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Evolutional heterochromatin condensation delineates chromocenter formation and retrotransposon silencing in plants

Nature Plants volume 10, pages 1215–1230 (2024)Cite this article

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Abstract

Heterochromatic condensates (chromocenters) are critical for maintaining the silencing of heterochromatin. It is therefore puzzling that the presence of chromocenters is variable across plant species. Here we reveal that variations in the plant heterochromatin protein ADCP1 confer a diversity in chromocenter formation via phase separation. ADCP1 physically interacts with the high mobility group protein HMGA to form a complex and mediates heterochromatin condensation by multivalent interactions. The loss of intrinsically disordered regions (IDRs) in ADCP1 homologues during evolution has led to the absence of prominent chromocenter formation in various plant species, and introduction of IDR-containing ADCP1 with HMGA promotes heterochromatin condensation and retrotransposon silencing. Moreover, plants in the Cucurbitaceae group have evolved an IDR-containing chimaera of ADCP1 and HMGA, which remarkably enables formation of chromocenters. Together, our work uncovers a coevolved mechanism of phase separation in packing heterochromatin and silencing retrotransposons.

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Fig. 1: Variations in ADCP1 are associated with chromocenter formation in plants.
Fig. 2: High mobility group protein HMGA interacts with ADCP1.
Fig. 3: HMGA is required for the localization of ADCP1 in heterochromatin.
Fig. 4: HMGA promoted phase separation of ADCP1 with heterochromatin.
Fig. 5: ADCP1-HMGA promotes heterochromatic condensates formation in non-chromocenter plant species relying on the presence of IDRs.
Fig. 6: Introduction of IDR-containing AtADCP1 with AtHMGA promotes heterochromatin condensation and TE silencing.
Fig. 7: A model for the role of ADCP1-HMGA condensator in regulating heterochromatic phase separation and chromocenter organization.

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Data availability

All sequencing data generated in this study are available on the NCBI GEO under accession no. GSE233265. Source data are provided with this paper.

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Acknowledgements

We thank all members of the Sun laboratory for valuable discussion; C. Dean, H. Zhang, G. Li, Q. Zhang, Y. Guo and D. Ren for constructive comments on the work; Y. Qi and Z. Fu for assistance with growing plant materials; Q. Chen and X. Yang for sharing CRISPR-Cas9 vectors; L. Yang for guidance in the preparation and transformation of cucumber protoplasts; Y. Lv for help in painting plant models; the Core Facility of the Center of Biomedical Analysis (Tsinghua University) for assistance with mass spectrometry and confocal microscopy analysis; and Y. Li of the Cryo-EM Facility (Tsinghua University) for help in the immuno-TEM samples preparation. This work was supported by grants from the National Natural Science Foundation of China (31822028 and 91940306 to Q.S., 32070651 to W.Z.) and the Ministry of Science and Technology of China (2016YFA0500800 to Q.S.). The Sun Lab is supported by Tsinghua-Peking Center for Life Sciences, and W.Z. is supported by the China Postdoctoral Science Foundation Project (2019M660610) and the postdoctoral fellowship from Tsinghua-Peking Center for Life Sciences.

Author information

Authors and Affiliations

  1. Center for Plant Biology, School of Life Sciences, Tsinghua University, Beijing, China

    Weifeng Zhang, Lingling Cheng, Kuan Li, Xue Lei, Anjie Jiang & Qianwen Sun

  2. Tsinghua-Peking Center for Life Sciences, Beijing, China

    Weifeng Zhang, Lingling Cheng, Kuan Li, Leiming Xie, Jinyao Ji, Xue Lei, Anjie Jiang, Chunlai Chen, Haitao Li, Pilong Li & Qianwen Sun

  3. State Key Laboratory of Membrane Biology, Beijing Frontier Research Center for Biological Structure, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China

    Leiming Xie, Jinyao Ji, Chunlai Chen & Pilong Li

  4. State Key Laboratory of Molecular Oncology, MOE Key Laboratory of Protein Sciences, Beijing Frontier Research Center for Biological Structure, SXMU-Tsinghua Collaborative Innovation Center for Frontier Medicine, School of Medicine, Tsinghua University, Beijing, China

    Haitao Li

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Contributions

Q.S. and W.Z. conceived the study. Q.S., P.L., H.L., C.C. and W.Z. designed the experiments. L.C. generated the pADCP1::ADCP1-eGFP/FLAG transgenic lines and performed the ADCP1 mass spectrometry analysis. K.L. analysed the TE distribution of plant species and calculated Gini coefficients. L.X. constructed the nucleosomal arrays. C.C. and J.J. quantitatively measured and analysed the ADCP1-HMGA-H3K9me3 puncta formation in vitro. X.L. conducted the protoplast truncated ADCP1 and HMGA Co-IP assays. A.J. performed ITC experiments and predicted the ADCP1-HMGA protein complex structure. W.Z. performed the rest of the experiments. W.Z. and Q.S. wrote the manuscript with input from all authors, and all authors read and approved the final manuscript.

