SPOCD1 is an essential executor of piRNA-directed de novo DNA methylation

Abstract

In mammals, the acquisition of the germline from the soma provides the germline with an essential challenge: the need to erase and reset genomic methylation1. In the male germline, RNA-directed DNA methylation silences young, active transposable elements2,3,4. The PIWI protein MIWI2 (PIWIL4) and its associated PIWI-interacting RNAs (piRNAs) instruct DNA methylation of transposable elements3,5. piRNAs are proposed to tether MIWI2 to nascent transposable element transcripts; however, the mechanism by which MIWI2 directs the de novo methylation of transposable elements is poorly understood, although central to the immortality of the germline. Here we define the interactome of MIWI2 in mouse fetal gonocytes undergoing de novo genome methylation and identify a previously unknown MIWI2-associated factor, SPOCD1, that is essential for the methylation and silencing of young transposable elements. The loss of Spocd1 in mice results in male-specific infertility but does not affect either piRNA biogenesis or the localization of MIWI2 to the nucleus. SPOCD1 is a nuclear protein whose expression is restricted to the period of de novo genome methylation. It co-purifies in vivo with DNMT3L and DNMT3A, components of the de novo methylation machinery, as well as with constituents of the NURD and BAF chromatin remodelling complexes. We propose a model whereby tethering of MIWI2 to a nascent transposable element transcript recruits repressive chromatin remodelling activities and the de novo methylation apparatus through SPOCD1. In summary, we have identified a previously unrecognized and essential executor of mammalian piRNA-directed DNA methylation.

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Fig. 1: Definition of the MIWI2 interactome and identification of SPOCD1 from gonocytes undergoing de novo genome methylation.
Fig. 2: SPOCD1 is required for spermatogenesis and LINE1/IAP silencing.
Fig. 3: SPOCD1 is required for de novo DNA methylation of transposable element loci but not piRNA expression.
Fig. 4: SPOCD1 is a nuclear protein that associates with the de novo DNA methylation machinery and repressive chromatin remodelling complexes.

Data availability

All mRNA expression data that support the findings of this study have been deposited at Array Express under accession number E-MTAB-7985. The Methyl-seq data generated in this study have been deposited at ArrayExpress under the accession number E-MTAB-7997. The sRNA-seq and RNA-seq data generated in this study have been deposited at the Gene Expression Omnibus under the accession number GSE131377. Data for the IP-MS experiments were deposited at ProteomeXchange under the accession number PXD016701Source data are provided with this paper.

Code availability

Scripts used for the Methyl-seq, RNA-seq and sRNA-seq analysis are available on github (https://github.com/rberrens/SPOCD1-piRNA_directed_DNA_met).

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Acknowledgements

This research was supported by Welcome Trust funding to D. O'C. (106144), R. V. B. (213612), A. G. C. (200898), J. R. (103139), R. C. A. (095021, 200885) and the Wellcome Centre for Cell Biology (203149) and a multi-user equipment grant (108504). D. O'C.’s laboratory is also supported by the European Union H2020 program grant GermAge. A. Z. was funded by a German Research Foundation fellowship (DFG, award ZO 376/1-1). We acknowledge the EMBL GeneCore facility in Heidelberg, Germany for preparing the microarray data set and RNA-seq data set of E16.5 gonocytes and sequencing all next-generation sequencing libraries, and specifically F. Jung at GeneCore for preparing the Methyl-seq libraries.

Author information

Affiliations

Authors

Contributions

A. Z. contributed to the design, execution and analysis of most experiments. T. A. helped established IP conditions and performed mass spectrometry analysis under the guidance of J. R. and R. C. A. R. B. V. and Y. K. performed bioinformatic analysis of Methyl-seq data or sRNA-seq as well as RNA-seq data, respectively. T. S. prepared sRNA-seq libraries and, with Y. K., RNA-seq libraries of P20 testes. M. H and A. G. C. performed homology alignment of the SPOC and TFIIS-M domains. L. V. performed immunofluorescence staining of HA-MIWI2 and generated the gonocyte microarray dataset. Y. A. P-R. performed phylogenetic analysis under guidance of A. S. D. O’C. conceived and supervised this study. D. O’C. and A. Z. wrote the final version of the manuscript.

