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piRNA gene density and SUMOylation organize piRNA transcriptional condensate formation

Nature Structural & Molecular Biology (2025)Cite this article

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Abstract

Piwi-interacting RNAs (piRNAs) are essential for maintaining genome integrity and fertility in various organisms. In flies and nematodes, piRNA genes are encoded in heterochromatinized genomic clusters. The molecular mechanisms of piRNA transcription remain intriguing. Through small RNA sequencing and chromatin editing, we discovered that spatial aggregation of piRNA genes enhances their transcription in nematodes. The facultative heterochromatinized piRNA genome recruits the piRNA upstream sequence transcription complex (USTC; including PRDE-1, SNPC4, TOFU-4 and TOFU-5) and the H3K27me3 reader UAD-2, which phase-separate into droplets to initiate piRNA transcription. We searched for factors that regulate piRNA transcription and isolated the SUMO E3 ligase GEI-17 as inhibiting and the SUMO protease TOFU-3 as promoting piRNA transcription foci formation, thereby regulating piRNA production. Our study revealed that spatial aggregation of piRNA genes, phase separation and deSUMOylation may benefit the organization of functional biomolecular condensates to direct piRNA transcription in the facultative heterochromatinized genome.

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Fig. 1: Clustered piRNA genes promote piRNA expression in C. elegans.
Fig. 2: Chromosomal editing alters piRNA clustering and expression.
Fig. 3: UAD-2 forms a liquid droplet-like condensate at piRNA cluster loci.
Fig. 4: IDRs of UAD-2 have a key role in condensate formation.
Fig. 5: Forward genetic screening identifies GEI-17 suppressing piRNA expression.
Fig. 6: TOFU-3 is required for UAD-2 condensate formation and piRNA production.
Fig. 7: SUMOylation suppresses cluster I piRNA expression.
Fig. 8: A working model for piRNA transcription in C. elegans.

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Data availability

The raw sequence data reported in this paper were deposited to the Genome Sequence Archive in National Genomics Data Center, China National Center for Bioinformation and Beijing Institute of Genomics, CAS under accession numbers CRA012796 and CRA018177. Source data are provided with this paper.

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Acknowledgements

We are grateful to the members of the S.G. lab for their comments. We are grateful to the International C. elegans Gene Knockout Consortium and the National Bioresource Project for providing the strains. Some strains were provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440). This work was supported by grants from the National Natural Science Foundation of China (32230016 to S.G., 32270583 to C.Z., 32070619 to X.F., 2023M733425 and 32300438 to X.H. and 32090044 and 31971128 to S.X.) and the National Key R&D Program of China (2022YFA1302700 to S.G. and 2019YFA0508403 to S.X.). This work was supported by the Research Funds of Center for Advanced Interdisciplinary Science and Biomedicine of IHM (QYPY20230021 to S.G.). This study was supported in part by the Fundamental Research Funds for the Central Universities. This work was also supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (CAS; XDB 37040202 to S.X.) and the CAS Project for Young Scientists in Basic Research (YSBR-068 to S.X.).

Author information

Author notes

  1. These authors contributed equally: Chengming Zhu, Xiaoyue Si.

Authors and Affiliations

  1. Department of Obstetrics and Gynecology, The First Affiliated Hospital of USTC, The USTC RNA Institute, Ministry of Education Key Laboratory for Membraneless Organelles & Cellular Dynamics, Hefei National Research Center for Physical Sciences at the Microscale, Center for Advanced Interdisciplinary Science and Biomedicine of IHM, School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China

    Chengming Zhu, Xiaoyue Si, Xinhao Hou, Panpan Xu, Jianing Gao, Yao Tang, Chenchun Weng, Mingjing Xu, Qi Yan, Qile Jin, Jiewei Cheng, Ke Ruan, Ying Zhou, Ge Shan, Demin Xu, Xiangyang Chen, Shengqi Xiang, Xinya Huang & Shouhong Guang

  2. School of Life Sciences, Anhui Medical University, Hefei, China

    Chengming Zhu

  3. School of Basic Medical Sciences, Anhui Medical University, Hefei, China

    Xuezhu Feng

  4. CAS Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Hefei, China

    Shouhong Guang

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Contributions

S.X., X. Huang, X.F. and S.G. conceptualized the research. C.Z., X. Huang, X.F. and S.G. designed the research. C.Z., X.S., P.X., Y.T., Q.J., K.R., Y.Z., G.S., D.X., X.C. and X. Huang performed the research. C.W., M.X., Q.Y. and J.C. contributed new reagents. C.Z., X. Hou and J.G. contributed analytic tools and performed the bioinformatics analysis. C.Z., X.F. and S.G. wrote the paper.

