Abstract
The mouse PIWI-interacting RNA (piRNA) pathway provides sustained anti-transposon immunity to the developing male germline by directing transposon DNA methylation1,2,3. The first step in this process is the recruitment of SPOCD1 to young LINE1 loci4. Thereafter, piRNA-mediated tethering of the PIWI protein MIWI2 (also known as PIWIL4) to the nascent transposon transcript recruits the DNA methylation machinery5,6. The piRNA pathway needs to methylate all active transposon copies but how this is achieved remains unknown. Here we show that nuclear piRNA and de novo methylation factors are all euchromatic, exposing constitutive heterochromatin as a genomic blind spot for the piRNA pathway. We discover a ‘nowhere-to-hide’ mechanism that enables piRNA pathway-mediated LINE1 surveillance of the entire genome. We find that SPOCD1 directly interacts with the nuclear pore component TPR, which forms heterochromatin exclusion zones adjacent to nuclear pores7. In fetal gonocytes undergoing piRNA-directed DNA methylation, TPR is found both at the nuclear periphery and throughout the nucleoplasm. We find that the SPOCD1–TPR interaction is required for complete non-stochastic piRNA-directed LINE1 methylation. The loss of the SPOCD1–TPR interaction results in a fraction of SPOCD1 and other chromatin-bound piRNA factors relocalizing to constitutive heterochromatin where they are no longer accessible to MIWI2 and the de novo methylation machinery. In summary, the piRNA pathway has co-opted TPR to guarantee that LINE1s are accessible to the piRNA and de novo methylation machineries.
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Data availability
The EM-seq data generated in this study have been deposited on ArrayExpress under the accession number E-MTAB-14862. Data for the IP–MS experiments have been deposited at ProteomeXchange under the accession number PXD060850. The CL–MS data have been deposited under PXD060851. EM-seq data for Spocd1−/− and Miwi2−/− P14 spermatogonia were retrieved from E-MTAB-7997, whereas that for C19orf84−/− spermatogonia were retrieved from E-MTAB-11612. The following Uniprot accession IDs for SPOCD1 proteins were used for conservation analyses: mouse (M. musculus, B1ASB6), golden hamster (M. auratus, A0A3Q0D6B7), Ord’s kangaroo rat (D. ordii, A0A1S3FIT4), western European hedgehog (E. europaeus, A0A1S3WPZ3), rabbit (O. cuniculus, G1SPR0), aardvark (O. afer afer, A0A8B7AXN8), bison (B. bison bison, A0A6P3HA20), bovine (B. taurus, F1MG39), goat (C. hircus, A0A452FMH8), sheep (O.aries, W5NRM3), pig (S. scrofa, F1SV96), horse (E. caballus, F6YBJ1), alpaca (V. pacos, A0A6J3AYV9), California sealion (Z. californianus, A0A6J2C2W2), northern fur seal (C. ursinus, A0A3Q7MZA7), Atlantic bottle-nosed dolphin (T. truncatus, A0A6J3PXS9), sperm whale (P. macrocephalus, A0A455B8T1), blue whale (B. musculus, A0A8B8W162), great Himalayan leaf-nosed bat (H. armiger, A0A8B7SLV8), large flying fox (P. vampyrus, A0A6P3Q928), leopard (P. pardus, A0A6P4UE11), cat (F. catus, A0A5F5XDK8), red fox (V. vulpes, A0A3Q7T0D7), dog (C. lupus familiaris, A0A8P0P5S7), polar bear (U. maritimus, A0A8M1EZU5), greater bamboo lemur (P. simus, A0A8C8YEI0), giant panda (A. melanoleuca, G1MHH0), rhesus macaque (M. mulatta, F7G2T4), northern white-cheeked gibbon (N. leucogenys, G1QNN7), Sumatran orangutan (P. abelii, H2N866), gorilla (G. gorilla gorilla, G3RKR7), chimpanzee (P. troglodytes, H2R1B9), human (H. spaiens, Q6ZMY3), American alligator (A. mississippiensis, A0A151MMW3), Chinese alligator (A. sinensis, A0A1U8CWC7), snapping turtle (C. serpentina, A0A8T1SPX3), central bearded dragon (P. vitticeps, A0A6J0UYI0), mainland tiger snake (N. scutatus, A0A6J1UEZ9), Indian cobra (N. naja, A0A8C6YBU2), American chameleon (A. carolinensis, H9GI50), western clawed frog (X. tropicalis, A0A8J0T0T7) and African clawed frog (X. laevis, A0A1L8HFK1). Source data are provided with this paper.