Corresponding author

Correspondence to Dónal O’Carroll.

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The authors declare no competing interests.

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Peer review information Nature thanks Déborah Bourc’his, Falk Butter, Matthew Lorincz and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Expression pattern and presence of nuclear localization signals for novel MIWI2 interactors.

a, b, Relative expression of indicated transcripts as measured by Affymetrix microarray in E16.5 gonocytes (n = 2), adult spermatogonia (n = 3), spermatocytes (n = 3), MEFs (n = 3) and bone marrow (n = 2). Data are mean and s.e.m. NLS indicates presence of a nuclear localization signal as predicted by cNLS mapper. Source data

Extended Data Fig. 2 Homology alignment of SPOCD1 SPOC and TFIIS-M domains.

a, Multiple sequence alignment of the SPOC domain from SPOCD1 with representative vertebrate sequences from PHF3, DIDO1 and SPEN orthologues. The numbering for mouse SPOCD1 is shown above. Secondary structure elements for the human SHARP SPOC domain (PDBid 1OW1; SHARP is the human SPEN orthologue) are shown below the sequence, with dark rectangles for alpha helices and lighter arrows for beta strands. b, Multiple sequence alignment of TFIIS-M domain of SPOCD1 with equivalent sequences from TFIIS, PHF3 and DIDO1. Secondary structure elements from human PHF3 (PDBid 2DME) and human TFIIS (PDBid 3NDQ) are shown below, using the same annotation as in a. Sequences are coloured according to sequence identity.

Extended Data Fig. 3 Phylogeny of SPOCD1.

Bayesian phylogeny of Spocd1 (blue) and its vertebrate paralogs Phf3 (red) and Dido1 (green) inferred from cDNA sequences. Posterior probabilities of splits are shown as node labels. Branch lengths measure the expected substitutions per site as indicated in the scale bar.

Extended Data Fig. 4 Generation and characterization of the Spocd1null mouse allele.

a, Schematic representation of the Spocd1 locus and the encoded 1015 amino acids (aa) protein (transcript XM_017320505.1) as well as design of the sgRNA targeting Spocd1 exon 7, which harbours part of the TFIIS-M domain. b, Schematic representation and sequencing trace of the part of Spocd1 exon 7 harbouring the mutation site. The mutated site, highlighted in red, contains two premature stop codons and causes a frameshift. Sequencing was repeated with identical results on n = 3 animals. c, Representative image of genotyping results for Spocd1+/+, Spocd1+/− and Spocd1−/− animals. Similar results were obtained for all animals of the Spocd1 line. d, Numbers of E16.5 embryos per plug from matings of mice with the indicated Spocd1 genotypes. Mean and s.e.m. from n = 7 Spocd1+/+ dams mated to n = 3 Spocd1+/+ studs and n = 12 Spocd1−/− dams mated to n = 5 Spocd1+/− studs is plotted. NS, nonsignificant difference (P 0.98), two-tailed Student’s t-test. e, Representative PAS and haematoxylin stained histological testis sections of different stages of the seminiferous cycle are shown of (n = 3) Spocd1+/+ and Spocd1−/− animals, indicating a germ cell differentiation arrest at the early pachytene stage. Scale bar, 5 μm. eP, early pachytene; RS, round spermatids, eS(13), elongating spermatids (step 13); PL, pre-leptotene; P, pachytene; L, leptotene; Z, zygotene; m2, secondary meiocytes. f, Representative images of zygotene spermatocytes in wild-type and Spocd1−/− adult testis sections stained for the synaptonemal complex proteins SCP1 (red) and SCP3 (green). DNA stained with DAPI (blue). Scale bar, 1 μm. The representative images presented in panels e and f are from n = 3 mice per genotype. g, h, Analysis of transposable element expression in P20 testes from n = 3 wild-type, Spocd1−/− and Miwi2−/− mice by RNA-seq. g, Comparison of transposable element expression in Miwi2−/− and wild-type testes is shown. Transposable elements with a significantly different (P < 0.01, BenjaminiHochberg-adjusted two-sided Wald’s test) change in expression (>2-fold) are highlighted in red and the top 12 most upregulated transposable elements in Miwi2−/− testes are labelled. h, Comparison of transposable element expression in Spocd1−/− and wild-type testes is shown. Transposable elements with a significantly different (P < 0.01, BenjaminiHochberg-adjusted two-sided Wald’s test) change in expression (>2-fold) are highlighted in red and the same transposable elements as in a are labelled. i, Comparison of transposable element expression in Spocd1−/− and Miwi2−/− testes is shown. Transposable elements with a significantly different (P < 0.01, BenjaminiHochberg-adjusted two-sided Wald’s test) change in expression (>2-fold) are highlighted in red. Transposable elements that are significantly upregulated in Miwi2−/− relative to wild type are highlighted in black. Source data

