Intrinsic mesoscale properties of a Polycomb protein underpin heterochromatin fidelity.
Journal
Nature structural & molecular biology
ISSN: 1545-9985
Titre abrégé: Nat Struct Mol Biol
Pays: United States
ID NLM: 101186374
Informations de publication
Date de publication:
07 2023
07 2023
Historique:
received:
28
03
2022
accepted:
17
04
2023
medline:
19
7
2023
pubmed:
23
5
2023
entrez:
22
5
2023
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 37217653
doi: 10.1038/s41594-023-01000-z
pii: 10.1038/s41594-023-01000-z
doi:
Substances chimiques
Heterochromatin
0
Histones
0
Polycomb-Group Proteins
0
Chromatin
0
Drosophila Proteins
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
891-901Subventions
Organisme : NIH HHS
ID : S10 OD028511
Pays : United States
Informations de copyright
© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.
Références
Janssen, A., Colmenares, S. U. & Karpen, G. H. Heterochromatin: guardian of the genome. Annu. Rev. Cell Dev. Biol. 34, 265–288 (2018).
pubmed: 30044650
Allshire, R. C. & Madhani, H. D. Ten principles of heterochromatin formation and function. Nat. Rev. Mol. Cell Biol. 19, 229–244 (2018).
pubmed: 29235574
Larson, A. G. et al. Liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin. Nature 547, 236–240 (2017).
pubmed: 28636604
pmcid: 5606208
Strom, A. R. et al. Phase separation drives heterochromatin domain formation. Nature 547, 241–245 (2017).
pubmed: 28636597
pmcid: 6022742
Grau, D. J. et al. Compaction of chromatin by diverse Polycomb group proteins requires localized regions of high charge. Genes Dev. 25, 2210–2221 (2011).
pubmed: 22012622
pmcid: 3205590
Lau, M. S. et al. Mutation of a nucleosome compaction region disrupts Polycomb-mediated axial patterning. Science 355, 1081–1084 (2017).
pubmed: 28280206
pmcid: 5503153
Plys, A. J. et al. Phase separation of Polycomb-repressive complex 1 is governed by a charged disordered region of CBX2. Genes Dev. 33, 799–813 (2019).
pubmed: 31171700
pmcid: 6601514
Tatavosian, R. et al. Nuclear condensates of the Polycomb protein chromobox 2 (CBX2) assemble through phase separation. J. Biol. Chem. 294, 1451–1463 (2019).
pubmed: 30514760
Kim, J. & Kingston, R. E. The CBX family of proteins in transcriptional repression and memory. J. Biosci. 45, 16 (2020).
pubmed: 31965994
Gibson, B. A. et al. Organization of chromatin by intrinsic and regulated phase separation. Cell 179, 470–484.e21 (2019).
pubmed: 31543265
pmcid: 6778041
Strickfaden, H. et al. Condensed chromatin behaves like a solid on the mesoscale in vitro and in living cells. Cell 183, 1772–1784.e13 (2020).
pubmed: 33326747
Wang, L. et al. Histone modifications regulate chromatin compartmentalization by contributing to a phase separation mechanism. Mol. Cell 76, 646–659.e6 (2019).
pubmed: 31543422
Narlikar, G. J. Phase-separation in chromatin organization. J. Biosci. 45, 5 (2020).
pubmed: 31965983
pmcid: 9107952
Erdel, F. et al. Mouse heterochromatin adopts digital compaction states without showing hallmarks of HP1-driven liquid–liquid phase separation. Mol. Cell 78, 236–249.e7 (2020).
pubmed: 32101700
pmcid: 7163299
Dumesic, P. A. et al. Product binding enforces the genomic specificity of a yeast polycomb repressive complex. Cell 160, 204–218 (2015).
pubmed: 25533783
Hyman, A. A., Weber, C. A. & Jülicher, F. Liquid–liquid phase separation in biology. Annu. Rev. Cell Dev. Biol. 30, 39–58 (2014).
pubmed: 25288112
Banani, S. F., Lee, H. O., Hyman, A. A. & Rosen, M. K. Biomolecular condensates: organizers of cellular biochemistry. Nat. Rev. Mol. Cell Biol. 18, 285–298 (2017).
