Tdrkh is essential for spermatogenesis and participates in primary piRNA biogenesis in the germline
2013; Springer Nature; Volume: 32; Issue: 13 Linguagem: Inglês
10.1038/emboj.2013.121
ISSN1460-2075
AutoresJonathan P. Saxe, Mengjie Chen, Hongyu Zhao, Haifan Lin,
Tópico(s)Plant Disease Resistance and Genetics
ResumoArticle28 May 2013free access Tdrkh is essential for spermatogenesis and participates in primary piRNA biogenesis in the germline Jonathan P Saxe Jonathan P Saxe Yale Stem Cell Center, Yale University, New Haven, CT, USA Department of Cell Biology, Yale University, New Haven, CT, USA Search for more papers by this author Mengjie Chen Mengjie Chen Yale Stem Cell Center, Yale University, New Haven, CT, USA Department of Cell Biology, Yale University, New Haven, CT, USA Department of Biostatistics, Yale University, New Haven, CT, USA Program of Computational Biology and Bioinformatics, Yale University, New Haven, CT, USA Search for more papers by this author Hongyu Zhao Hongyu Zhao Department of Biostatistics, Yale University, New Haven, CT, USA Program of Computational Biology and Bioinformatics, Yale University, New Haven, CT, USA Search for more papers by this author Haifan Lin Corresponding Author Haifan Lin Yale Stem Cell Center, Yale University, New Haven, CT, USA Department of Cell Biology, Yale University, New Haven, CT, USA Department of Biostatistics, Yale University, New Haven, CT, USA Program of Computational Biology and Bioinformatics, Yale University, New Haven, CT, USA Search for more papers by this author Jonathan P Saxe Jonathan P Saxe Yale Stem Cell Center, Yale University, New Haven, CT, USA Department of Cell Biology, Yale University, New Haven, CT, USA Search for more papers by this author Mengjie Chen Mengjie Chen Yale Stem Cell Center, Yale University, New Haven, CT, USA Department of Cell Biology, Yale University, New Haven, CT, USA Department of Biostatistics, Yale University, New Haven, CT, USA Program of Computational Biology and Bioinformatics, Yale University, New Haven, CT, USA Search for more papers by this author Hongyu Zhao Hongyu Zhao Department of Biostatistics, Yale University, New Haven, CT, USA Program of Computational Biology and Bioinformatics, Yale University, New Haven, CT, USA Search for more papers by this author Haifan Lin Corresponding Author Haifan Lin Yale Stem Cell Center, Yale University, New Haven, CT, USA Department of Cell Biology, Yale University, New Haven, CT, USA Department of Biostatistics, Yale University, New Haven, CT, USA Program of Computational Biology and Bioinformatics, Yale University, New Haven, CT, USA Search for more papers by this author Author Information Jonathan P Saxe1,2, Mengjie Chen1,2,3,4, Hongyu Zhao3,4 and Haifan Lin 1,2,3,4 1Yale Stem Cell Center, Yale University, New Haven, CT, USA 2Department of Cell Biology, Yale University, New Haven, CT, USA 3Department of Biostatistics, Yale University, New Haven, CT, USA 4Program of Computational Biology and Bioinformatics, Yale University, New Haven, CT, USA *Corresponding author. Yale Stem Cell Center, Department of Cell Biology and Program of Computational Biology and Bioinformatics, Yale University School of Medicine, 10 AMistad Street, Room 237, New Haven, CT 06519, USA. Tel.:+1 203 785 6239; Fax:+1 203 785 4305; E-mail: [email protected] The EMBO Journal (2013)32:1869-1885https://doi.org/10.1038/emboj.2013.121 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Piwi proteins and Piwi-interacting RNAs (piRNAs) repress transposition, regulate translation, and guide epigenetic programming in the germline. Here, we show that an evolutionarily conserved Tudor and KH domain-containing protein, Tdrkh (a.k.a. Tdrd2), is required for