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Correspondence to Qianwen Sun.

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Extended data

Extended Data Fig. 1 Microscopic images of H3K9me2 and H3K27me1 immunostaining in nuclei of different plant species.

Colors indicated the signals of H3K9me2 (green), H3K27me1 (magenta), and DNA stain (DAPI, grey). At least 50 independent nuclei were observed each species, and the representative ones were imaged. Scale bars, 5 μm. The models of corresponding ADCP1 proteins are shown in the right.

Extended Data Fig. 2 Variations of TE distribution and chromocenter formation in plants.

a,b, The distribution of TEs in plant species with (a) and without (b) chromocenters. The green lines indicated the positions of TEs in the presented chromosomes. Blue-lined boxes showed the heterochromatin regions that concentrate the heterochromatic elements in chromocenter-containing species. c, Gini coefficient is calculated by the numbers of TEs in 100kb-windows to represent the differences in distributions of TEs across the chromosome. The species with and without chromocenters are separated by a black dotted line. Error bars denote standard deviation (SD) of chromosomes.

Source data

Extended Data Fig. 3 ADCP1 interacts specifically with HMGA but not with its paralogs.

a, Phylogenetic tree and ___domain architecture of Arabidopsis HMGA proteins. b, The sequence alignment of Arabidopsis HMGA proteins. The N-terminal GH1 ___domain is highlighted in blue, and the four AT-hooks are highlighted in orange. The HMGA peptides found by ADCP1 IP-MS were shown by green boxes. c, The BiFC assays in Arabidopsis show that ADCP1 interacts specifically with HMGA but not with its homologous proteins HMGA1, HMGA2 and HMGA4. Scale bar, 10 μm. d, BiFC assays in Arabidopsis using ___domain truncated ADCP1-YFPn shown in left and full-length HMGA-YFPc. Scale bar, 5 μm. c,d, At least 50 independent cells were observed in each experiment, and the representative ones were imaged. e, Arabidopsis protoplasts were used to co-express MYC-HMGA-GFP with full length or truncated FLAG-ADCP1-mCherry to conduct Co-IP assays. f, The prediction result of ADCP1-HMGA complex using AlphaFold2.

Source data

Extended Data Fig. 4 HMGA promotes ADCP1-depended chromocenter formation.

a, Transient expression of ADCP1-GFP in Col-0 protoplasts. The zoom-in images of the boxed area are shown in Fig. 2h. Scale bar, 5 μm. b, The ___location of ADCP1 in hmga mutant protoplasts. The zoom-in images of the boxed area are shown in Fig. 2i. Scale bar, 5 μm. c, The nucleus of Arabidopsis transgenic plant leaf expressing mCherry-HMGA. Right image is line scans at the position depicted by the white line. Scale bar, 5 μm. d, The nucleus of Arabidopsis transgenic plant root expressing ADCP1-GFP. Right image is line scans at the position depicted by the white line. Scale bar, 5 μm. e, The Arabidopsis mCherry-HMGA and ADCP1-GFP co-transgenic plants showed ADCP1 and HMGA were co-localized in the chromocenters. Right image is line scans at the position depicted by the white line. Scale bar, 5 μm. At least 50 independent nuclei were observed in each line, and the representative ones were imaged.

Source data

Extended Data Fig. 5 HMGA helps ADCP1 to localize in heterochromatic regions.

a, Scatter plots showing the weaken of ADCP1 binding capacity on H3K9me2-marked regions in hmga mutant. b, The classification of ADCP1-targeted TEs in Col-0 and hmga background, the classification of all TEs in TAIR10 annotation is displayed as a control. c, The HMGA binding motifs found by MEME-ChIP analysis. d, H3K9me2 (red), ADCP1 in wild type (green) and hmga (blue), and HMGA (purple) ChIP-seq enrichment along Arabidopsis chromosomes 2-5. e, Snapshots of H3K9me2 (red), ADCP1 in wild type (green) and hmga (blue), and HMGA (purple) ChIP-seq signals on Arabidopsis LTR-TEs sites. ChIP-seq signals are showed as reads per kilobase per million mapped reads (RPKM).

Source data

Extended Data Fig. 6 The phase separation activity of ADCP1 and HMGA.

a,b, In vitro phase separation assay of full-length ADCP1-GFP and mC-HMGA proteins with H3K9me3 NA. The images in panels and show the DAPI (a) and merged (b) signals, respectively. Scale bars, 20 μm. c,d, ADCP1 (c) and HMGA (d) ___domain architecture and predictor of natural disordered regions (PONDR) score for intrinsic disorder regions (IDRs) (black line), >0.5 is considered disordered. e, Full length and truncated ADCP1-GFP and mCherry-HMGA proteins used in phase separation assays. f, The puncta formed by ADCP1-HMGA-H3K9me3 NA were quantitatively measured by Fluorescence Correlation Spectroscopy (FCS) system. FCS curves (left) and fluorescence trajectories (right) of condensates formed by H3K9me NA with ADCP1 and HMGA are shown. g, Hydrodynamic radii calculated from FCS curves shown in Fig. 4g. Error bars denote standard deviation (SD) of replicates.