pubmed: 28225081
pmcid: 7434221
Alberti, S., Gladfelter, A. & Mittag, T. Considerations and challenges in studying liquid–liquid phase separation and biomolecular condensates. Cell 176, 419–434 (2019).
pubmed: 30682370
pmcid: 6445271
Patel, S. S., Belmont, B. J., Sante, J. M. & Rexach, M. F. Natively unfolded nucleoporins gate protein diffusion across the nuclear pore complex. Cell 129, 83–96 (2007).
pubmed: 17418788
Zhang, Q. et al. Visualizing dynamics of cell signaling in vivo with a phase separation-based kinase reporter. Mol. Cell 69, 334–346.e4 (2018).
pubmed: 29307513
pmcid: 5788022
Qamar, S. et al. FUS phase separation is modulated by a molecular chaperone and methylation of arginine cation-π interactions. Cell 173, 720–734.e15 (2018).
pubmed: 29677515
pmcid: 5927716
Wang, J. et al. A molecular grammar governing the driving forces for phase separation of prion-like RNA binding proteins. Cell 174, 688–699.e16 (2018).
pubmed: 29961577
pmcid: 6063760
Simon, J. R., Carroll, N. J., Rubinstein, M., Chilkoti, A. & López, G. P. Programming molecular self-assembly of intrinsically disordered proteins containing sequences of low complexity. Nat. Chem. 9, 509–515 (2017).
pubmed: 28537592
pmcid: 5597244
Choi, J.-M., Holehouse, A. S. & Pappu, R. V. Physical principles underlying the complex biology of intracellular phase transitions. Annu. Rev. Biophys. 49, 107–133 (2020).
pubmed: 32004090
Brangwynne, C. P., Tompa, P. & Pappu, R. V. Polymer physics of intracellular phase transitions. Nat. Phys. 11, 899–904 (2015).
Harmon, T. S., Holehouse, A. S., Rosen, M. K. & Pappu, R. V. Intrinsically disordered linkers determine the interplay between phase separation and gelation in multivalent proteins. eLife 6, e30294 (2017).
pubmed: 29091028
pmcid: 5703641
Ranganathan, S. & Shakhnovich, E. I. Dynamic metastable long-living droplets formed by sticker-spacer proteins. eLife 9, e56159 (2020).
pubmed: 32484438
pmcid: 7360371
Boeynaems, S. et al. Spontaneous driving forces give rise to protein−RNA condensates with coexisting phases and complex material properties. Proc. Natl Acad. Sci. USA 116, 7889–7898 (2019).
pubmed: 30926670
pmcid: 6475405
Fan, Y. & Lin, X. Multiple applications of a transient CRISPR–Cas9 coupled with electroporation (TRACE) system in the Cryptococcus neoformans species complex. Genetics 208, 1357–1372 (2018).
pubmed: 29444806
pmcid: 5887135
Huang, M. Y. et al. Short homology-directed repair using optimized Cas9 in the pathogen Cryptococcus neoformans enables rapid gene deletion and tagging. Genetics 220, iyab180 (2022).
pubmed: 34791226
Walther, T. C. et al. The conserved Nup107–160 complex Is critical for nuclear pore complex assembly. Cell 113, 195–206 (2003).
pubmed: 12705868
Ebrahimi, H. & Cooper, J. P. Finding a place in the SUN: telomere maintenance in a diverse nuclear landscape. Curr. Opin. Cell Biol. 40, 145–152 (2016).
pubmed: 27064212
pmcid: 6284805
McQuin, C. et al. CellProfiler 3.0: next-generation image processing for biology. PLoS Biol. 16, e2005970 (2018).
pubmed: 29969450
pmcid: 6029841
Weissmann, F. et al. biGBac enables rapid gene assembly for the expression of large multisubunit protein complexes. Proc. Natl Acad. Sci. USA 113, E2564–E2569 (2016).
pubmed: 27114506
pmcid: 4868461
Sanulli, S. et al. HP1 reshapes nucleosome core to promote phase separation of heterochromatin. Nature 575, 390–394 (2019).
pubmed: 31618757
pmcid: 7039410
Keenen, M. M. et al. HP1 proteins compact DNA into mechanically and positionally stable phase separated domains. eLife 10, e64563 (2021).