spermatogenesis and involved in piRNA biogenesis. Tdrkh partners with Miwi and Miwi2 via symmetrically dimethylated arginine residues in Miwi and Miwi2. Tdrkh is a mitochondrial protein often juxtaposed to pi-bodies and piP-bodies and is required for Tdrd1 cytoplasmic localization and Miwi2 nuclear localization. Tdrkh mutants display meiotic arrest at the zygotene stage, attenuate methylation of Line1 DNA, and upregulate Line1 RNA and protein, without inducing apoptosis. Furthermore, Tdrkh mutants have severely reduced levels of mature piRNAs but accumulate a distinct population of 1′U-containing, 2′O-methylated 31–37 nt RNAs that largely complement the missing mature piRNAs. Our results demonstrate that the primary piRNA biogenesis pathway involves 3′→5′ processing of 31–37 nt intermediates and that Tdrkh promotes this final step of piRNA biogenesis but not the ping-pong cycle. These results shed light on mechanisms underlying primary piRNA biogenesis, an area in which information is conspicuously absent. Introduction The establishment and maintenance of the germline are essential for passing genetic information from one generation to the next. The Piwi proteins, a subfamily of the Piwi/Argonaute family (Cox et al, 1998), play a central role in germline development and gametogenesis (Thomson and Lin, 2009; Siomi et al, 2011). There are three murine Piwi proteins, Mili, Miwi, and Miwi2. Mili and Miwi2 act in concert to mediate DNA methylation of transposons. Spermatogenic arrest is coincident with loss of DNA methylation of mobile genetic elements and their resultant activation in Mili and Miwi2 mutants (Aravin et al, 2007; Kuramochi-Miyagawa et al, 2008). Piwi-interacting RNAs (piRNAs) bind to Piwi proteins and are hypothesized to act as sequence-specific guides to recruit epigenetic machinery to genomic sites complementary to their sequences (Lin and Yin, 2008; Watanabe et al, 2011b; Huang et al, 2013). They are derived from cellular transcripts, including gene-coding mRNAs and retrotransposons, and specific intergenic loci (Aravin et al, 2006; Girard et al, 2006; Grivna et al, 2006a; Lau et al, 2006). Mili functions in both primary and secondary piRNA biogenesis, whereas Miwi2 functions mainly in the secondary biogenesis pathway (also referred to as the ping-pong cycle; Siomi et al, 2011). In the primary pathway, cellular transcripts are recruited and processed through a mostly uncharacterized mechanism that relies on the scaffolding protein Tdrd1 for proper function (Reuter et al, 2009; Mathioudakis et al, 2012). Mature primary piRNAs are bound by Mili and Miwi (Beyret et al, 2012). Recent reports have demonstrated a complete lack of piRNAs following mutations of Mov10l1 and MitoPLD (the mouse homologue of Drosophila Zuc), implicating these genes in primary piRNA processing (Zheng et al, 2010; Watanabe et al, 2011a). Here, we report that Tdrkh, a Tudor and KH domain-containing protein, interacts with Miwi and Miwi2. Mutation of tdrkh results in male sterility due to meiotic defects with concurrent loss of retrotransposon silencing. piRNA populations are severely reduced in Tdrkh mutants, with piRNA biogenesis blocked at a novel intermediate state resulting in accumulation of a class of previously uncharacterized precursor RNAs. Our data demonstrate that Tdrkh is a component of the primary piRNA processing pathway and facilitates processing and maturation of the 3′ ends of piRNAs. Results Tdrkh interacts with Miwi and Miwi2 in vitro and in vitro Tdrkh was identified as an interacting partner of mouse Miwi, but not Mili (Chen et al, 2009; Vagin et al, 2009; Wang et al, 2009). Tdrkh and its fly homologue Papi (Liu et al, 2011) are 24% identical and 37% similar, with