin Biological Mass Bendak, K., Loughlin, F. E., Cheung, V., O’connell, M. R., Crossley, M. and Spectrometry Facility to be analyzed by LC/MS/MS (Taplin, Boston, MA). Mackay, J. P. (2012). A rapid method for assessing the RNA-binding potential of a protein. Nucleic Acids Res. 40, e105. Small RNA IP and purification Bestor, T. H. and Bourc’his, D. (2004). Transposon silencing and imprint TEX19 libraries were prepared as described elsewhere (Reuter et al., 2009). establishment in mammalian germ cells. Cold Spring Harb. Symp. Quant. Biol. 69, 381-387. Beyret, E., Liu, N. and Lin, H. 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