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Panoramix SUMOylation on chromatin connects the piRNA pathway to the cellular heterochromatin machinery

Nature Structural & Molecular Biology volume 29, pages 130–142 (2022)Cite this article

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Abstract

Nuclear Argonaute proteins, guided by small RNAs, mediate sequence-specific heterochromatin formation. The molecular principles that link Argonaute-small RNA complexes to cellular heterochromatin effectors on binding to nascent target RNAs are poorly understood. Here, we explain the mechanism by which the PIWI-interacting RNA (piRNA) pathway connects to the heterochromatin machinery in Drosophila. We find that Panoramix, a corepressor required for piRNA-guided heterochromatin formation, is SUMOylated on chromatin in a Piwi-dependent manner. SUMOylation, together with an amphipathic LxxLL motif in Panoramix’s intrinsically disordered repressor domain, are necessary and sufficient to recruit Small ovary (Sov), a multi-zinc-finger protein essential for general heterochromatin formation and viability. Structure-guided mutations that eliminate the Panoramix–Sov interaction or that prevent SUMOylation of Panoramix uncouple Sov from the piRNA pathway, resulting in viable but sterile flies in which Piwi-targeted transposons are derepressed. Thus, Piwi engages the heterochromatin machinery specifically at transposon loci by coupling recruitment of a corepressor to nascent transcripts with its SUMOylation.

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Fig. 1: An amphipathic LxxLL motif in the Panx IDR binds Sov.
Fig. 2: Sov is required for Piwi and Panx-mediated heterochromatin formation.
Fig. 3: Structural basis of the Panx–Sov interaction.
Fig. 4: Panx is SUMOylated.
Fig. 5: A SUMOylation-dependent dual mode interaction between Panx and Sov.
Fig. 6: SUMOylation of Panx at chromatin depends on Piwi.
Fig. 7: Direct SUMOylation of Panx by Ubc9 is independent of Su(var)2-10.

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Data availability

Coordinates and structure factors of Sov NTD in complex with the Panx LxxLL peptide were deposited in the Protein Data Bank (PDB accession 7MKK). Sequencing data sets were deposited in the NCBI GEO archive (accession GSE173237). The proteomics data were deposited in the ProteomeXchange Consortium via the PRIDE partner repository (data set PXD025437). The Drosophila melanogaster genome dm6 version was used throughout this work. Source data are provided with this paper.

Code availability

Standard methods were used and are cited and described in the Methods section. Source data are provided with this paper.

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Acknowledgements

We thank the VBCF core facilities (Protein Technologies, NGS, VDRC) for support and the IMBA Fly House for generating transgenic and CRISPR-edited fly lines. The GMI/IMBA/IMP Scientific Service units, especially the mass spectrometry unit (K. Mechtler and team) provided outstanding support. The Max Perutz Laboratories Monoclonal Antibody facility generated the Sov and Su(var)2-10 hybridoma cell line. We thank the Brennecke laboratory for help throughout this project, and P. Batalski for generating amino acid mapping scripts. C. Lima (MSKCC) and U. Hohmann (IMBA, IMP) gave valuable feedback on the manuscript. Funding statement: this research was funded by the Austrian Academy of Sciences, the European Community (grant no. ERC-2015-CoG-682181) and the Austrian Science Fund (grant nos. F4303 and W1207). For the purpose of Open Access, the author has applied for a CC BY public copyright licence to any Author Accepted Manuscript (AAM) version arising from this submission. X-ray diffraction studies were conducted at the Advanced Photon Source on the Northeastern Collaborative Access Team beamlines, supported by the National Institute of General Medical Sciences, grant no. P30 GM124165 and US Department of Energy grant no. DE-AC02-06CH11357. The Eiger 16M detector on the 24-ID-E beamline is funded by a NIH-ORIP HEI grant (no. S10OD021527). This work was supported in part by the Maloris Foundation (D.J.P.). The Memorial Sloan Kettering Cancer Center structural biology core facility is supported by National Cancer Institute Core grant no. P30-CA008748. C.Y. and M.G. are supported by the VIP2 Post-Doctoral fellowship program as part of the EU Horizon 2020 research and innovation program (Marie Skłodowska-Curie grant no. 847548). L.B. and G.S. were funded by Boehringer Ingelheim PhD fellowships.