similarity approaching 56% within the conserved Tudor domain (Supplementary Figure 1). Miwi was preferentially pulled down by Tdrkh when co-transfected in 293T cells with five different Tudor domain-containing proteins (Tdrds; Figure 1A). In the reciprocal experiment, Tdrkh was specifically pulled down by Miwi and Miwi2 (Figure 1B); only a slight interaction with Mili was noted. In adult testes, Tdrkh and Miwi reciprocally co-immunoprecipitated each other (Figure 1C and D), but no in vivo interaction between Mili and Tdrkh was noted (Figure 1C). Furthermore, Tdrkh and Miwi2 co-localize in pre-natal testes (Supplementary Figure 3h). Thus, Tdrkh specifically interacts with Miwi and Miwi2. Figure 1.Tdrkh interacts with Miwi and Miwi2. (A) FLAG-tagged Miwi and myc-tagged Tdrd proteins were co-transfected into 293T cells followed by immunoprecipitation of the myc-tagged proteins. Tdrkh is also known as Tdrd2. (B) Myc-tagged Tdrkh was co-transfected with FLAG-tagged Piwi and Ago proteins into 293T cells, followed by immunoprecipitation of the FLAG tag. (C) Co-immunoprecipitation of Miwi and Mili followed by western blot detection of Tdrkh from 2-month-old CD-1 mouse testis extract. (D) Co-immunoprecipitation of Miwi by Tdrkh from 2-month-old CD-1 mouse testis extract treated with or without RNase A. (E) Domain mapping of the Miwi–Tdrkh interaction by co-transfection with FLAG-tagged Miwi truncations and myc-Tdrkh. (F) Domain mapping of the Miwi–Tdrkh interaction by co-transfection with myc-tagged Tdrkh truncations and FLAG-Miwi. Arrows indicate specific western blot bands. (G) Co-immunoprecipitation of myc-Tdrkh by FLAG-Miwi2 containing point mutations to prevent symmetric dimethylation. (H) Co-immunoprecipitation of myc-Tdrkh by FLAG-Miwi containing point mutations to prevent symmetric dimethylation. (I) siRNA-mediated knockdown of PRMT5 prevents co-immunoprecipitation of myc-Tdrkh by FLAG-Miwi and FLAG-Miwi2. (J) Biotinylated Miwi peptides corresponding to Miwi residues 2–17, either with no methylated residues (Miwi2–17) or with symmetric dimethylation on arginine 4 (R4(me2s)) or arginine 14 (R14(me2s)), were incubated with adult C57 testis lysate prepared with 150 mM salt followed by western blot analysis for Tdrkh. Download figure Download PowerPoint The interaction between Tdrkh and Miwi does not require RNA, because digestion of testicular extracts with RNase A had no effect on the ability of Tdrkh to co-immunoprecipitate Miwi (Figure 1D; Supplementary Figure 2a). Furthermore, mutating residues the KH domains critical for RNA binding had no effect on binding (Supplementary Figure 2b). The Tudor domain of Tdrkh interacts with Miwi and Miwi2 via symmetrically dimethylated arginines We then identified protein domains that mediate these interactions by co-immunoprecipitation assay of various Miwi and Tdrkh variants in 293 T cells (see Supplementary Materials). The N-terminus of Miwi interacts with the Tudor domain of Tdrkh (Figure 1E and F). This is consistent with findings that the Tudor domain of Tdrd proteins interacts with the N-terminus of Piwi proteins (Chen et al, 2009; Kirino et al, 2009; Reuter et al, 2009; Vagin et al, 2009). Some Tdrd proteins are known to interact with Piwi proteins by binding to symmetrically dimethylated arginine residues in the N-terminus of Piwi proteins (Kirino et al, 2009; Reuter et al, 2009; Vagin et al, 2009). We tested whether this mechanism also mediates the interaction between Tdrkh and Miwi/Miwi2. Miwi2 contains four arginines that could be symmetrically dimethylated; they lie in a GRARVRARG motif near the N-terminus of the protein. Mutating these four arginines abolished the interaction between Miwi2 and Tdrkh (Figure 1G). For