Code availability
The scripts used for the EM-seq analysis are available on GitHub (https://github.com/tamchow/spocd1_pirna-directed-dna-met-variance/ (version of the record deposited on Zenodo54, https://doi.org/10.5281/zenodo.17162836) and https://github.com/rberrens/SPOCD1-piRNA_directed_DNA_met (version of the record deposited on Zenodo55, https://doi.org/10.5281/zenodo.10509247)). The Fiji/ImageJ2 plugin used to calculate Pearson’s correlation coefficients in germ cell nuclei is also available on GitHub (https://github.com/COIL-Edinburgh/ROI_NucleusColocalisation; a version of the record has been deposited on Zenodo41, https://doi.org/10.5281/zenodo.17200734).
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Acknowledgements
This research was supported by the Welcome Trust funding to D.O. (225237), A.G.C (200898), the Wellcome Centre for Cell Biology (203149) and the multi-user equipment grants (108504 and 092076). This work was supported by funding for the Wellcome Discovery Research Platform for Hidden Cell Biology (226791), and we acknowledge support from the Microscopy, Proteomics and Bioinformatics cores. T.C. is funded by the Darwin Trust of Edinburgh. A.Z. was funded by a German Research Foundation fellowship (DFG; award ZO 376/1-1). S.B. and W.A.B. are funded by MRC University Unit grant MC_UU_00035/7 and by a Wellcome Trust Investigator Award 217120/Z/19/Z. This work utilized the University of Edinburgh Protein Production Facility (EPPF), the Wellcome Centre for Cell Biology Centre Optical Instrumentation Laboratory/Light Microscopy Core, Proteomics and Bioinformatics Core Platforms as well as the Centre for Regenerative Medicine’s FACS facility. We also acknowledge the EMBL’s GeneCore facility for preparing the methyl-seq libraries and sequencing all libraries.
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Contributions
T.C. contributed to the design, execution and analysis of most experiments. S.B. performed the FISH experiments. A.Z. performed the IP–MS experiments. X.X. performed the bioinformatic analysis of the EM-seq data. M.D.M. and H.F. performed the initial biochemical experiments and prepared the mESCs expressing SPOCD1–HA. J.Z. performed the analysis of the CL–MS data. C.S. analysed the IP–MS data. D.K. developed the plugin to calculate the colocalization coefficients. D.O., A.G.C. and W.A.B. supervised the study. D.O. conceived the study. D.O. and T.C. wrote the final version of the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Nuclear piRNA factors and the de novo methylation machinery are euchromatic.
a, Representative E16.5 gonocyte, somatic cell and corresponding seminiferous cord stained for DAPI, HP1B (red) and H3K9me3 (green, top row) or HP1A (green, bottom row) in foetal testis sections from wild-type mice. Scale bars, 2 μm (top row), 10 μm (bottom row). White rectangles on images of seminiferous cords highlight cells shown zoomed-in. b-e, Representative E16.5 seminiferous cord stained for HA (green), HP1B (red, top row) or H3K4me3 (red, bottom row) and DAPI (blue) in foetal testis sections from Spocd1HA/+ (b), Miwi2HA/+ (c), Dnmt3lHA/HA (d) and Dnmt3cHA/HA (e) mice. f-g, Representative E16.5 seminiferous cord stained for C19ORF84 (f) or SPIN1 (g) (both green), HP1B (red, top row) or H3K4me3 (red, bottom row) and DAPI (blue) in foetal testis sections from wild-type mice. Also shown in b-g are the channel intensity profiles (RFU, relative fluorescence units) along the indicated line passing through major heterochromatin domains (for HP1B) or avoiding nucleoli (for H3K4me3) for the representative gonocytes presented in Fig. 1. Images shown in both rows of f are from the same quadruple stain (DAPI, C19ORF84, H3K4me3 and HP1B) of a seminiferous cord. Images shown in both rows of g are from another quadruple stain (DAPI, SPIN1, H3K4me3 and HP1B) of a seminiferous cord. Images in a through g are representative of n = 4 (b, c, e) or n = 3 (a, d, f, g) biological replicates; scale bars, 10 μm for seminiferous cords and 2 μm for single gonocytes. White rectangles highlight cells shown in Fig. 1 and in the profile plots.