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Author notes

  1. These authors contributed equally: Veselin I. Andreev, Changwei Yu.

Authors and Affiliations

  1. Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter (VBC), Vienna, Austria

    Veselin I. Andreev, Changwei Yu, Jakob Schnabl, Laszlo Tirian, Maja Gehre, Dominik Handler, Peter Duchek, Maria Novatchkova, Lisa Baumgartner, Katharina Meixner, Grzegorz Sienski & Julius Brennecke

  2. Vienna BioCenter PhD Program, Doctoral School of the University of Vienna and Medical University of Vienna, Vienna, Austria

    Veselin I. Andreev, Jakob Schnabl, Lisa Baumgartner & Grzegorz Sienski

  3. Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Juncheng Wang & Dinshaw J. Patel

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Contributions

J.W. undertook X-ray studies on the Sov NTD–Panx LxxLL peptide complex and ITC assays under the supervision of D.J.P. V.I.A. and C.Y. performed all molecular biology experiments, except those where J.S. identified the LxxLL motif and purified recombinant SFiNX complex and J.B., L.T., L.B. and P.D. performed the fly experiments, M.G. performed the H3K9me3 Cut&Run experiments. G.S. generated GRO-seq data. D.H. and C.Y. performed computational analyses. M.N. performed the phylogenetic analysis of the Panx LxxLL peptide. L.B. established sov RNAi lines and characterized the Sov antibody. K.M. generated OSC cell lines. The project was supervised by J.B. and D.J.P. The paper was written by V.I.A., C.Y. and J.B. with input from all authors.

Corresponding author

Correspondence to Julius Brennecke.

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Nature Structural and Molecular Biology thanks Alla Kalmykova, Phillip Zamore and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Anke Sparmann and Carolina Perdigoto were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1

a, Western blot analysis showing expression levels of indicated Gal4-DBD fusion proteins following transient transfection in OSCs (Ponceau S-levels indicate protein loading; related to Fig. 1b). b, H3K9me3 levels (normalized to heterochromatic light locus) at the reporter locus (amplicon indicated in Fig. 1a) determined by ChIP-qPCR from OSCs expressing indicated Gal4-DBD fusion proteins (data are presented as mean of n = 2 biological replicates). c, Western blot analysis showing expression levels of Gal4-DBD fusion proteins with indicated Panx IDR fragments (amino acid boundaries indicated). Ponceau S levels indicate protein loading; related to Fig. 1d. d, Western blot analysis showing expression levels of Gal4-DBD fusions with indicated WT and LxxLL mutant Panx fragments. Ponceau S levels indicate protein loading; related to Fig. 1e. e, To the left: Boxplots showing GFP reporter levels in OSCs following transfection with plasmids encoding Gal4-DBD fusions of Panx peptides (wildtype versus alanine mutant; numbers indicate median fold repression normalized to control for two biological replicates, boxplots indicate median, first and third quartiles (box), whiskers show 1.5× interquartile range; outliers were omitted; n = 10,000 cells). To the right: Western blot analysis showing expression levels of Gal4-DBD fusion proteins with indicated Panx peptides. Ponceau S levels indicate protein loading.

Source data

Extended Data Fig. 2

a, Confocal image of egg chambers with indicated germline-specific knockdown (GLKD) stained for Sov (greyscale; scale bar: 20 μm). b, H3K9me3 Cut&Run signal from OSCs with indicated knockdowns at indicated transposons (The Doc retroelement serves as a control transposon as it is not targeted by the piRNA pathway in OSCs). c, Sov-GFP ChIP–Seq signal at indicated transposon families from OSCs expressing Sov-GFP or not (control) (The Doc retroelement serves as a control transposon family as it is not targeted by the piRNA pathway in OSCs). d, Western blot analysis showing expression of Gal4-DBD-FLAG-Sov following plasmid transfection in the OSC reporter cell line (related to Fig. 2f,g).

