Abstract
Little is understood about how the two major types of heterochromatin domains (HP1 and Polycomb) are kept separate. In the yeast Cryptococcus neoformans, the Polycomb-like protein Ccc1 prevents deposition of H3K27me3 at HP1 domains. Here we show that phase separation propensity underpins Ccc1 function. Mutations of the two basic clusters in the intrinsically disordered region or deletion of the coiled-coil dimerization domain alter phase separation behavior of Ccc1 in vitro and have commensurate effects on formation of Ccc1 condensates in vivo, which are enriched for PRC2. Notably, mutations that alter phase separation trigger ectopic H3K27me3 at HP1 domains. Supporting a direct condensate-driven mechanism for fidelity, Ccc1 droplets efficiently concentrate recombinant C. neoformans PRC2 in vitro whereas HP1 droplets do so only weakly. These studies establish a biochemical basis for chromatin regulation in which mesoscale biophysical properties play a key functional role.
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Data availability
ChIP–seq data were deposited in the Gene Expression Omnibus under accession no. GSE195824. Live cell imaging data to quantify nuclear condensates were deposited in Figshare (https://figshare.com/s/ecee627ee1c7d05a91b0). Source data are provided with this paper.
Code availability
The customized Cellprofiler pipeline is described in Supplementary Table 1 and available at https://github.com/madhanicode/sujinlee_cellprofiler. The scripts used to analyze and generate graphs for ChIP-seq data are available at https://github.com/madhanicode/sujinlee_chipseq.
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Acknowledgements
We thank members of the Madhani laboratory for helpful discussions and G. J. Narlikar, R. Pappu, D. Canzio and P. A. Dumesic for critical reading of the manuscript. We thank S. Lei for help with making a figure, T. Lou for help with condensate assays, M. Jaime-Garza for help with the mass photometer, K. Herrington and S.Y. Kim at the Center for Advanced Light Microscopy (UCSF) for technical advice. Sequencing was performed at the UCSF CAT, supported by UCSF PBBR, RRP IMIA and National Institutes of Health (NIH; grant no. 1S10OD028511-01). This work was supported by the US NIH (grant no. R01GM71801 to H.D.M.).
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S.L. and H.D.M. designed the experiments and wrote the manuscript. H.D.M. supervised all aspects of this work. S.L. performed most of the experiments and analyzed the data. S.A.-A. and K.-J.A. performed and supervised purification of the recombinant PRC2 and histone methyltransferase assay. A.G. generated a customized CellProfiler pipeline to analyze nuclear foci. D.S.P. performed analysis of nuclear foci and ChIP–seq. M.Y.H. analyzed ChIP–seq. B.R. helped with protein purification and ChIP. J.K.D., J.J.M. and J.R.Y. performed, analyzed and supervised MS.
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Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Sara Osman and Dimitris Typas were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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Extended data
Extended Data Fig. 1 Ccc1 is not a tightly bound core subunit of PRC2 complex.
(a) Expression of endogenous Ccc1 and Ezh2 with a C-terminal CBP-2XFLAG tag, as assessed by western blotting using the antibodies indicated on the left. H3 serves as a loading control. Data are representative of two independent experiments. Protein interaction partners of (b) Ccc1 and (c) Ezh2. Each bait protein was purified by tandem affinity purification following DNase treatment and its protein interaction partners were determined by mass spectrometry. Subunits of the PRC2 complex are indicated in bold. Likely contaminants have been excluded (Supplementary Table 4).
Extended Data Fig. 2 Determination of oligomerization status of purified Ccc1 wild-type and mutants.
(a) Purified 6XHis-Ccc1 wild-type, C-terminally truncated Ccc1-CC∆ (1-434 amino acids), and IDR mutants. Data are representative of three independent experiments. Mass photometry analysis of (b) Ccc1 wild-type, (c) Ccc1-CC∆, (d) Ccc1 IDR mutants in 20 mM HEPES, pH 7.5, and 250 mM NaCl.
Extended Data Fig. 3 Ccc1 undergoes phase separation in vitro and forms liquid-like droplets.
(a) DIC images of concentration-dependent wild-type Ccc1 condensate formation in 20 mM HEPES, pH 7.5, and 250 mM NaCl. Scale bars, 10 µm. (b) Ccc1 condensate formation with or without prior nuclease treatment. For nuclease treatment, 20 µl of 20 µM Ccc1 was incubated with 1 µl of TURBO DNase (2 U/µl) and 1 µl of RNase A (10 mg/ml) at RT for 1 h. Scale bars, 10 µm. (c) Ccc1 condensate formation in the presence of 2.7 kbp DNA. Scale bars, 10 µm. (d) Salt-dependent Ccc1 condensate formation. Phase separation of 5 µM Ccc1 was induced for 30 min in 20 mM HEPES, pH 7.5, buffer containing 500, 250, and 150 mM NaCl. Scale bars, 10 µm. (e) Salt-dependent reversibility of Ccc1 condensate formation. After 10 min induction of condensate formation in 20 mM HEPES, pH 7.5, and 250 mM NaCl, NaCl concentration of buffer was adjusted to 500 mM. Scale bars, 10 µm. (f) After 10 min induction of condensate formation in 20 mM HEPES, pH 7.5, and 150 mM NaCl, NaCl concentration of buffer was adjusted to 250 mM. Scale bars, 10 µm. Data are representative of three (a, d, e, f) or two (b, c) independent experiments.