Extended Data Fig. 2 piRNA pathway factors are euchromatic from initiation of expression.
a-c, Representative seminiferous cords stained for HA (SPOCD1-HA) (a), MIWI2 (b) and DNMT3L (c), H3K4me3 and DAPI from Spocd1HA/+ (a) or wild-type mice (b-c) at the indicated developmental stages. Shown to the right are boxplots of Pearson’s correlation coefficients of SPOCD1 (a), MIWI2 (b) and DNMT3L (c) with H3K4me3 in gonocyte nuclei at the indicated developmental stages. MIWI2 and DNMT3L are not expressed at E14.5, so co-localization was not calculated for this developmental stage. Scale bars, 10 μm. Images in a through c are representative of n = 3 biological replicates. Box indicates interquartile range, central line represents the mean, and whiskers extend to median ± 1.5× the interquartile range or data limits, whichever is smaller.
Extended Data Fig. 3 TPR is highly expressed in male foetal germ cells undergoing de novo genome methylation.
a, Volcano plot showing enrichment (log2(mean LFQ ratio of anti-HA immunoprecipitates from Miwi2HA/HA/wild-type E16.5 foetal testis lysates) and statistical confidence of proteins co-purifying with HA-MIWI2 (n = 4 with 50 testes per replicate, from previously published data19). b, Volcano plot showing enrichment (log2(mean LFQ ratio of anti-C19ORF84 immunoprecipitated/anti-rabbit serum immunoprecipitated from wild-type E16.5 foetal testis lysates) and statistical confidence of proteins co-purifying with C19ORF84 (n = 3 with 25 testes per replicate, from previously published data20). c, Single representative E16.5 germ and somatic cells stained for TPR (red) and DAPI (blue) in Spocd1HA/+ foetal testis sections. Scale bars, 2 μm. d, Representative E16.5 seminiferous cord stained for HA (green), TPR (red) and DAPI in foetal testis sections from Spocd1HA/+ mice. White rectangles highlight cells shown in Fig. 2f and Extended Data Fig. 3c. Scale bars, 10 μm. e, Representative gonocytes stained for TPR (red) and DAPI (blue) from wild-type mice at the indicated developmental stages. Scale bars, 2 μm. f, Representative seminiferous cords stained for TPR (red) and DAPI (blue) from wild-type mice at the indicated developmental stages. White rectangles highlight cells shown in Extended Data Fig. 3e. Scale bars, 10 μm. Images in c through f are representative of n = 3 biological replicates.
Extended Data Fig. 4 SPOCD1-K464A does not interact with TPR.
a, Multiple sequence alignment of the SPOCD1 TFIIS-M domains from the indicated species. Red rectangles highlight lysine residues making direct crosslinks with TPR-M in the CL-MS data. Sequence identity conservation is shown by depth of colour. b, Representative Coomassie-stained gel image of n = 3 MBP pull-down experiments with the indicated recombinant mouse SPOCD1 and human TPR fragments. For uncropped source gel image, see Supplementary Fig. 1d.