Corresponding authors

Correspondence to Shengqi Xiang, Xinya Huang, Xuezhu Feng or Shouhong Guang.

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Nature Structural & Molecular Biology thanks Wen Tang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Sara Osman, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 UAD-2 and USTC form distinct foci corresponding to piRNA Cluster II during spermatogenesis in hermaphrodites and males.

a, Genome-wide distribution of piRNA genes (n = 15365) in C. elegans. b, Diagram illustrating LacI::tagRFP driven by the rps-2 promoter and unc-54 UTR inserted at the LG IV ttti5605 site. c. Diagram illustrating 256x LacO inserted into piRNA cluster II (LG IV position: 15,809,942, contributed by the Christian Frøkjær-Jensen lab). d. Images showing the subcellular localization of UAD-2::GFP and LacI::tagRFP in the nuclei of pachytene germ cells from wild-type animals. All images were taken by the Leica THUNDER imaging System and deconvoluted using Leica Application Suite X software (version 3.7.4.23463). All images are representative of more than three animals (see also in Extended Data Fig. 1e-o). e. Images showing the subcellular localization of TOFU-4::GFP and LacI::tagRFP in the nuclei of pachytene germ cells from wild-type animals. f. Images showing the subcellular localization of UAD-2::GFP and LacI::tagRFP in the nuclei of pachytene germ cells from animals with 256xLacO insertions. g. Images showing the subcellular localization of TOFU-4::GFP and LacI::tagRFP in the nuclei of pachytene germ cells from animals with 256xLacO insertions. h-k. Images displaying the subcellular localization of UAD-2 and USTC components alongside the chromatin marker mCherry::H2B in the spermiogenesis region of the germline in L4 stage hermaphrodite animals. l-o. Images displaying the subcellular localization of UAD-2 and USTC components alongside the chromatin marker mCherry::H2B of the germline in adult male animals.

Extended Data Fig. 2 ChIP-seq profiles of UAD-2 and USTC in C. elegans; piRNA gene localization correlates with expression in C. briggsae.

a. Heatmap of ChIP-seq binding profiles of UAD-2 around type I and type II piRNA transcription start sites (TSSs). b. Heatmap of ChIP-seq binding profiles of TOFU-4 around type I and type II piRNA transcription start sites (TSSs). c. Heatmap of ChIP-seq binding profiles of TOFU-5 around type I and type II piRNA transcription start sites (TSSs). d, A flowchart for UMI piRNA sequencing. e, Genome-wide distribution of piRNA genes (n = 25883) in C. briggsae. f, Boxplots presenting log2(mean of piRNA reads from two biological replicates) for Cbr-PRG-1-associated piRNAs across Cluster 1, Cluster 2, Cluster 3, and out-Cluster in C. briggsae. The box itself represented the interquartile range between 5% and 95%, thereby encompassing the middle 90% of the data set. The central horizontal line and the adjacent numeric value within the box represent the mean value. The star within the box represents the median value. Error bars represent the mean ± 1.5 standard deviations (SD), indicating the variability of the data around the mean. Outliers are individually marked as points, representing data points that fall outside the range of mean±1.5 SD. Statistical significance was determined using a two-sample t test. g, Graphs presenting the fractional distribution of piRNA genes and their corresponding expression reads (normalized as reads per million from two biological replicates) across Cluster 1, Cluster 2, Cluster 3, and out-Cluster in C. briggsae. Source data are provided as a Source Data file.

