Intrinsically disordered sequences in the Polycomb protein Polyhomeotic regulate condensate formation

Polycomb Protein ◽

Size Number ◽

Condensate Formation ◽

Polycomb Bodies

The Polycomb group (PcG) complex PRC1 localizes in the nucleus in the form of condensed structures called Polycomb bodies. The PRC1 subunit Polyhomeotic (Ph) contains a polymerizing sterile alpha motif (SAM) that is implicated in both PcG body formation and chromatin organization in Drosophila and mammalian cells. A truncated version of Ph containing the SAM (mini-Ph), forms phase separated condensates with DNA or chromatin in vitro, suggesting PcG bodies may form by phase separation. In cells, Ph forms multiple condensates, while mini-Ph forms a single large nuclear condensate. We therefore hypothesize that sequences outside of mini-Ph are required for proper condensate formation. We identified three distinct Intrinsically Disordered Regions (IDRs) in Ph based on sequence composition and complexity. We tested the role of each IDR in Ph condensates using live imaging of transfected Drosophila S2 cells. We find that each IDR uniquely affects Ph SAM-dependent condensate size, number, and morphology.

Intrinsically disordered sequences in the Polycomb protein Polyhomeotic regulate condensate formation

10.21203/rs.3.rs-955684/v1 ◽

2021 ◽

Author(s):

Ibani Kapur ◽

Elodie Boulier ◽

Nicole Francis

Keyword(s):

Mammalian Cells ◽

Polycomb Group ◽

Sequence Composition ◽

Polycomb Protein ◽

Size Number ◽

Condensate Formation ◽

Polycomb Bodies

Abstract The Polycomb group (PcG) complex PRC1 localizes in the nucleus in the form of condensed structures called Polycomb bodies. The PRC1 subunit Polyhomeotic (Ph) contains a polymerizing sterile alpha motif (SAM) that is implicated in both PcG body formation and chromatin organization in Drosophila and mammalian cells. A truncated version of Ph containing the SAM (mini-Ph), forms phase separated condensates with DNA or chromatin in vitro, suggesting PcG bodies may form by phase separation. In cells, Ph forms multiple condensates, while mini-Ph forms a single large nuclear condensate. We therefore hypothesize that sequences outside of mini-Ph are required for proper condensate formation. We identified three distinct Intrinsically Disordered Regions (IDRs) in Ph based on sequence composition and complexity. We tested the role of each IDR in Ph condensates using live imaging of transfected Drosophila S2 cells. We find that each IDR uniquely affects Ph SAM-dependent condensate size, number, and morphology.

Nuclear condensates of the Polycomb protein chromobox 2 (CBX2) assemble through phase separation

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.006620 ◽

2018 ◽

Vol 294 (5) ◽

pp. 1451-1463 ◽

Cited By ~ 92

Author(s):

Roubina Tatavosian ◽

Samantha Kent ◽

Kyle Brown ◽

Tingting Yao ◽

Huy Nguyen Duc ◽

...

Keyword(s):

Phase Separation ◽

Polycomb Group ◽

Site Directed Mutagenesis ◽

Developmental Defects ◽

Heterochromatin Formation ◽

Polycomb Protein ◽

Condensate Formation

Polycomb group (PcG) proteins repress master regulators of development and differentiation through organization of chromatin structure. Mutation and dysregulation of PcG genes cause developmental defects and cancer. PcG proteins form condensates in the cell nucleus, and these condensates are the physical sites of PcG-targeted gene silencing via formation of facultative heterochromatin. However, the physiochemical principles underlying the formation of PcG condensates remain unknown, and their determination could shed light on how these condensates compact chromatin. Using fluorescence live-cell imaging, we observed that the Polycomb repressive complex 1 (PRC1) protein chromobox 2 (CBX2), a member of the CBX protein family, undergoes phase separation to form condensates and that the CBX2 condensates exhibit liquid-like properties. Using site-directed mutagenesis, we demonstrated that the conserved residues of CBX2 within the intrinsically disordered region (IDR), which is the region for compaction of chromatin in vitro, promote the condensate formation both in vitro and in vivo. We showed that the CBX2 condensates concentrate DNA and nucleosomes. Using genetic engineering, we report that trimethylation of Lys-27 at histone H3 (H3K27me3), a marker of heterochromatin formation produced by PRC2, had minimal effects on the CBX2 condensate formation. We further demonstrated that the CBX2 condensate formation does not require CBX2–PRC1 subunits; however, the condensate formation of CBX2–PRC1 subunits depends on CBX2, suggesting a mechanism underlying the assembly of CBX2–PRC1 condensates. In summary, our results reveal that PcG condensates assemble through liquid–liquid phase separation (LLPS) and suggest that phase-separated condensates can organize PcG-bound chromatin.