Extended Data Fig. 4 Ccc1 phase separation is programmed by two basic charged clusters in IDR.
(a) Ccc1 protein sequence. IDR, chromodomain, coiled-coil, and IDR mutation sites are as indicated. (b) Ccc1 charge distribution. (c) Concentration-dependent condensate formation of Ccc1 wild-type and IDR mutants in 20 mM HEPES, pH 7.5, and 250 mM NaCl after 2 h of plating. Scale bars, 10 µm. Data are representative of three independent experiments.
Extended Data Fig. 5 Ccc1 foci are enriched for Ezh2.
(a) Live cell images of Ccc1-2XEGFP foci in the wild-type and ezh2∆. Scale bars, 5 µm. (b) Live cell images of cells expressing 2XmNeonGreen-Ezh2 and Ccc1 variants tagged with 2XmCherry. Scale bars, 5 µm. Data in a and b are representative of three independent experiments.
Extended Data Fig. 6 Ccc1 foci are colocalized with H3K27me3.
(a) Live cell images of Ccc1 variants tagged with 2XmCherry. Scale bars, 5 µm. (b) Distribution of H3K27me3 in cells expressing Ccc1 variants tagged with 2XmCherry. Scale bars, 2 µm. Data in a and b are representative of three independent experiments.
Extended Data Fig. 7 ChIP-seq analysis of the replicate sequencing libraries.
(a) Average centromeric H3K27me3. (b) Average subtelomeric H3K27me3. (c) H3K27me3 at subtelomeric (blue bar) versus centromeric regions (green bar) as measured by ChIP-seq. Density (RPKM) of signal above background is reported. (d) ChIP-seq traces of H3K27me3 signal across chromosome 13 in cells expressing Ccc1 variants or cells lacking Ccc1 or Ezh2.
Extended Data Fig. 8 Purification of catalytically active PRC2 complex and Swi6.
(a) Schematic representation of the PRC2 coexpression construct. Each gene expression cassette contains a polyhedrin promoter (PPH), a cDNA of the PRC2 component, and an SV40 terminator (term). The cDNAs are tagged as follows: 3XStrep-TagII-HRV3C-EZH2, 9XHis-HRV3C-EED1, FLAG-HRV3C-BND1, and 6XHis-TEV-MSL1. (b) Agarose gel of SwaI-digested construct in (a). (c) Coomassie stained SDS-polyacrylamide gel of the purified recombinant PRC2. (d) (Left) SAH standard curve (Right) Histone Methyltransferase assay of the recombinant PRC2 on the nucleosome assembled with Xenopus histone H3 (28SAPAT32 replaced for 28QTTTSAAA34 of C. neoformans histone H3). (Mean ± SEM, n = 3 independent replicates) (e) Purified 6XHis-Swi6. (f) Mass photometry analysis of Swi6 in 20 mM HEPES, pH 7.5, and 150 mM NaCl. (g) DIC images of concentration-dependent Swi6 condensate formation in 20 mM HEPES, pH 7.5, and 125 mM NaCl 30 min after plating. Scale bars, 10 µm. (h) Condensate fusion of 189 µM Swi6 at indicated time points. Scale bars, 10 µm. Data in b, c, e, g, and h are representative of three independent experiments.
Supplementary information
Supplementary Information
Supplementary Table 2.
Supplementary Video 1
In vitro condensate formation of 23 µM Ccc1 wild-type over 2 h.
Supplementary Video 2
In vitro condensate formation of 23 µM Ccc1-4KRA over 2 h.
Supplementary Table 1
Supplementary Tables 1, 3 and 4.
Supplementary Video 3
The 3D images of Swi6-2xmCherry foci and Ccc1-2xEGFP foci in a single cell.
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Lee, S., Abini-Agbomson, S., Perry, D.S. et al. Intrinsic mesoscale properties of a Polycomb protein underpin heterochromatin fidelity. Nat Struct Mol Biol 30, 891–901 (2023). https://doi.org/10.1038/s41594-023-01000-z
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DOI: https://doi.org/10.1038/s41594-023-01000-z