Source data

Extended Data Fig. 3 CRISPR/Cas9-mediated chromosome inversion and translocation alter piRNA expression.

a, Diagram showing sequence details of intrachromosomal inversion in LG IV. b, Graphs presenting the expression levels of total piRNAs from Cluster I of the indicated animals, based on average reads from two biological replicates, normalized as reads per million+1. c-e, Scatter plots displaying the expression levels of Cluster I, Cluster II, and out-Cluster piRNAs in the wild-type (x-axis) and inversion strain (y-axis), based on the log2(average piRNA reads of two biological replicates), normalized as reads per million+1. f, Boxplot revealing log2(average piRNA reads of two biological replicates) for both the wild-type and inversion strains across Cluster I, Cluster II, and out-Cluster loci. Normalized as reads per million+1. The box itself represented the interquartile range between 5% and 95%, thereby encompassing the middle 90% of the data set. The central horizontal line and the adjacent numeric value within the box represent the mean value. The star within the box represents the median value. Error bars represent the mean ± 1.5 standard deviations (SD), indicating the variability of the data around the mean. Outliers are individually marked as points, representing data points that fall outside the range of mean±1.5 SD. Statistical significance was determined using a paired-sample t test.

Extended Data Fig. 4 UAD-2 and the USTC complex form liquid droplet-like condensates at piRNA cluster loci.

a, The upper panel shows the localization of UAD-2::GFP and mCherry::H2B in pachytene cells. The lower panel shows the localization of TOFU-5::GFP and mCherry::H2B in pachytene cells. All images were taken by the Leica THUNDER imaging System and deconvoluted using Leica Application Suite X software (version 3.7.4.23463). All images are representative of more than three animals (see also in Extended Data Fig. 4c-f). b, Statistical analysis of the focus area for UAD-2::GFP and TOFU-5::GFP in pachytene cells. Quantification of size data from n = 3 independent animals, error bars represent the mean ± 1.5 standard deviations (SD), indicating the variability of the data around the mean. Source data are provided as a Source Data file. c-f, The images reflect the subcellular localization of the mentioned proteins in pachytene cells under the indicated culture conditions.

Source data

Extended Data Fig. 5 UAD-2 is required for PDRE-1 foci formation and piRNA expression.

a and b. Images displaying the subcellular localization of PRDE-1::GFP and the chromatin marker mCherry::H2B in the germline from wild-type animals and uad-2(ust200) mutants. c. Scatter plots displaying the expression levels of Cluster I, Cluster II, and out-Cluster piRNAs in the wild type (x-axis) and uad-2(ust200) mutants (y-axis), based on the log2(reads per million+1) from a single biological replicate. d. Boxplots revealing total piRNA reads per million of the indicated adult animals from a single biological replicate. e. Boxplots revealing log2(piRNA reads per million+1) of the indicated adult animals form a single biological replicate. The box itself represented the interquartile range between 5% and 95%, thereby encompassing the middle 90% of the data set. The central horizontal line and the adjacent numeric value within the box represent the mean value. Error bars represent the mean ± 1.5 standard deviations (SD), indicating the variability of the data around the mean. Outliers are individually marked as points, representing data points that fall outside the range of mean±1.5 SD. Statistical significance was determined using a paired-sample t test.

Extended Data Fig. 6 Predicted UAD-2 IDRs are important for PRDE-1 foci formation and piRNA expression.