Miwi, however, mutating arginines in the conserved GRARVRARG motif in its N-terminal domain had no effect on Miwi–Tdrkh interaction (Figure 1H). Miwi contains two additional symmetrically dimethylatable arginines in the 12th and 14th position (Vagin et al, 2009). Mutating them to lysine compromised Miwi–Tdrkh interaction (Figure 1H). Thus, the methylation of arginine residues in the N-terminal domain of Piwi proteins mediates their interaction with Tdrkh. We then directly tested whether this interaction required symmetric dimethylation. siRNA-mediated knockdown of PRMT5, which catalyses symmetric dimethylation of arginines at RA/RG motifs, abolished the Miwi2-Tdrkh interaction and severely attenuated the Miwi–Tdrkh interaction (Figure 1I). We also performed pulldown assays with biotinylated peptides encompassing residues 2–17 of the N-terminal of Miwi that contains six RA/RG motifs. We analysed arginines 4 and 14, which interact with Tudor domain-containing proteins or are symmetrically dimethylated, respectively (Vagin et al, 2009; Liu et al, 2010). Symmetric dimethylation of R4 enhances the ability of the N-terminal of Miwi to pull down Tdrkh by two-fold (Figure 1I). In contrast, symmetric dimethylation of R14 severely weakened the interaction. This pattern occurred regardless of salt concentration, using either cytoplasmic testicular lysate or Tdrkh overexpressed in rabbit reticulocyte lysate. As a whole, these results strongly argue that symmetric dimethylation of specific arginine residues within the N-terminal domain of Miwi and Miwi2 mediate their interaction with the Tudor domain of Tdrkh. Tdrkh is associated with mitochondria in the germline Tdrkh is expressed quite ubiquitously and is highly enriched in the brain and the testis (Supplementary Figure 3a). In situ RNA hybridization (Supplementary Figure 4a) and immunofluorescence (Supplementary Figure 3b) revealed its expression in spermatogonia, spermatocytes, and round spermatids, but not in elongating spermatids (Supplementary Figure 3b). Tdrkh is cytoplasmic and localized in punctuate structures. Immunofluorescence microscopy revealed that Tdrkh is extensively co-localized with the mitochondrial marker CoxIV (Supplementary Figure 3c). Biochemical fractionation further revealed that Tdrkh is highly enriched in the mitochondrial matrix (Supplementary Figure 3d). Immuno-electron microscopy confirmed the mitochondrial localization of Tdrkh and observed enrichment within the intermitochondrial cement (IMC), a form of the nuage (Supplementary Figure 3e). In sum, these data show that Tdrkh is a mitochondrion-associated protein. Tdrkh foci overlap with pi-bodies and piP-bodies Recent studies have described distinct cytoplasmic organelles as sites of piRNA biogenesis and function (Aravin et al, 2009; Shoji et al, 2009). Pi-bodies contain Mili, Tdrd1, and Mvh and localize to IMC in pro-spermatogonia and postnatal germ cells. IMC and associated pi-bodies are required for primary piRNA biogenesis (Watanabe et al, 2011a). PiP-bodies are larger structures containing Miwi2, Tdrd9, Mael, and P-body components. Cross-talk between pi- and piP-bodies is thought to be responsible for ping-pong-based piRNA biogenesis. As Tdrkh also localizes to IMC, we examined potential co-localization of Tdrkh and piRNA pathway components. In 18dpc testis sections, Tdrkh shows identical granular cytoplasmic localization as in adults and is often adjacent to and partially co-localizes with the pi-body components Mili (Supplementary Figure 3f) and Tdrd1 (Supplementary Figure 3g). Tdrkh was also found to overlap with cytoplasmic Miwi2 (Supplementary Figure 3h) and Mael (Supplementary Figure 3i) in piP-bodies, and occasionally co-localizes with