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A Pandas complex adapted for piRNA-guided transcriptional silencing and heterochromatin formation

Nature Cell Biology volume 21, pages 1261–1272 (2019)Cite this article

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Abstract

The repression of transposons by the Piwi-interacting RNA (piRNA) pathway is essential to protect animal germ cells. In Drosophila, Panoramix enforces transcriptional silencing by binding to the target-engaged Piwi–piRNA complex, although the precise mechanisms by which this occurs remain elusive. Here, we show that Panoramix functions together with a germline-specific paralogue of a nuclear export factor, dNxf2, and its cofactor dNxt1 (p15), to suppress transposon expression. The transposon RNA-binding protein dNxf2 is required for animal fertility and Panoramix-mediated silencing. Transient tethering of dNxf2 to nascent transcripts leads to their nuclear retention. The NTF2 domain of dNxf2 competes dNxf1 (TAP) off nucleoporins, a process required for proper RNA export. Thus, dNxf2 functions in a Panoramix–dNxf2-dependent TAP/p15 silencing (Pandas) complex that counteracts the canonical RNA exporting machinery and restricts transposons to the nuclear peripheries. Our findings may have broader implications for understanding how RNA metabolism modulates heterochromatin formation.

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Fig. 1: dNxf2 functions as a cofactor of Panx in the piRNA pathway.
Fig. 2: The UBA domain of dNxf2 interacts directly with Panx.
Fig. 3: Structure of dNxf2UBA in complex with PanxNIR.
Fig. 4: The UBA domain of dNxf2 lost NPC binding capability.
Fig. 5: dNxf2 preferentially binds to piRNA-targeted transposons.
Fig. 6: dNxf2 is required for Panx to associate with chromatin.
Fig. 7: The NTF2 domain of dNxf2 interacts directly with dNxf1.
Fig. 8: The Pandas complex is required for piRNA-guided transposon silencing.

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Data availability

The raw files of all sequencing libraries generated for this study have been submitted to the Gene Expression Omnibus under accession number GSE130042. The coordinates for the structures reported in this paper have been deposited in the PDB under accession number 6IEW (dNxt2UBA and PanxNIR complex) and 6IHJ (dNxt1NTF2 and dNxt1 complex). The proteomics data of the binding proteins for Panx and the cross-link mass spectrometry data have been deposited in ProteomeXchange with the primary accession codes PXD014926 and PXD014884.

The structure data collection and refinement statistics have been provided as Supplementary Table 1. The DNA oligo sequences have been provided as Supplementary Table 2. The source data for Figs. 1–7 and Supplementary Figs. 1,2,4,5 have been provided as Supplementary Table 3. All other data supporting the findings of this study are available from the corresponding authors on request.

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Acknowledgements

We thank the National Facility for Protein Science in Shanghai Zhangjiang Lab and the Shanghai Science Research Center for their instrumental support and technical assistance. We thank the staff from the BL19U1 beamline at the Shanghai Synchrotron Radiation Facility for assistance with data collection. We thank S. Li from the Center for Biological Imaging and Y. Wang from the Protein Science Core Facility at the Institute of Biophysics, CAS for their technical support and assistance with data collection. This work was supported in part by grants from the Ministry of Science and Technology of China (grant no. 2017YFA0504200 to Y.Y.), the Strategic Priority Research Program of the Chinese Academy of Sciences (grant no. XDB19000000 to Y.Y.), the National Natural Science Foundation of China (grant nos 91640105 and 31770875 to Y.Y., and 91640102 and 31870741 to Y.H.), the National Postdoctoral Program for Innovative Talents (grant no. BX20190081 to Y.H.Z.) and the China Postdoctoral Science Foundation Grant (grant no. 2019M653166 to J.Q.G.).

Author information

Author notes

  1. These authors contributed equally: Kang Zhao, Sha Cheng, Na Miao, Ping Xu, Xiaohua Lu, Yuhan Zhang.

Authors and Affiliations

  1. Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Kang Zhao, Na Miao, Ping Xu, Xiaohua Lu, Ming Wang, Xuan Ouyang, Weiwei Liu, Xin Lu, Peng Zhou, Jiaqi Gu, Yiqun Zhang, Ding Qiu & Yang Yu

  2. University of Chinese Academy of Sciences, Beijing, China

    Kang Zhao, Sha Cheng, Na Miao, Xiaohua Lu, Xuan Ouyang, Xun Yuan, Xin Lu, Peng Zhou, Ding Qiu, Zhaohui Jin, Hong Cheng, Ying Huang & Yang Yu

  3. State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China

    Sha Cheng, Yuhan Zhang, Xun Yuan, Zhaohui Jin, Hong Cheng & Ying Huang

  4. National Engineering Laboratory of AIDS Vaccine, Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, School of Life Sciences, Jilin University, Changchun, China