Extended Data Fig. 5 CpG methylation analysis of different genomic features and transposable element families.

Analysis of genomic CpG methylation of undifferentiated P14 spermatogonia from (n = 3) wild-type, Spocd1−/− and Miwi2−/− mice is presented. a, b, Scatter plots comparing CpG methylation levels for the respective genomic features between wild-type and Spocd1−/− or Miwi2−/− (a) and between Spocd1−/− or Miwi2−/− spermatogonia (b). c, d, Scatter plots comparing CpG methylation levels for the respective transposable element families between wild-type and Spocd1−/− or Miwi2−/− (c) and between Spocd1−/− or Miwi2−/− spermatogonia (d.) Data are mean from n = 3 biological replicates per genotype and shown as individual data points (grey) overlaid by a density map. Source data

Extended Data Fig. 6 Methylation analysis of transposable element families.

Analysis of genomic CpG methylation of undifferentiated P14 spermatogonia from (n = 3) wild-type, Spocd1−/− and Miwi2−/− mice. a, Metaplots of CpG methylation over L1Md_A, IAPEy and MMERVK10C elements and adjacent 2 kb. Below, schematic representation of the element. b, Metaplots of mean CpG methylation over LINE1 elements and adjacent 1 kb. The Methyl-seq data sets of P14 wild-type, Miwi2−/− and Spocd1−/− spermatogonia are compared to WGBS data sets of adult Mili−/− spermatocytes (Molaro et al. 20144) and P10 Dnmt3c+/−, Dnmt3c−/−, Dnmt3l+/− and Dnmt3l−/− germ cells (Barau et al. 201612). Below, schematic representation of LINE1. c, Correlation analysis of mean CpG methylation loss relative to wild type over individual elements of the indicated transposable element family in relation to their divergence from the consensus sequence for Miwi2−/− and Spocd1−/− spermatogonia. Source data

Extended Data Fig. 7 piRNA analysis.

piRNA analyses of small RNAs sequenced from E16.5 testis from (n = 3) Spocd1+/− and Spocd1−/− mice are presented. a, Relative frequency of piRNAs mapping to LINE1 and IAP families from Spocd1+/− and Spocd1−/− E16.5 testes. Plots are shown for all piRNA or antisense piRNAs. Data are mean and s.e.m. Adjusted P-values are listed; P  1.0 values are denoted as NS (Bonferroni-adjusted two-sided Student’s t-test). b, Scatter plots showing mean expression of all (n = 124,411) piRNAs. The identity line is shown in red. r, Pearson’s correlation coefficient. c, Nucleotide features of piRNA from Spocd1+/− and Spocd1−/− E16.5 testes. Frequency of mapped piRNAs with a U at position 1 (1U) and with an A at position 10 (10A) are shown for L1Md_T elements. Data represent the mean and s.e.m. Adjusted P-values are shown (Bonferroni-adjusted two-sided Student’s t-test) d, Ping-pong analysis of piRNAs from Spocd1+/− and Spocd1−/− E16.5 testis. Relative frequencies of the distances between 5′ ends of complementary piRNAs are shown for the indicated LINE1 and IAP families. e, Nucleotide features of piRNA from Spocd1+/− and Spocd1−/− E16.5 testis. Relative frequencies of piRNAs with a U at position 1 (1U) and with an A at position 10 (10A) are shown for respective elements shown in (d). Data are mean and s.e.m. Adjusted P-values are listed; P  1.0 values are denoted as NS (Bonferroni-adjusted two-sided Student’s t-test) f, Positions of piRNAs mapped to the consensus sequence of L1Md_T. Positive and negative values indicate sense and antisense piRNAs, respectively. Schematic representation of L1Md_T is shown above. Source data