Polycomb Cbx2 Condensates Assemble through Phase Separation

10.1101/468926 ◽

2018 ◽

Cited By ~ 1

Author(s):

Roubina Tatavosian ◽

Samantha Kent ◽

Kyle Brown ◽

Tingting Yao ◽

Huy Nguyen Duc ◽

...

Keyword(s):

Phase Separation ◽

Polycomb Group ◽

Developmental Defects ◽

Condensate Formation ◽

Development And Differentiation ◽

Polycomb Repressive Complex 1 ◽

Liquid Liquid Phase Separation

AbstractPolycomb group (PcG) proteins are master regulators of development and differentiation. Mutation and dysregulation of PcG genes cause developmental defects and cancer. PcG proteins form condensates in the nucleus of cells and these condensates are the physical sites of PcG-targeted gene silencing. However, the physiochemical principles underlying the PcG condensate formation remain unknown. Here we show that Polycomb repressive complex 1 (PRC1) protein Cbx2, one member of the Cbx family proteins, contains a long stretch of intrinsically disordered region (IDR). Cbx2 undergoes phase separation to form condensates. Cbx2 condensates exhibit liquid-like properties and can concentrate DNA and nucleosomes. We demonstrate that the conserved residues within the IDR promote the condensate formation in vitro and in vivo. We further indicate that H3K27me3 has minimal effects on the Cbx2 condensate formation while depletion of core PRC1 subunits facilitates the condensate formation. Thus, our results reveal that PcG condensates assemble through liquid-liquid phase separation (LLPS) and suggest that PcG-bound chromatin is in part organized through phase-separated condensates.

Molecular determinants of phase separation forDrosophilaDNA replication licensing factors

10.1101/2021.04.12.439485 ◽

2021 ◽

Author(s):

Matthew W. Parker ◽

Jonchee Kao ◽

Alvin Huang ◽

James M. Berger ◽

Michael R. Botchan

Keyword(s):

Phase Separation ◽

Self Assembly ◽

A Priori ◽

Initiation Factor ◽

Sequence Composition ◽

Partner Proteins

ABSTRACTLiquid-liquid phase separation (LLPS) of intrinsically disordered regions (IDRs) in proteins can drive the formation of membraneless compartments in cells. Phase-separated structures enrich for specific partner proteins and exclude others. We have shown that the IDRs of metazoan DNA replication initiators drive DNA-dependent phase separationin vitroand chromosome bindingin vivo, and that initiator condensates selectively recruit specific partner proteins. How initiator IDRs facilitate LLPS and maintain compositional specificity is unknown. UsingD. melanogaster (Dm)Cdt1 as a model initiation factor, we show that phase separation results from a synergy between electrostatic DNA-bridging interactions and hydrophobic inter-IDR contacts. Both sets of interactions depend on sequence composition (but not sequence order), are resistant to 1,6- hexanediol, and do not depend on aromaticity. These findings demonstrate that distinct sets of interactions drive self-assembly and condensate specificity across different phase-separating systems and advance efforts to predict IDR LLPS propensity and specificitya priori.

Molecular determinants of phase separation for Drosophila DNA replication licensing factors

eLife ◽

10.7554/elife.70535 ◽

2021 ◽

Vol 10 ◽

Author(s):

Matthew W Parker ◽

Jonchee A Kao ◽

Alvin Huang ◽

James M Berger ◽

Michael R Botchan

Keyword(s):

Phase Separation ◽

Dna Replication ◽

A Priori ◽

Initiation Factor ◽

Sequence Composition ◽

Partner Proteins

Liquid-liquid phase separation (LLPS) of intrinsically disordered regions (IDRs) in proteins can drive the formation of membraneless compartments in cells. Phase-separated structures enrich for specific partner proteins and exclude others. Previously, we showed that the IDRs of metazoan DNA replication initiators drive DNA-dependent phase separation in vitro and chromosome binding in vivo, and that initiator condensates selectively recruit replication-specific partner proteins (Parker et al., 2019). How initiator IDRs facilitate LLPS and maintain compositional specificity is unknown. Here, using D. melanogaster (Dm) Cdt1 as a model initiation factor, we show that phase separation results from a synergy between electrostatic DNA-bridging interactions and hydrophobic inter-IDR contacts. Both sets of interactions depend on sequence composition (but not sequence order), are resistant to 1,6-hexanediol, and do not depend on aromaticity. These findings demonstrate that distinct sets of interactions drive condensate formation and specificity across different phase-separating systems and advance efforts to predict IDR LLPS propensity and partner selection a priori.