Source data

Extended Data Fig. 3

Coomassie stained SDS-PAGE showing an in vitro pulldown experiment with streptavidin-bound wildtype Panx LxxLL peptide or two mutant peptides (NxxQQ and AxxAA variants) and recombinant GFP-tagged Sov NTD (14-90 aa) fragment as prey (asterisk indicates a background band from Streptavidin beads).

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Extended Data Fig. 4

a, Volcano plot showing fold enrichment of proteins determined by quantitative mass spectrometry in GFP-FLAG-Sov co-immunoprecipitates versus control (n = 3 biological replicates; statistical significance of differentially enriched proteins was derived from a two-sided t-test and p-values were subsequently corrected for multiple testing (Benjamini-Hochberg)). b, Confocal image of egg chamber expressing GFP-Smt3 (greyscale) under the smt3 regulatory control elements (scale bar: 20 μm).

Extended Data Fig. 5

a, Western blot analysis showing expression of indicated GFP-FLAG tagged Sov NTD variants following transient transfection in OSCs (related to Fig. 5a). b, Western blot analysis showing expression levels of Gal4-DBD-FLAG fusions with indicated Panx IDR variants following transient transfection in the OSC reporter line. Staining with Ponceau S serves as loading control (related to Fig. 5b).

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Extended Data Fig. 6

a, GFP-Panx ChIP-Seq signal from OSCs expressing GFP-Panx or not (control) at gypsy (piRNA targeted) or Doc transposons (not targeted). b, Heatmap of GRO-seq signal (left), GFP-Panx ChIP-Seq signal (middle) and chromatin-enriched GFP-Panx ChIP-Seq signal (right) around transcription start sites (TSSs) of expressed genes in OSCs (all heatmaps sorted for maximal GRO-seq signal at the TSS; plots above heatmaps depict the corresponding meta-profiles). c, GFP-Panx ChIP-Seq signal from pre-extracted OSCs expressing GFP-Panx or not (control) at gypsy (piRNA targeted) or Doc transposons (not targeted). d, Heatmap showing GRO-seq signal at genomic regions flanking 381 piRNA-targeted transposon insertions (vertical line) in OSCs depleted for indicated factors.

Extended Data Fig. 7

a, Transcript levels of lwr, measured by RT–qPCR with two amplicons, following siRNA transfection (n = 2 biological replicates; 96 h time point; similar results were obtained after 48 h and 72 h). To the right, panx transcript levels measured by RT-qPCR with two different amplicons (panx-1 and -2; data presented as mean of n = 3 biological replicates, error bars: standard deviation). b, Coomassie-stained SDS-PAGE showing recombinant Drosophila Uba2/His6-Aos1, Lwr and Smt3 used in the in vitro SUMOylation assays. c, Western blot analysis showing endogenous Nxf2 (left) and Panx (right) proteins in OSC whole cell lysate prepared with or without N-ethylmaleimide (NEM). Tubulin and staining with Ponceau S serve as loading control for Panx and Nxf2 blots, respectively. d, Western blot analysis showing impact of increasing concentration of Sov NTD variants lacking SIM1 or SIM2 on the efficiency of Panx IDR in vitro SUMOylation. e, Western blot analysis showing the enhancement of Panx HA-IDR in vitro SUMOylation by the Sov NTD in an LxxLL binding-dependent manner. f, Western blot analysis showing extent of endogenous Panx SUMOylation in OSCs depleted for Sov with two different siRNAs. g, Cartoon model of how the Panx–Sov-NTD interaction enhances Panx SUMOylation. h, Phylogenetic distribution of Sov NTD sequence homologs. Identified Sov NTD homologs belong either to the Sov or to the Med15 ortholog groups in the same database (phylogenetic tree based on NCBI taxonomy using iTOL v.6).

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Supplementary information

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Supplementary Data 1

Mass spectrometry data lists.

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Fly stocks.

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Guide RNA sequences.

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siRNA sequences.

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Antibodies.

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qPCR oligos.

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Next generation sequencing data.

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Andreev, V.I., Yu, C., Wang, J. et al. Panoramix SUMOylation on chromatin connects the piRNA pathway to the cellular heterochromatin machinery. Nat Struct Mol Biol 29, 130–142 (2022). https://doi.org/10.1038/s41594-022-00721-x

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