the P-body markers Ge-1 (Supplementary Figure 3j) and Ddx6 (Supplementary Figure 3k). This suggests that Tdrkh can alternatively interact with both the Mili and Miwi2 protein complexes. Tdrkh is required for male meiosis To investigate the function of Tdrkh in spermatogenesis, we generated a Tdrkh-null allele by gene targeting (Figure 2A; Supplementary Figure 5a–d). Tdrkh−/− females generated litters of normal size and appearance upon crossing with C57 males, but Tdrkh−/−males are sterile, even though vaginal plugs are present in the mated females. Testes from 8-week-old homozygous nulls were ∼1/4 the size of control littermates (Figure 2B; Supplementary Figure 5e). Histological analysis revealed that spermatogenesis in tdrkh mutant animals was arrested in meiosis; no haploid spermatid was observed (Figure 2C). Figure 2.Tdrkh is required for male fertility. (A) Western blot analysis of tdrkh +/+, +/−, and −/− animal testis lysate. (B) Two-month-old testis from tdrkh +/− and −/− animals, ticks=0.1″. (C) H&E staining of 2-month-old +/− and −/− testis sections, scale bar=50 μm. (D) Quantitation of 14dpp spermatocyte spreads, N(+/−)=1821, N(−/−)=848. (E) Labelling of spermatocyte spreads from 14dpp +/− and −/− testis stained with Sycp1 and Sycp3. L, leptotene; Z, zygotene; P, pachytene, scale bar=10 μm. (F) Double labelling of 14dpp spermatocyte spreads with Rad51 (red) and Sycp3 (green). L, leptotene; Z, zygotene; Z/P, zygotene/pachytene; P, pachytene. Scale bar=10 μm. (G) Double labelling of 14dpp spermatocyte spreads with γH2A.X (red) and Sycp3 (green). L, leptotene; Z, zygotene; P, pachytene, scale bar=10 μm. (H) Staining of 14dpp germ cell spreads. Spermatogonia stained with anti-phospho-S25 53BP1, spermatocytes stained with total anti-53BP1, scale bar=50 μm. (I) Staining of 2-month-old CD-1 testis sections with anti-phospho-S25 53BP1 (green) and laminin (red), scale bar=75 μm. (J) TUNEL labelling (green) of 2-month-old +/− and −/− testis sections, scale bar=75 μm. (K) Western blot analysis of 11dpp Tdrkh +/− and −/− lysates. C, UV-irradiated CCE ES cell extract. Download figure Download PowerPoint To determine the sub-stage of meiotic arrest, we stained spermatocyte spreads from tdrkh+/− and tdrkh−/−littermates with Sycp3 and Sycp1 at 14dpp, a time point at which a substantial portion of spermatocytes have progressed to pachytene stage (36%, Figure 2D). No pachytene spermatocytes were observed in tdrkh−/−spreads (Figure 2C–E). Mutant spermatocytes showed major defects in forming synaptonemal complexes, as lateral elements marked by Sycp3 were severely fragmented in the zygotene stage and transverse elements marked by Sycp1 did not assemble properly (Figure 2E). Thus, the tdrkh−/− spermatocytes are arrested at the zygotene stage. Tdrkh deletion causes massive DNA damage without apoptosis We next performed staining with Rad51 to determine whether double-stranded break (DSB) foci had formed properly in tdrkh−/−mutants. In tdrkh+/− males, Rad51 foci were numerous in leptotene stage, coalescing onto Sycp3-labelled chromatin in zygotene stage, and become 2–3 sites/chromosome of crossover in pachytene stage (Figure 2F). In contrast, tdrkh−/− spreads ranged from a relatively normal appearance to widespread Rad51 staining along chromosomes. This was often continuous with, yet separate from, Sycp3-positive regions (Figure 2F). Staining for γH2A.X revealed further defects in mutant spermatocytes. γH2A.X foci appear in leptotene as DSBs form, and disappear by zygotene as the majority of DSBs resolve (Figure 2G; staining is retained on the XY body, however). Tdrkh−/− spermatocytes display extremely high levels of γH2A.X staining. Mutant leptotene spermatocytes initiate γH2A.X foci similarly to the controls, but these foci spread extensively and do not resolve in zygotene stage (Figure 2G). These data indicate defects in recognition of homologous pairing and a high level of DNA damage. Tdrkh mutants activate a robust DNA damage response. 