Cavin1 intrinsically disordered domains are essential for fuzzy electrostatic interactions and caveola formation

Nature Communications ◽

10.1038/s41467-021-21035-4 ◽

2021 ◽

Vol 12 (1) ◽

Cited By ~ 3

Author(s):

Vikas A. Tillu ◽

James Rae ◽

Ya Gao ◽

Nicholas Ariotti ◽

Matthias Floetenmeyer ◽

...

Keyword(s):

Electrostatic Interactions ◽

Membrane Lipid ◽

Membrane Curvature ◽

Caveolin 1 ◽

Solution Generation ◽

Endosomal System ◽

Disordered Regions

AbstractCaveolae are spherically shaped nanodomains of the plasma membrane, generated by cooperative assembly of caveolin and cavin proteins. Cavins are cytosolic peripheral membrane proteins with negatively charged intrinsically disordered regions that flank positively charged α-helical regions. Here, we show that the three disordered domains of Cavin1 are essential for caveola formation and dynamic trafficking of caveolae. Electrostatic interactions between disordered regions and α-helical regions promote liquid-liquid phase separation behaviour of Cavin1 in vitro, assembly of Cavin1 oligomers in solution, generation of membrane curvature, association with caveolin-1, and Cavin1 recruitment to caveolae in cells. Removal of the first disordered region causes irreversible gel formation in vitro and results in aberrant caveola trafficking through the endosomal system. We propose a model for caveola assembly whereby fuzzy electrostatic interactions between Cavin1 and caveolin-1 proteins, combined with membrane lipid interactions, are required to generate membrane curvature and a metastable caveola coat.

The CTCF Insulator Protein Is Posttranslationally Modified by SUMO

Molecular and Cellular Biology ◽

10.1128/mcb.00825-08 ◽

2008 ◽

Vol 29 (3) ◽

pp. 714-725 ◽

Cited By ~ 81

Author(s):

Melissa J. MacPherson ◽

Linda G. Beatty ◽

Wenjing Zhou ◽

Minjie Du ◽

Paul D. Sadowski

Keyword(s):

Posttranslational Modifications ◽

Posttranslational Modification ◽

Zinc Finger Protein ◽

Finger Protein ◽

Ability To Act ◽

Polycomb Protein ◽

P2 Promoter ◽

Polycomb Bodies

ABSTRACT The CTCF protein is a highly conserved zinc finger protein that is implicated in many aspects of gene regulation and nuclear organization. Its functions include the ability to act as a repressor of genes, including the c-myc oncogene. In this paper, we show that the CTCF protein can be posttranslationally modified by the small ubiquitin-like protein SUMO. CTCF is SUMOylated both in vivo and in vitro, and we identify two major sites of SUMOylation in the protein. The posttranslational modification of CTCF by the SUMO proteins does not affect its ability to bind to DNA in vitro. SUMOylation of CTCF contributes to the repressive function of CTCF on the c-myc P2 promoter. We also found that CTCF and the repressive Polycomb protein, Pc2, are colocalized to nuclear Polycomb bodies. The Pc2 protein may act as a SUMO E3 ligase for CTCF, strongly enhancing its modification by SUMO 2 and 3. These studies expand the repertoire of posttranslational modifications of CTCF and suggest roles for such modifications in its regulation of epigenetic states.

A new class of disordered elements controls DNA replication through initiator self-assembly

eLife ◽

10.7554/elife.48562 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 22

Author(s):

Matthew W Parker ◽

Maren Bell ◽

Mustafa Mir ◽

Jonchee A Kao ◽

Xavier Darzacq ◽

...

Keyword(s):

Dna Replication ◽

Self Assembly ◽

Origin Recognition Complex ◽

Replication Initiation ◽

Linear Arrangement ◽

New Class ◽

Liquid Liquid Phase Separation

The initiation of DNA replication in metazoans occurs at thousands of chromosomal sites known as origins. At each origin, the Origin Recognition Complex (ORC), Cdc6, and Cdt1 co-assemble to load the Mcm2-7 replicative helicase onto chromatin. Current replication models envisage a linear arrangement of isolated origins functioning autonomously; the extent of inter-origin organization and communication is unknown. Here, we report that the replication initiation machinery of D. melanogaster unexpectedly undergoes liquid-liquid phase separation (LLPS) upon binding DNA in vitro. We find that ORC, Cdc6, and Cdt1 contain intrinsically disordered regions (IDRs) that drive LLPS and constitute a new class of phase separating elements. Initiator IDRs are shown to regulate multiple functions, including chromosome recruitment, initiator-specific co-assembly, and Mcm2-7 loading. These data help explain how CDK activity controls replication initiation and suggest that replication programs are subject to higher-order levels of inter-origin organization.