Source data

Extended Data Fig. 7 The ADCP1-HMGA recognition pair triggers the formation of chromocenter-like condensates in rice and maize protoplasts.

a, BiFC assays using ADCP1 and HMGA homologs from soybean and tobacco in Col-0 protoplasts. Scale bar, 5 μm. b, Schematic diagram of the ADCP1-HMGA pair expression in rice and maize protoplasts. The Arabidopsis, rice and maize plant models were created with BioRender.com. c,d, The distribution change of ADCP1-mCherry, HMGA-GFP, and DAPI signals in the nuclei of rice (c) and maize (d) protoplasts over time. e-g, Transformation of ADCP1 protein alone does not affect the pattern of DAPI staining in the nuclei of tobacco (e), rice (f), or maize (g). The images were taken at 72h after transformation. Scale bar, 5 μm. At least 50 independent nuclei were observed in each experiment, and the representative ones were imaged. h, Relative expression of LTR-TEs in tobacco 84h after co-transformed ADCP1-GFP and mCherry-HMGA. The NbActin3 gene was used as the internal reference. Error bars denote standard deviation (SD) of three replicates. Two-tailed t tests.

Source data

Extended Data Fig. 8 The structure feature of CsaADCP1.

a, Domain predication results of CsaADCP1 from SMART (http://smart.embl-heidelberg.de), and snapshots showed the hidden HMG-like ___domain in CsaADCP1. b, Sequence alignment of the hidden HMG-like ___domain in CsaADCP1 and HMG proteins. The sequences alignment shows HMG-like ___domain in CsaADCP1 protein owns similar structure features with HMG proteins. c, Three examples from tobacco infection assays using AT-hooks-truncated CsaADCP1. Scale bar, 5 μm. d, CRISPR mediated knock out the CsaADCP1 in cucumber to confirm its function in chromocenter formation. The right panels show the enlarged nuclei, and the numbers mean percentage of nuclei that display DAPI distribution patterns as in the figures. Scale bars, 5 μm.

Extended Data Fig. 9 Introduction of AtADCP1 and AtHMGA promotes heterochromatin condensation and TE silencing.

a, Formation of chromocenter-like condensates in the AtADCP1 and AtHMGA co-transgenic plants of tomato. At least 50 independent nuclei were observed, and the representative ones were imaged. Scale bar, 2 μm. Right image shows line scans at the position depicted by white line. b, Nuclei of tomato without transgene as a control. Scale bar, 2 μm. Right image shows line scans at the position depicted by white line. c, Heatmaps of H3K9me2 and AtADCP1 ChIP-seq enrichment around their binding sites. d,e, ChIPseq snapshots show the binding of H3K9me2 and AtADCP1 at LTR-TE regions of tomato transgenic plants. ChIP-seq signals are showed as reads per kilobase per million mapped reads (RPKM). f, Relative expression of ADCP1 targeted TEs in tomato transgenic plants. The SlActin7 gene was used as the internal reference. Error bars denote standard deviation (SD) of three replicates. Two-tailed t tests. g, H3K9me2 immunostaining in nucleus of the AtADCP1 and AtHMGA co-transgenic plants of tobacco. Colors indicated the signals of ADCP1 (green), HMGA (magenta), H3K9me2 (cyan), and DNA stain (DAPI, grey). Scale bar, 2 μm. h, FISH with the Copia481 repeats in nucleus of the AtADCP1 and AtHMGA co-transgenic plants of tobacco. Colors indicated the signals of Copia481 (magenta) and DNA stain (DAPI, grey). Scale bar, 2 μm.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2, and Tables 1–4.

Reporting Summary

Supplementary Video 1

FRAP assay of the ADCP1 foci in ADCP1-GFP transgenic plant of wild-type background.

Supplementary Video 2

FRAP assay of the ADCP1 foci in ADCP1-GFP transgenic plant of hmga background.

Supplementary Video 3

FRAP assay of the HMGA foci in mCherry-HMGA transgenic plant of wild-type background.

Supplementary Video 4

FRAP of the ADCP1-HMGA complex foci in BiFC assay.

Source data

Source Data Figs. 2–6 and Extended Data Figs. 2, 4–7 and 9

Summary of all statistical source data.

Source Data Fig. 2

Unprocessed western blots of Fig. 2e.

Source Data Extended Data Fig. 3

Unprocessed western blots of Extended Data Fig. 3e.

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Zhang, W., Cheng, L., Li, K. et al. Evolutional heterochromatin condensation delineates chromocenter formation and retrotransposon silencing in plants. Nat. Plants 10, 1215–1230 (2024). https://doi.org/10.1038/s41477-024-01746-4

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