    Ping Xu & Youzhong Wan

  5. State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai, China

    Yuhan Zhang, Jiaqi Gu & Jinbiao Ma

  6. National Facility for Protein Science in Shanghai, Zhangjiang Lab, Shanghai Science Research Center, Chinese Academy of Sciences, Shanghai, China

    Chen Su & Chao Peng

  7. Graduate School of Peking Union Medical College and Chinese Academy of Sciences of Medical Sciences, Beijing, China

    Jian-Hua Wang

  8. National Institute of Biological Sciences, Beijing, China

    Meng-Qiu Dong

  9. Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, Beijing, China

    Meng-Qiu Dong

  10. Shanghai Key Laboratory of Biliary Tract Disease Research, Shanghai Research Center of Biliary Tract Disease, Department of General Surgery, Xinhua Hospital, affiliated with Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Ying Huang

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  1. Kang Zhao
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Contributions

Y.Y. and Y.H. conceived the project and wrote the manuscript. K.Z. constructed the dNxf2–Halo knock-in OSCs and established the dNxf2 mutant. K.Z., P.X., W.L., X.L. and D.Q. performed the co-immunoprecipitations, tethering assays, transgenic fly constructions, RNA-seq and RT–qPCR experiments. S.C. and X.Y. performed the structural studies, β-gal activity assays and isothermal titration calorimetry experiments. S.C. and Yuhan Zhang performed the GST pull-down assays. K.Z., Z.J., P.Z., X.O., J.G. and P.X. performed the cloning. S.C. and X.L. performed the Y2H assays. K.Z. and X.O. performed the FACS analysis. N.M. performed the FISH and RNAi experiments. M.W. and Yiqun Zhang performed the bioinformatics analysis. C.S., C.P., J.-H.W. and M.-Q.D. performed the mass spectrometry and analysed the data. Y.W., J.M. and H.C. provided critical reagents and advice. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Ying Huang or Yang Yu.

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Integrated supplementary information

Supplementary Figure 1 dNxf2 binds to Panx and is required for transposon silencing.

a, A network graph showing proteins co-immunoprecipitated with Flag-tagged Panx from the DPiM data downloaded from flybase.org. b, Scatter plots showing enrichment values for proteins co-purified with GFP-Panx (n=2) from ovary lysates (the two most enriched proteins are labelled). c, Western blots and Halo-ligand staining showing co-immunoprecipitation of GFP-tagged Panx with Halo-tagged dNxf2 from OSC cell lysates. GFP serves as a negative control. Three biologically independent experiments were repeated with similar results. d, The expression profiles (Y-axis) of Panx and dNxf2 across different adult tissues (X-axis) are shown (blue, Panx; red, dNxf2). The mean affy2 probeset expression value (X-axis) is based on FlyAtlas anatomical microarray data downloaded from flybase.org. e, RT-qPCR results showing relative steady-state RNA levels of the indicated transposons for the germline specific (nanos-GAL4) knockdown of the indicated genes. The attp2 is used as a control. Fold changes are calculated as rp49-normalized RNA levels divided by that of the corresponding controls. Mean values ± s.d. from 3 biologically independent experiments are shown. The source data for e are provided in Supplementary Table 3. Unprocessed gels for c are provided in Supplementary Fig. 8.

Supplementary Figure 2 The dNxf2 mutant resembles the loss of function Panx mutant.

a, A schematic of CRISPR/Cas9-induced mutation in the open reading frame of dNxf2 is shown. Arrowheads indicate the cleavage sites targeted by the gRNA with the corresponding PAM site highlighted in light blue. The resulting mutant is shown below the wild-type sequence with the mutated/deleted region shown in red. b, Vials showing pupae from equal number of either heterozygous or mutant dNxf2 female flies crossed with OregonR males. c, Representative immunostaining of endogenous Piwi protein (green) from stage 5 ovaries of either heterozygous or mutant dNxf2. DAPI staining (blue) was used to indicate nuclei. d, Western blots showing protein expression level of endogenous dNxf2, Panx and Piwi from either heterozygous or mutant dNxf2 ovary lysates. Beta-tubulin serves as a loading control. For b-d, three biologically independent experiments were repeated with similar results. e, RT-qPCR results showing relative steady-state RNA levels of the indicated transposons for the mutant or heterozygous dNxf2 ovaries, compared to the wildtype control (w1118). Fold changes are calculated as rp49-normalized RNA levels divided by that of the corresponding controls. Mean values ± s.d. from 3 biologically independent experiments are shown. f, Comparison of steady-state RNA levels are shown as reads per million (rpm) mapping to the sense strands of each transposon consensus from dNxf2 mutant versus w1118 wild-type fly ovaries. Dashed lines indicate two-fold changes. The average of two replicates is shown. Red dots indicate transposon elements with significant changes (P value<0.05, Wald test). g, Comparison of steady-state RNA levels (RNA-seq; shown as RPM) mapping to the sense strands of each transposon consensus from dNxf2 mutant versus Panx mutant fly ovaries. Red dots indicate transposon elements with significant changes from f (P value<0.05, Wald test). h, RT-qPCR results showing relative steady-state RNA levels of the indicated transposons for the mutant or heterozygous dNxf2 ovaries, compared to the wildtype control while overexpressing λN-Flag-Panx driven by a ubiquitin promoter. Fold changes are calculated as rp49-normalized RNA levels divided by that of the corresponding controls. Mean values ± s.d. from 3 biologically independent experiments are shown. i, Comparison of steady-state RNA levels are shown as reads per million (rpm) mapping to the sense strands of each transposon consensus from dNxf2 mutant versus wild-type fly ovaries when overexpressing ubiquitin-driven λN-Flag-Panx. Dashed lines indicate two-fold changes. The average of two replicates is shown. Red dots indicate transposon elements with significant changes (P value<0.05, Wald test). The source data for e and h are provided in Supplementary Table 3. Unprocessed gels for d are provided in Supplementary Fig. 8.