Extended Data Fig. 5 The Spocd1K464A mouse allele.
a, Schematic representations of the mouse Spocd1 locus and encoded 1015 amino acid protein are shown, along with a schematic of the CRISPR targeting strategy showing the location of single-stranded oligo DNA donor (ssODN) and homology arms (HA) used. The sgRNA used for generation of the Spocd1K464A allele (red), adjacent PAM sites (red) and the primers used for genotyping (blue) are indicated, along with a representative sequencing trace of Spocd1K464A exon 7 harbouring the 3 bp mutation encoding the K464A mutation (highlighted in red). Sequencing was performed on n = 3 F1 animals. b, Representative image of PCR genotyping result for Spocd1+/+, Spocd1+/K464A and Spocd1K464A mice. PCR genotyping was performed for over 600 mice with similar results. c, Volcano plot showing enrichment (LFQ ratio of anti-SPOCD1 immunoprecipitates from wild-type/Spocd1K464A E16.5 foetal testis lysates) and statistical confidence (−log10(P-value of two-sided Student’s t-test)) of proteins co-purifying with wild-type SPOCD1 (right quadrant) or SPOCD1-K464A (left quadrant) (n = 3 with 24 foetal testes per replicate per genotype). All identified proteins meeting the enrichment cut-off are listed in Supplementary Table 2. d, Representative adult testis sections of n = 3 wild type, Spocd1K464A and Spocd1−/− stained in blue for DAPI and red for the DNA damage marker γH2AX (top) or apoptotic cells by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay (bottom). Scale bars, 50 μm.
Extended Data Fig. 6 The SPOCD1-TPR interaction ensures that SPOCD1-SPIN1-C19ORF84 avoids heterochromatin.
a-c, e-g, Representative E16.5 seminiferous cord stained for DAPI, HP1B (red) and SPOCD1 (a), SPIN1 (b), C19ORF84 (c), MIWI2 (e), DNMT3L (f), or HA (HA-DNMT3C) (g) (all green) stains in wild-type (top row) or Spocd1K464A (bottom row) foetal testis sections from littermates. Images are representative of n = 8 (a), n = 4 (c) or n = 3 (b, d-f) biological replicates of each genotype. White rectangles highlight cells shown zoomed-in in Fig. 4a–f. d, Representative E16.5 gonocyte (top two rows) and seminiferous cords (bottom two rows) stained for DAPI (blue), H3K4me3 (red or green), SPOCD1 (green) and HP1B (red) stains in wild-type (first and third row) or Spocd1K464A (second and last row) foetal testis sections from littermates. Images are representative of n = 4 biological replicates of each genotype. White rectangles in the last two rows highlight cells shown in the first two rows. Also shown below are channel intensity profiles (RFU, relative fluorescence units) along the indicated line passing through major heterochromatic domains in the representative gonocyte. Shown to the right are boxplots of Pearson’s correlation coefficients of SPOCD1 with H3K4me3 in gonocyte nuclei of the indicated genotypes. Box indicates interquartile range, central line represents the mean, and whiskers extend to median ± 1.5× the interquartile range or data limits, whichever is smaller. P-values are from unpaired, two-tailed Student’s t-tests. h, Representative E16.5 seminiferous cord stained for DAPI, SPOCD1 and LINE1 DNA in foetal testis sections of the indicated genotypes. Images are representative of n = 6 biological replicates of each genotype. White rectangles highlight cells shown in Fig. 4g. a-h, Scale bars, 10 μm for seminiferous cords and 2 μm for single gonocytes.
Extended Data Fig. 7 The SPOCD1-TPR interaction is necessary for Rasgrf1 methylation.
a, Heatmap presentation of mean CpG methylation of imprinted loci in P14 undifferentiated spermatogonia of the indicated genotypes. The imprint control region (ICR) of Rasgrf1 is shown in detail on the bottom. b, Cartoon representation of piRNA-directed epigenetic silencing of LINE1 elements in mice. TPR ensures SPOCD1-targeted LINE1 loci are present in euchromatin and accessible to the piRNA-directed DNA methylation machinery.
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Identified cross-links between SPOCD1-TFIISM and TPR-M. Table listing all Xi-validated cross-linked peptides detected in and between SPOCD1-TFIISM and TPR-M for n=2 cross-linking reactions (analysed together), relevant to Fig. 2e.
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Chowdhury, T., Boyle, S., Zoch, A. et al. A nowhere-to-hide mechanism ensures complete piRNA-directed DNA methylation. Nature 650, 779–785 (2026). https://doi.org/10.1038/s41586-025-09940-w
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DOI: https://doi.org/10.1038/s41586-025-09940-w