53BP1 is a DNA damage checkpoint protein with sensation and transduction functions. In 14dpp tdrkh mutant spermatocyte spreads, we observed a re-distribution from a diffuse nuclear pattern to a distinct punctuate pattern indicative of 53BP1 activation in spermatogonia (Figure 2H, upper panels) and spermatocytes (Figure 2H, lower panels). In wild-type adult testes, 53BP1 expression largely mirrors γH2A.X, with nuclear-wide expression in leptotene, which dissipates in zygotene, and is restricted to the sex body in pachytene spermatocytes (Figure 2I). In contrast, tdrkh mutants have high and abnormal activation of 53BP1, indicating a widespread DNA damage response (Figure2I). We next asked whether this response led to apoptosis. Surprisingly, TUNEL staining in 11dpp and 2-month-old animals did not reveal apoptosis in the tdrkh mutant (Figure 2J). No activation of Caspase-3 or PARP cleavage was detected in the mutant, either (Figure 2K). This is highly surprising, as nearly all piRNA pathway mutants display robust TUNEL labelling (Kuramochi-Miyagawa et al, 2004; Carmell et al, 2007; Shoji et al, 2009; Frost et al, 2010; Watanabe et al, 2011a). Thus, Tdrkh may have additional downstream functions in response to DNA damage. Alternatively, spermatogenic arrest in Tdrkh mutants may be a result of a hyperactive chromosomal asynapsis checkpoint and not a result of DNA damage. Thirteen-month-old tdrkh mutant testes are highly atrophic and depleted of mature cells (Supplementary Figure 5f). Thus, abnormal cells are eliminated during ageing, likely through a necrotic process, and are not replaced as the animal ages. Interestingly, Tdrkh mutant spermatogonia were occasionally positive for the mitotic marker phospho-H3 (Supplementary Figure 5g). It is unclear if this indicates ongoing mitosis or arrest during mitosis. In sum, our data are consistent with overall arrest of mitosis and meiotic progression, resulting in the reduced size of tdrkh−/− testes. Tdrkh represses Line-1 retrotransposon expression One mechanism through which DNA damage could occur is by elevated transposon activity as in other piRNA pathway mutants (Siomi et al, 2011). To investigate if Tdrkh represses transposition, we first examined localization of pi- and piP-body markers in 18dpc testes of tdrkh+/− and tdrkh−/− littermates. Pi-body markers Mili and Mvh are localized similarly in tdrkh+/− and tdrkh−/− testes (Figure 3A). In contrast, Tdrd1 is lost from most pi-bodies in tdrkh−/− testes. The remaining pi-bodies contain weaker Tdrd1 staining. Thus, Tdrkh may recruit Tdrd1 to intact pi-bodies (Figure 3A). The piP-body components Miwi2 and Mael are in the nucleus and the cytoplasm, respectively (Figure 3A). In tdrkh−/− testes, however, Miwi2 becomes exclusively cytoplasmic and displays weak piP-body staining (Figure 3A). Mael remains diffusely cytoplasmic, with its signal in piP-bodies similarly weaker (Figure 3A). These results imply that Tdrkh is required for proper function of the Miwi2 in the nucleus and the piP-body. Electron microscopy on 11dpp tdrkh+/− and tdrkh−/− testes did not reveal ultrastructural changes in tdrkh−/− testes. In particular, IMC was still observed (Figure 3B), which indicates that Tdrkh does not contribute to the structural integrity of IMC. Figure 3.Tdrkh represses Line1 expression. (A) Localization of pi-body (Mili, Tdrd1), piP-body (Miwi2, Mael), and pan-nuage (Mvh) components in 18dpc tdrkh +/− and −/− testis sections, scale