a, IUPred2 prediction of UAD-2 and the USTC components using the IUPred2 long disorder option. b, qRT-PCR analysis of UAD-2 mRNA in wild-type animals and uad-2 deletion mutants. The levels were normalized to eft-3. Quantification of qRT-PCR data from n = 3 independent animals, presented as Mean ± SD. Statistical significance was determined using a paired-sample t test. Source data are provided as a Source Data file. c. Images showing the subcellular localization of the indicated UAD-2::GFP variants and mCherry::PRDE-1 in pachytene cells. All images were taken by the Leica THUNDER imaging System and deconvoluted using Leica Application Suite X software (version 3.7.4.23463). All images are representative of more than three animals (see also in Extended Data Fig. 6f). d, Graphs presenting the relative fluorescence unit intensity (RFU) of mCherry::PRDE-1 for wild-type animals and the indicated UAD-2 variants. The fluorescence intensity threshold is set to 8–255. Three worms were analyzed for each strain. Statistical significance was determined using a two-sample t test. Source data are provided as a Source Data file. e. Graphs presenting the relative fluorescence unit intensity (RFU) of wild-type animals and the indicated UAD-2 variants, marked by the dashed line. f. Images showing the subcellular localization of the indicated UAD-2::GFP variants and TOFU-4:mCherry in pachytene cells. g-j. Scatter plots displaying the second biological replicate expression levels of Cluster I, Cluster II, and out-Cluster piRNAs in the wild-type (x-axis) and indicated mutants (y-axis), based on the log2(reads per million+1). k. Boxplots revealing log2(piRNA reads per million+1) of the indicated adult animals of the second biological replicate. The box itself represented the interquartile range between 5% and 95%, thereby encompassing the middle 90% of the data set. The central horizontal line and the adjacent numeric value within the box represent the mean value. Error bars represent the mean ± 1.5 standard deviations (SD), indicating the variability of the data around the mean. Outliers are individually marked as points, representing data points that fall outside the range of mean±1.5 SD. Statistical significance was determined using a paired-sample t test.

Source data

Extended Data Fig. 7 Forward genetic screening identifies factors suppressing UAD-2 foci formation.

a, Forward genetic screening procedures for UAD-2::GFP regulators at 20 °C and 25 °C. b, Complementation test for two alleles of gei-17 at 25 °C. All images were taken by the Leica THUNDER imaging System and deconvoluted using Leica Application Suite X software (version 3.7.4.23463). All images are representative of more than three animals (see also in Extended Data Fig. 7g and h). c, Expression levels of total piRNAs from LG IV in WT worms fed L4440 and smo-1 dsRNA at 25 °C, normalized as reads per million+1. d, Boxplots illustrating log2(piRNA reads per million+1) for WT worms fed with L4440 and smo-1 dsRNA at 25 °C from a single biological replicate. The box itself represented the interquartile range between 5% and 95%, thereby encompassing the middle 90% of the data set. The central horizontal line and the adjacent numeric value within the box represent the mean value. Error bars represent the mean ± 1.5 standard deviations (SD), indicating the variability of the data around the mean. Outliers are individually marked as points, representing data points that fall outside the range of mean±1.5 SD (see also in Extended Data Fig. 7e and f). Statistical significance was determined using a paired-sample t test. e and f, Boxplots illustrating log2(pre-piRNA reads per million+1) for WT worms and gei-17 mutants at 25 °C. Statistical significance was determined using a paired-sample t test. g and h, Images displaying GFP::FLAG::Degron::GEI-17 (green) and mCherry::H2B (magenta) in embryos and larval animals.

Extended Data Fig. 8 Forward genetic screening identified TOFU-3 as required for UAD-2 condensate formation and piRNA production.

a. Schematic representation summarizing piRNA genes and alleles identified from the two forward genetic screens. b. Images of the meiotic region of the indicated adult animal germline grown at 20 °C. All images were taken by the Leica THUNDER imaging System and deconvoluted using Leica Application Suite X software (version 3.7.4.23463). All images are representative of more than three animals (see also in Extended Data Fig. 8c, d and f). c, Complementation test of two alleles of tofu-3 at 20 °C. d, Images of pachytene cells of the indicated adult animals grown at 20 °C. mCherry::TOFU-3 rescued UAD-2::GFP foci formation in tofu-3(tm3068) cells. e, Sequence alignment of the TOFU-3 (ULP-5) protein. f, Subcellular localization of UAD-2::GFP in pachytene cells in eri-1(mg366);UAD-2::GFP worms fed the indicated RNAi bacteria. n, the number of independent animals tested. Source data are provided as a Source Data file. g, Deep sequencing of total small RNAs of the indicated adult animals from one biological replicate. Blue dashed lines indicate piRNAs. h, Scatter plots depicting the expression levels of Cluster I, Cluster II, and out-Cluster piRNAs in the wild-type (x-axis) and tofu-3 mutant strains (y-axis) from one biological replicate, normalized as reads per million+1.