Supplementary Figure 3 Structural analysis of dNxf1NTF2 in complex with dNxt1.

a, Cartoon representation of dNxf1 NTF2 domain (orange) in complex with dNxt1 (green). b, Electrostatic surface potential representation of dNxt1, highlighting hydrophobic and electrostatic surfaces continuously from acidic (red) to non-polar (grey), to basic (blue). Key residues of dNxf1 are shown in stick mode (orange). The corresponding residues in dNxf2 (yellow) are modelled in Coot by mutation operation based on the dNxf1 structure in this study. c, Western blots and Halo-ligand staining showing co-immunoprecipitation of GFP-tagged dNxt1 with Halo-tagged dNxf1 or dNxf2 from OSC cells. GFP serves as a negative control. Three biologically independent experiments were repeated with similar results. d, Sequence alignment of the NTF2 domains of dNxf1, dNxf2, human TAP (Q9UBU9), and ceNxf2 (Q9XVS8). The secondary structure annotation of dNxf1 is shown on the top. Residue positions of the reference sequence (dNxf1) are also indicated above the sequences. Arrows and spirals indicate beta-strands and alpha-helices, respectively. Red letters indicate similar residue; white letters in red shading indicate identical residues; blue boxes indicate conserved positions. e, Sequence alignment of the region of Panx that interacted with dNxf2. Residues involved in the interactions are marked by teal diamond. f, Sequence alignment of the UBA domains of NXF family proteins. Residues within the dNxf2-Panx interaction interface are indicated in brown diamond. Residues that blocks the entry of the FG peptides are indicated in green circle. Residues that are identical are shown in white with red shading. Residues with high similarities are shown in red. Secondary structural elements are indicated above. The source data for c are provided in Supplementary Table 3.

Supplementary Figure 4 dNxf2 specifically binds to transposons in vivo.

a, A Schematic of the knock-in strategy is displayed. Agarose gel showing PCR results confirming a correct integration of Halo-tag to the C-terminus of the dNxf2 locus. PCR primers amplifying the indicated regions are shown on the top and two sgRNAs targeting near the stop codon of dNxf2 are indicated at the bottom of the schematic. Two biologically independent experiments were repeated with similar results. b, Western blots and Halo-ligand staining showing depletion of dNxf2-Halo or Halo alone by Halo pull down. Beta-tubulin blots serve as a loading control. The asterix indicates non-specific bands stained by Halo-ligand. Three biologically independent experiments were repeated with similar results. c, RT-qPCR results showing relative steady-state RNA levels of the indicated transposons for OSC from the H1+HP1a double siRNA knockdown, compared to a control siRNA knockdown. Fold changes are calculated as rp49-normalized RNA levels divided by that of the corresponding controls. Mean values ± s.d. from 3 biologically independent experiments are shown. d, FACS sorting showing counts and fluorescence of GFP positive cells gated by high RFP expressing OSCs electro-transfected with the indicated plasmids expressing different λN-fusion proteins. OSCs (grey), autofluorescence of OSCs without any GFP reporter; Tethered (red), GFP reporter OSCs transfected with the indicated expression plasmids; GFP-10×BoxB (green), GFP reporter cells with no transfection. Three biologically independent experiments were repeated with similar results. Two representative replicates were shown. The source data for c are provided in Supplementary Table 3. Unprocessed gels for a-b are provided in Supplementary Fig. 8.