bar=10 μm. (B) Electron microscopy of 11dpp tdrkh +/− and −/− spermatocytes. Arrowheads indicate nuage/intermitochondrial cement. Scale bar=300 nm. (C) RT-qPCR analysis of transposon expression in three independent pairs of 12dpp testis for each genotype. Error bars indicate standard deviation. (D) Line1 Orf1p expression (green) in 18dpc, 7dpp, 11dpp, and adult tdrkh +/− and −/− testis sections. Scale bar=75 μm. Download figure Download PowerPoint We then assessed transposon RNA expression in tdrkh+/− and tdrkh−/− testes during development. Immunostaining revealed that the Line1 protein Orf1p is barely detectable in 18dpc tdrkh+/− gonocytes due to the establishment of DNA methylation (Aravin et al, 2009). In contrast, 18dpc tdrkh−/− gonads display drastic upregulation of Orf1p staining. At 7dpp, Orf1p expression decreases in tdrkh+/− testes, consistent with its RNA decrease as revealed by qPCR analysis (Supplementary Figure 6b). At 11dpp, immediately before the terminal arrest of mutant spermatocytes, Orf1p remained undetectable in tdrkh+/− testes but was drastically upregulated in the mutant testes and persists at high levels in the arrested tubules at 2 months (Figure 3D). Notably, only Line1 RNA but not other transposon RNAs is upregulated (Figure 3C; Supplementary Figure 6a). Thus, deletion of tdrkh results in robust Line1 expression during embryogenesis when DNA would be methylated and again during meiosis. Interestingly, we failed to observe cytoplasmic inclusions indicative of Line1 particle formation (for instance, Shoji et al, 2009) in our EM analysis of 11dpp tdrkh−/− animals (Figure 3B, data not shown), suggesting that mature particles may not form. Deletion of Tdrkh results in CpG hypomethylation at Line1 promoters To examine whether the activation of Line1 expression in tdrkh−/− testes is due to the loss of DNA methylation, we performed bisulphite sequencing to determine the methylation of transposonic DNA sequences. Line1 consist of five distinct subfamilies; two of them, L1MdA and L1MdGf, are capable of re-integration. DNA from tdrkh−/− postnatal spermatogonia shows severe defects in CpG methylation of L1MdA sequences and obvious defects in L1MdGf sequences (Supplementary Figure 7a). No change was observed in methylation of IAP sequences (Supplementary Figure 7b). Thus, DNA hypomethylation in tdrkh−/− testes is specific to Line1. Embryonic piRNA biogenesis is compromised in Tdrkh−/− mice DNA methylation of repetitive elements is thought to be guided in a sequence-specific manner by piRNAs (Siomi et al, 2011). The observed DNA demethylation phenotype indicates that Tdrkh may play an important role in piRNA biogenesis. To address this hypothesis, we first analysed global piRNA populations at 18dpc, when germ cell-specific DNA methylation is active and Tdrkh partially co-localizes with Mili and Miwi2 (Supplementary Figure 3f and h). Known mutations in piRNA pathway, such as tdrd1, tdrd9, and mael, result only in skewing of otherwise mostly normal piRNA populations (Aravin et al, 2009; Reuter et al, 2009; Shoji et al, 2009). However, deletion of tdrkh causes nearly complete elimination of global piRNA levels (Figure 4A; Supplementary Table 1). In all, 25–31 bp reads were reduced by ∼80% in the mutant library, indicating severe defects in piRNA biogenesis. Concordantly, unique piRNAs mapped to the top 19 embryonic piRNA-generating clusters were decreased by 85% compared to wild-type testes (Figure 4B). Figure 4.Tdrkh functions in primary piRNA biogenesis. (A) Size distribution of small RNA libraries from 18dpc tdrkh +/− and −/− testes, without normalization. (B) Ratio of 18dpc piRNA levels in Tdrkh mutant testes derived from the top 19 embryonic piRNA