pubmed: 33661100
pmcid: 7932698
Fan, H. et al. BAHCC1 binds H3K27me3 via a conserved BAH module to mediate gene silencing and oncogenesis. Nat. Genet. 52, 1384–1396 (2020).
pubmed: 33139953
pmcid: 8330957
Seif, E. et al. Phase separation by the polyhomeotic sterile alpha motif compartmentalizes Polycomb group proteins and enhances their activity. Nat. Commun. 11, 5609 (2020).
pubmed: 33154383
pmcid: 7644731
Huseyin, M. K. & Klose, R. J. Live-cell single particle tracking of PRC1 reveals a highly dynamic system with low target site occupancy. Nat. Commun. 12, 887 (2021).
pubmed: 33563969
pmcid: 7873255
Eeftens, J. M., Kapoor, M., Michieletto, D. & Brangwynne, C. P. Polycomb condensates can promote epigenetic marks but are not required for sustained chromatin compaction. Nat. Commun. 12, 5888 (2021).
pubmed: 34620850
pmcid: 8497513
Grau, D. et al. Structures of monomeric and dimeric PRC2:EZH1 reveal flexible modules involved in chromatin compaction. Nat. Commun. 12, 714 (2021).
pubmed: 33514705
pmcid: 7846606
Zhang, Y. et al. Nuclear condensates of p300 formed though the structured catalytic core can act as a storage pool of p300 with reduced HAT activity. Nat. Commun. 12, 4618 (2021).
pubmed: 34326347
pmcid: 8322156
Darzacq, X. & Tjian, R. Weak multivalent biomolecular interactions: a strength versus numbers tug of war with implications for phase partitioning. RNA 28, 48–51 (2022).
pubmed: 34772790
pmcid: 8675282
McSwiggen, D. T., Mir, M., Darzacq, X. & Tjian, R. Evaluating phase separation in live cells: diagnosis, caveats, and functional consequences. Genes Dev. 33, 1619–1634 (2019).
pubmed: 31594803
pmcid: 6942051
Chun, C. D. & Madhani, H. D. Applying genetics and molecular biology to the study of the human pathogen Cryptococcus neoformans. Methods Enzymol. 470, 797–831 (2010).
pubmed: 20946836
pmcid: 3611884
Burke, J. E. et al. Spliceosome profiling visualizes operations of a dynamic RNP at nucleotide resolution. Cell 173, 1014–1030.e17 (2018).
pubmed: 29727661
pmcid: 5940017
Erdős, G., Pajkos, M. & Dosztányi, Z. IUPred3: prediction of protein disorder enhanced with unambiguous experimental annotation and visualization of evolutionary conservation. Nucleic Acids Res. 49, W297–W303 (2021).
pubmed: 34048569
pmcid: 8262696
Jones, P. et al. InterProScan 5: genome-scale protein function classification. Bioinformatics 30, 1236–1240 (2014).
pubmed: 24451626
pmcid: 3998142
Simm, D., Hatje, K. & Kollmar, M. Waggawagga: comparative visualization of coiled-coil predictions and detection of stable single α-helices (SAH domains). Bioinformatics 31, 767–769 (2015).
pubmed: 25338722
Trigg, J., Gutwin, K., Keating, A. E. & Berger, B. Multicoil2: predicting coiled coils and their oligomerization states from sequence in the twilight zone. PLoS ONE 6, e23519 (2011).
pubmed: 21901122
pmcid: 3162000
Holehouse, A. S., Das, R. K., Ahad, J. N., Richardson, M. O. G. & Pappu, R. V. CIDER: resources to analyze sequence-ensemble relationships of intrinsically disordered proteins. Biophys. J. 112, 16–21 (2017).
pubmed: 28076807
pmcid: 5232785
Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17, 10 (2011).
Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).
pubmed: 19261174
pmcid: 2690996
Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
pubmed: 19505943
pmcid: 2723002
Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).
pubmed: 20110278
pmcid: 2832824
Koulouras, G. et al. EasyFRAP-web: a web-based tool for the analysis of fluorescence recovery after photobleaching data. Nucleic Acids Res. 46, W467–W472 (2018).
pubmed: 29901776
pmcid: 6030846