Supplementary Figure 5 Het-A left nuclear peripheries upon loss of either Panx or dNxf2.

a, H3K9me3 mark enrichments at transposons comparing heterozygous and mutant for dNxf2, without Panx overexpression. Green and brown bar, polyclonal antibody specific to H3K9me3; Black and grey bar, pre-immune rabbit IgG. Mean values ± s.d. from 3 biologically independent experiments are shown. b, Left three columns showing 3D SIM super-resolution microscopy of DNA FISH (HeT-A, green) and Lamin (red) double staining from either Panx or dNxf2 heterozygote versus mutant ovaries. Right three columns, estimation of the positioning of clustered HeT-A signals relative to the nuclear membrane of nurse cells. 10-20 foci were quantified for each condition. The distance was displayed in a blue-red scale with red indicating longer distance between the green dots and nuclear membrane. c, Projections of the 3D videos on the XY surface either in combination (bottom right) or in split channels for DAPI (blue, upper right), Lamin (red, bottom left) and HeT-A (green, upper left) on the selected ovaries from b. See also Supplementary Movies 1-4. For b-c, three biologically independent experiments were repeated with similar results. The source data for a are provided in Supplementary Table 3.

Supplementary Figure 6 dNxf2 interacts with dNxf1 directly.

a, Western blots and Halo-ligand staining showing co-immunoprecipitation of GFP-tagged various truncated dNxf2 with Halo-tagged full length dNxf1 from OSCs. ΔRBD, dNxf2 lacking N-terminus RNA binding domains; NTF2, dNxf2NTF2 domain; UBA, dNxf2UBA domain. b, Western blots and Halo-ligand staining showing co-immunoprecipitation of GFP-tagged dNxf1NTF2 domain with Halo-tagged various truncated dNxf1 from S2 cells. ΔRBD, dNxf1 lacking all N-terminus RNA binding domains; NTF2, dNxf1NTF2 domain; UBA, dNxf1UBA domain. GFP serves as a negative control. c, The same as in c except the experiments were done using OSCs. d-f, GST pull-down assays showed the direct interactions between dNxf1 and dNxf2 (n = 3 biologically independent experiments with similar results). One dataset is shown. Each of 60 μg of purified GST-dNxf1NTF2/dNxt1 (d), GST-dNxf1UBA (e), GST-dNxf1NTF2+UBA/TS-dNxt1 complex (f) and GST protein was immobilized on GSH beads, respectively. Increasing amounts of dNxf2NTF2/dNxt1 complex (0, 15, 30, 60 μg) were added and incubated. The beads were washed and aliquots of the bound fractions (8%) were analysed by SDS-PAGE. Each input protein of 2 μg was loaded on SDS-PAGE as well. Positions of molecular weight markers are indicated on the left in kDa. For a-f, three biologically independent experiments were repeated with similar results. Unprocessed gels for a-c are provided in Supplementary Fig. 8.

Supplementary Figure 7 dNxf2 forms a ternary complex with dNxf1 and dNxt1.

a, OD280 trace of complexes formed by dNxf2NTF2+UBA, dNxt1, and PanxNIR in solution as shown on the size exclusion chromatography profile. b, SDS-PAGE shows the components of the peaks from a in the elution profile. c, Confocal microscopy of RNA FISH done the same as in Fig. 8c except that either FRB-Piwi or FRB-Asterix expression constructs were transfected. Top panel, RNA signal (red); upper-middle, DAPI staining (blue); lower-middle, Lamin staining (green); bottom, merge. The scale bars represent 2 µm in length. d, Confocal microscopy of Lamin immunostaining (green) with either the OSCs or the GFP-RanGAP-BoxB stable line pretreated with or without H2O2. For a-d, three biologically independent experiments were repeated with similar results.

Supplementary Figure 8 Full blots of figures.

The black sections indicate blot results shown in the indicated figures.

Supplementary information

Supplementary Information

Supplementary Figures 1–8, Supplementary Table titles/legends, Supplementary Video titles/legends

Reporting Summary

Supplementary Table 1

Data collection and refinement statistics.

Supplementary Table 2

Oligo information.

Supplementary Table 3

Source data for graphical representation.

Supplementary Video 1

3D SIM super-resolution microscopy of DNA FISH (HeT-A, green) and Lamin A (red) double staining from dNxf2 heterozygote ovaries.

Supplementary Video 2

3D SIM super-resolution microscopy of DNA FISH (HeT-A, green) and Lamin A (red) double staining from dNxf2 mutant ovaries.

Supplementary Video 3

3D SIM super-resolution microscopy of DNA FISH (HeT-A, green) and Lamin A (red) double staining from Panx heterozygote ovaries.

Supplementary Video 4

3D SIM super-resolution microscopy of DNA FISH (HeT-A, green) and Lamin A (red) double staining from Panx mutant ovaries.

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Zhao, K., Cheng, S., Miao, N. et al. A Pandas complex adapted for piRNA-guided transcriptional silencing and heterochromatin formation. Nat Cell Biol 21, 1261–1272 (2019). https://doi.org/10.1038/s41556-019-0396-0

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