clusters, relative to tdrkh +/− levels. (C) Percentage of total library reads mapping to indicated categories in 18dpc libraries. (D) Percentage of repeat-associated reads mapping to indicated transposon family in control (upper) and mutant (lower) 18dpc libraries. (E) Ratio of sense and antisense 18dpc piRNAs mapping to specified transposons, relative to control levels. (F) Ratio of 18dpc piRNA reads mapping to indicated transposon families, relative to control levels. (G) Levels of piRNAs mapping to indicated mRNA region, expressed as a percentage of total reads in each genotype. (H) Distances between 5′ complementary ends of piRNA mapping to IAP consensus sequence were measured to calculate a ping-pong biogenesis signature. (A–H) 18dpc libraries. (I) Size distribution of small RNA libraries from 11dpp tdrkh +/− and −/− testes, without normalization. (J) Ratio of 11dpp piRNA levels in Tdrkh mutant testes derived from the top 19 post-natal pre-pachytene piRNA clusters, relative to control reads. (K) Percentage of total library reads mapping to indicated categories in 11dpp libraries. (L) Percentage of repeat-associated reads mapping to indicated transposon family in control (upper) and mutant (lower) 11dpp libraries. (M) Ratio of sense and antisense 11dpp piRNAs mapping to specified transposons, relative to control levels. (N) Ratio of 11dpp piRNA reads mapping to indicated transposon families, relative to control levels. (O) Levels of 11dpp piRNAs mapping to indicated mRNA region, expressed as a percentage of total reads in each genotype. (I–O) 11dpp libraries. Download figure Download PowerPoint We then focused on the repeat-associated 18dpc piRNA population. While the percentage of repeat-associated reads within the residual piRNA population was unchanged (Figure 4C), the majority of these reads were shifted to Line1-derived piRNAs in the tdrkh-mutant library (Figure 4D). Levels of sense and antisense piRNAs mapped to Line1 and IAP were proportionally decreased (Figure 4E). piRNAs mapped to the five most abundant classes of transposons were also reduced (Figure 4F; Supplementary Table 2). These data suggest that the residual piRNA population responds to increased transposon activity and that loss of transposon piRNAs is reflective of a global decrease in primary piRNA biogenesis. Further analysis of residual 18dpc piRNA populations revealed abrupt changes in mRNA-derived piRNAs. Reads of 5′ and 3′UTR-derived piRNAs were increased by ∼1.8-fold in tdrkh mutants. In contrast, reads of the small population of piRNAs from coding sequences were further reduced by 40% and intron-derived piRNAs decreased by 60% (Figure 4G). These results suggest a role for Tdrkh in processing of piRNAs from transcripts, possibly through regulation of Tdrd1 localization (Figure 3A). We also noted a 2.3-fold increase in the reads of 22-bp RNAs in mutant testes, which is highly consistent with the 2.4-fold increase in reads mapped to mature miRNAs (Figure 4A; Supplementary Table 1). A similar change was observed in 11dpp libraries even though no change in abundant miRNAs was observed by northern blotting (Supplementary Figure 8a). Hence, this increase is likely an artifact of library construction. Therefore, normalization to either total reads or miRNAs is inappropriate. As such, we did not perform normalization between libraries so that piRNA levels can be directly compared. Secondary piRNA biogenesis is intact in tdrkh mutant testes We next asked whether secondary piRNA biogenesis was intact in these mutants. Secondary piRNA biogenesis, a.k.a. the ping-pong cycle, generates new piRNAs from transposons bound and cleaved by Piwi prot
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