Extended Data Fig. 8 Transposable element and gene expression in Spocd1−/− gonocytes.

Analysis of transposable element and gene expression in E16.5 Spocd1+/− and Spocd1−/− gonocytes by RNA-seq from n = 3 mice per genotype. a, Comparison of transposable element expression in Spocd1+/− and Spocd1−/− gonocytes is shown. Transposable elements upregulated in Miwi2−/− testes at P20 are highlighted in black. b, Comparison of gene expression in Spocd1+/− and Spocd1−/− gonocytes. Significantly expressed genes (P < 0.01, Benjamini–Hochberg-adjusted two-sided Wald’s test, >2-fold change) are highlighted in red. Source data

Extended Data Fig. 9 Generation of the Spocd1HA mouse allele.

a, Schematic representation of the SPOCD1 protein and Spocd1 locus as well as design of the sgRNA targeting the 3′ UTR near the translation termination site on Spocd1 exon 15. The Spocd1HA allele encodes for a carboxy-terminal GGGGS linker followed by the HA epitope tag. The protospacer adjacent motif (PAM) site was mutated to inhibit re-targeting of the Spocd1HA allele by the sgRNA-CAS9 complex. All inserted nucleotides and corresponding encoded amino acids are highlighted in red. The SPOCD1-HA protein is shown as a schematic representation. b, Schematic representation of the targeting strategy to generate the Spocd1HA allele with a short single-stranded DNA oligo donor (ssODN) of 200 nucleotides containing 5′ and 3′ homology arms (5′HA and 3′HA) of 72 nucleotides. c, Representative image of genotyping results for Spocd1+/+, Spocd1HA/+ and Spocd1HA/HA animals. Similar results were obtained for all animals of the Spocd1HA line. d, Sequencing trace of part of a PCR amplicon of the HA epitope tag insertion site from a Spocd1HA/HA animal. The experiment was repeated with identical results on n = 2 animals. e, f, g, Representative images of wild-type (e), Spocd1HA/+ (f) and Miwi2HA/+ (g) testis sections at the indicated developmental time point probed with anti-HA antibody in green. DNA stained with DAPI in blue. Scale bars, 10 μm. The representative images presented in eg are from experiments done in n = 3 mice as biological replicates with similar results.

Extended Data Fig. 10 Co-immunoprecipitation experiments of SPOCD1 and DNMT3A/L/C in HEK cells.

Western blot analysis of co-immunoprecipitation of SPOCD1-HA with DNMT3L-FLAG, DNMT3A-FLAG, DNMT3C-FLAG or GFP in HEK cells. Shown are lysate sample (L), control IP (protein G beads) (B) and anti-HA IP (IP) for 4 experiments. For uncropped source data, see Supplementary Fig. 1.

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Supplementary Information

Supplementary information containing uncropped scans of Western blot experiments shown in Extended Data Figure 10 (Supplementary Figure 1), the FACS gating strategy for sorting undifferentiated spermatogonia (Supplementary Figure 2) and seven additional data tables (Supplementary Table 1-7) relating to IPMS data, RNA-seq data, and the Methyl-seq datasets.

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Zoch, A., Auchynnikava, T., Berrens, R.V. et al. SPOCD1 is an essential executor of piRNA-directed de novo DNA methylation. Nature 584, 635–639 (2020). https://doi.org/10.1038/s41586-020-2557-5

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