BAR scaffolds drive membrane fission by crowding disordered domains

10.1101/276147 ◽

2018 ◽

Cited By ~ 1

Author(s):

Wilton T. Snead ◽

Wade F. Zeno ◽

Grace Kago ◽

Ryan W. Perkins ◽

J Blair Richter ◽

...

Keyword(s):

Cellular Membranes ◽

Bar Domain ◽

Membrane Fission ◽

Cell Assays ◽

Membrane Tubules ◽

Bar Domains ◽

Lipid Tubules

SummaryCylindrical protein scaffolds are thought to stabilize membrane tubules, preventing membrane fission. In contrast, Snead et al. find that when scaffold proteins assemble, bulky disordered domains within them become acutely concentrated, generating steric pressure that destabilizes tubules, driving fission.AbstractCellular membranes are continuously remodeled. The crescent-shaped bin-amphiphysinrvs (BAR) domains remodel membranes in multiple cellular pathways. Based on studies of BAR domains in isolation, the current paradigm is that they polymerize into cylindrical scaffolds that stabilize lipid tubules, preventing membrane fission. But in nature BAR domains are often part of multi-domain proteins that contain large intrinsically-disordered regions. Using in vitro and live cell assays, here we show that full-length BAR domain-containing proteins, rather than stabilizing membrane tubules, are instead surprisingly potent drivers of membrane fission. Specifically, when BAR scaffolds assemble at membrane surfaces, their bulky disordered domains become crowded, generating steric pressure that destabilizes lipid tubules. More broadly, we observe this behavior with BAR domains that have a range of curvatures. These data challenge the idea that cellular membranes adopt the curvature of BAR scaffolds, suggesting instead that the ability to concentrate disordered domains is the key requirement for membrane remodeling and fission by BAR domain-containing proteins.

Ubiquitin-independent protein degradation in proteasomes

Biomeditsinskaya Khimiya ◽

10.18097/pbmc20186402134 ◽

2018 ◽

Vol 64 (2) ◽

pp. 134-148 ◽

Cited By ~ 5

Author(s):

O.A. Buneeva ◽

A.E. Medvedev

Keyword(s):

Protein Degradation ◽

Mammalian Cells ◽

Intrinsically Disordered Proteins ◽

Amino Acid Sequences ◽

Disordered Proteins ◽

20S Proteasome ◽

Independent Manner ◽

Independent Protein

Proteasomes are large supramolecular protein complexes present in all prokaryotic and eukaryotic cells, where they perform targeted degradation of intracellular proteins. Until recently, it was generally accepted that prior proteolytic degradation in proteasomes the proteins had to be targeted by ubiquitination: the ATP-dependent addition of (typically four sequential) residues of the low-molecular ubiquitin protein, involving the ubiquitin-activating enzyme, ubiquitin-conjugating enzyme and ubiquitin ligase. The cytoplasm and nucleoplasm proteins labeled in this way are then digested in 26S proteasomes. However, in recent years it has become increasingly clear that using this route the cell eliminates only a part of unwanted proteins. Many proteins can be cleaved by the 20S proteasome in an ATP-independent manner and without previous ubiquitination. Ubiquitin-independent protein degradation in proteasomes is a relatively new area of studies of the role of the ubiquitin-proteasome system. However, recent data obtained in this direction already correct existing concepts about proteasomal degradation of proteins and its regulation. Ubiquitin-independent proteasome degradation needs the main structural precondition in proteins: the presence of unstructured regions in the amino acid sequences that provide interaction with the proteasome. Taking into consideration that in humans almost half of all genes encode proteins that contain a certain proportion of intrinsically disordered regions, it appears that the list of proteins undergoing ubiquitin-independent degradation will demonstrate further increase. Since 26S of proteasomes account for only 30% of the total proteasome content in mammalian cells, most of the proteasomes exist in the form of 20S complexes. The latter suggests that ubiquitin-independent proteolysis performed by the 20S proteasome is a natural process of removing damaged proteins from the cell and maintaining a constant level of intrinsically disordered proteins. In this case, the functional overload of proteasomes in aging and/or other types of pathological processes, if it is not accompanied by triggering more radical mechanisms for the elimination of damaged proteins, organelles and whole cells, has the most serious consequences for the whole organism.