Source data

Extended Data Fig. 9 UAD-2 is required for PDRE-1 foci formation and piRNA expression.

a and b. Images displaying the subcellular localization of PRDE-1::GFP and the chromatin marker mCherry::H2B in the germline from wild-type animals and tofu-3(tm3068) mutants. All images were taken by the Leica THUNDER imaging System and deconvoluted using Leica Application Suite X software (version 3.7.4.23463). All images are representative of more than three animals (see also in Extended Data Fig. 9d–f). c, Expression levels of total piRNAs from LG IV of the indicated adult animals cultured at 20 °C, normalized as reads per million+1 from one biological replicate. d-f, Subcellular localization of UAD-2::GFP and mCherry::H2B in pachytene cells from WT, tofu-3(ust235) and tofu-3(tm3068) mutant worms.

Extended Data Fig. 10 SMO-1 suppresses piRNA expression and increases UAD-2 mobility.

a-c, Subcellular localization of UAD-2::GFP, TOFU-4::GFP and TOFU-5::GFP with mCherry::H2B in pachytene cells of WT worms fed L4440 and smo-1 dsRNA at 20 °C. All images were taken by the Leica THUNDER imaging System and deconvoluted using Leica Application Suite X software (version 3.7.4.23463). All images are representative of more than three animals (see also in Extended Data Fig. 10m). d-f, Relative fluorescence unit intensity (RFU) indicated by dashed lines along chromosome IV of (a-c). Source data are provided as a Source Data file. g, Expression levels of total piRNAs from LG IV in specified adult animals grown at 20 °C, normalized as reads per million+1. h, Boxplots illustrating log2(piRNA reads per million+1) for the indicated adult animals from one biological replicate. The box itself represented the interquartile range between 5% and 95%, thereby encompassing the middle 90% of the data set. The central horizontal line within the box represents the mean value. Error bars represent the mean ± 1.5 standard deviations (SD), indicating the variability of the data around the mean. Outliers are individually marked as points, representing data points that fall outside the range of mean±1.5 SD (see also in Extended Data Fig. 10j). Statistical significance was determined using a paired-sample t test. i, qRT-PCR analysis of tofu-3 mRNA in wild-type animals and mCherry::TOFU-3 animals. The levels were normalized to eft-3. Quantification of qRT-PCR data from n = 3 independent animals, error bars represent the mean ± 1.5 standard deviations (SD), indicating the variability of the data around the mean. Statistical significance was determined using a paired-sample t test. Source data are provided as a Source Data file. j. Boxplots illustrating log2(pre-piRNA reads per million+1) for wild-type worms and mCherry::TOFU-3 animals at 25 °C from one biological replicate. Statistical significance was determined using a paired-sample t test. k, Fluorescence recovery after photobleaching (FRAP) of UAD-2::GFP under L4440 and smo-1 RNAi was conducted using a Zeiss LSM980 confocal microscope. All images are representative of 6 animals. l, Graphs presenting the relative fluorescence unit intensity (RFU) of the control area and bleached area of UAD-2 under L4440 and smo-1 RNAi. Quantification of FRAP data from n = 6 independent animals, error bars represent the mean ± 1.5 standard deviations (SD), indicating the variability of the data around the mean. Source data are provided as a Source Data file. m, Images showing germline nuclei expressing UAD-2::GFP. After release by needle disruption, the germline was imaged within 5 minutes using a Leica Thunder imaging system. Germlines were treated with 10% 1,6-hexanediol to prohibit phase separation.

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Supplementary information

Reporting Summary

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Supplementary Table 1

List of strains used in the work.

Supplementary Table 2

Sequences of sgRNAs for CRISPR–Cas9-mediated gene editing and primers for RT–qPCR.

Supplementary Table 3

piRNA gene reference.

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Source Data Figs. 1–3 and 7

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Source Data Extended Data Figs. 2, 4, 6, 8 and 10

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Zhu, C., Si, X., Hou, X. et al. piRNA gene density and SUMOylation organize piRNA transcriptional condensate formation. Nat Struct Mol Biol (2025). https://doi.org/10.1038/s41594-025-01533-5

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