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1.
Dev Cell ; 56(18): 2592-2606.e7, 2021 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-34508658

RESUMO

Membrane contact between intracellular organelles is important in mediating organelle communication. However, the assembly of molecular machinery at membrane contact site and its internal organization correlating with its functional activity remain unclear. Here, we demonstrate that a gel-like condensation of Cidec, a crucial protein for obesity development by facilitating lipid droplet (LD) fusion, occurs at the LD-LD contact site (LDCS) through phase separation. The homomeric interaction between the multivalent N terminus of Cidec is sufficient to promote its phase separation both in vivo and in vitro. Interestingly, Cidec condensation at LDCSs generates highly plastic and lipid-permeable fusion plates that are geometrically constrained by donor LDs. In addition, Cidec condensates are distributed unevenly in the fusion plate generating stochastic sub-compartments that may represent unique lipid passageways during LD fusion. We have thus uncovered the organization and functional significance of geometry-constrained Cidec phase separation in mediating LD fusion and lipid homeostasis.

2.
Cell Rep ; 36(5): 109482, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34297909

RESUMO

Bearing a relatively large single-stranded RNA genome in nature, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes sophisticated replication/transcription complexes (RTCs), mainly composed of a network of nonstructural proteins and nucleocapsid protein, to establish efficient infection. In this study, we develop an innovative interaction screening strategy based on phase separation in cellulo, namely compartmentalization of protein-protein interactions in cells (CoPIC). Utilizing CoPIC screening, we map the interaction network among RTC-related viral proteins. We identify a total of 47 binary interactions among 14 proteins governing replication, discontinuous transcription, and translation of coronaviruses. Further exploration via CoPIC leads to the discovery of extensive ternary complexes composed of these components, which infer potential higher-order complexes. Taken together, our results present an efficient and robust interaction screening strategy, and they indicate the existence of a complex interaction network among RTC-related factors, thus opening up opportunities to understand SARS-CoV-2 biology and develop therapeutic interventions for COVID-19.


Assuntos
COVID-19/virologia , Mapeamento de Interação de Proteínas/métodos , Proteoma , SARS-CoV-2/patogenicidade , Proteínas não Estruturais Virais/fisiologia , Animais , Células CACO-2 , Compartimento Celular , Linhagem Celular , Chlorocebus aethiops , Células HEK293 , Humanos , Mapas de Interação de Proteínas , Células Vero , Replicação Viral
3.
Dev Cell ; 56(9): 1313-1325.e7, 2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33891898

RESUMO

Cells sense and respond to extracellular mechanical cues through cell-matrix adhesions. Interestingly, the maturation of focal adhesions (FAs) is reciprocally force dependent. How biomechanical cues dictate the status of cell motility and how FAs spatial temporally coordinate force sensing and self-organization remain enigmatic. Here, we identify that LIMD1, a member of the LIM domain scaffolding proteins, undergoes force-sensitive condensation at the FAs. We also unveil that the multivalent interactions of LIMD1 intrinsically disordered region (IDR) and the LIM domains concertedly drive this phase transition under the regulation of phosphorylation. Intriguingly, formation of condensed LIMD1 protein compartments is sufficient to specifically enrich and localize late FA proteins. We further discover that LIMD1 regulates cell spreading, maintains FA dynamics and cellular contractility, and is critical for durotaxis-the ability of cells to crawl along gradients of substrate stiffness. Our results suggest a model that recruitment of LIMD1 to the FAs, via mechanical force triggered inter-molecular interaction, serves as a phase separation hub to assemble and organize matured FAs, thus allowing for efficient mechano-transduction and cell migration.


Assuntos
Movimento Celular , Matriz Extracelular/metabolismo , Adesões Focais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , Mecanotransdução Celular , Animais , Fenômenos Biomecânicos , Linhagem Celular Tumoral , Células HEK293 , Humanos , Camundongos , Modelos Biológicos , Paxilina/metabolismo , Fosforilação
5.
Biomed Pharmacother ; 138: 111520, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33765580

RESUMO

In the physical sciences, solid, liquid, and gas are the most familiar phase states, whose essence is their existence reflecting the different spatial distribution of molecular components. The biological molecules in the living cell also have differences in spatial distribution. The molecules organized in the form of membrane-bound organelles are well recognized. However, the biomolecules organized in membraneless compartments called biomolecular condensates remain elusive. The liquid-liquid phase separation (LLPS), as a new emerging scientific breakthrough, describes the biomolecules assembled in special distribution and appeared as membraneless condensates in the form of a new "phase" compared with the surrounding liquid milieu. LLPS provides an important theoretical basis for explaining the composition of biological molecules and related biological reactions. Mounting evidence has emerged recently that phase-separated condensates participate in various biological activities. This article reviews the occurrence of LLPS and underlying regulatory mechanisms for understanding how multivalent molecules drive phase transitions to form the biomolecular condensates. And, it also summarizes recent major progress in elucidating the roles of LLPS in chromatin organization and provides clues for the development of new innovative therapeutic strategies for related diseases.


Assuntos
Cromatina/isolamento & purificação , Cromatina/metabolismo , Extração Líquido-Líquido/métodos , Organelas/metabolismo , Transição de Fase , Animais , Fenômenos Biofísicos/fisiologia , Cromatina/química , Cromatina/genética , Humanos , Organelas/química , Organelas/genética
6.
Mol Cell ; 81(5): 896-898, 2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33667379

RESUMO

In this issue of Molecular Cell, Rawat et al. (2021) characterize novel stress-induced condensates of the negative elongation factor (NELF) as the nuclear counterparts of cytosolic stress granules. This provides a new perspective on transcription repression orchestrated by phase separation.


Assuntos
Núcleo Celular , Fatores de Transcrição , Núcleo Celular/metabolismo , Regulação para Baixo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
7.
Nat Commun ; 12(1): 1491, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33674598

RESUMO

Abnormally formed FUS/EWS/TAF15 (FET) fusion oncoproteins are essential oncogenic drivers in many human cancers. Interestingly, at the molecular level, they also form biomolecular condensates at specific loci. However, how these condensates lead to gene transcription and how features encoded in the DNA element regulate condensate formation remain unclear. Here, we develop an in vitro single-molecule assay to visualize phase separation on DNA. Using this technique, we observe that FET fusion proteins undergo phase separation at target binding loci and the phase separated condensates recruit RNA polymerase II and enhance gene transcription. Furthermore, we determine a threshold number of fusion-binding DNA elements that can enhance the formation of FET fusion protein condensates. These findings suggest that FET fusion oncoprotein promotes aberrant gene transcription through loci-specific phase separation, which may contribute to their oncogenic transformation ability in relevant cancers, such as sarcomas and leukemia.


Assuntos
Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Transcrição Genética , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , RNA Polimerase II/metabolismo , Proteína EWS de Ligação a RNA , Proteína FUS de Ligação a RNA
8.
Nat Chem Biol ; 17(5): 549-557, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33633378

RESUMO

How aerobic organisms exploit inevitably generated but potentially dangerous reactive oxygen species (ROS) to benefit normal life is a fundamental biological question. Locally accumulated ROS have been reported to prime stem cell differentiation. However, the underlying molecular mechanism is unclear. Here, we reveal that developmentally produced H2O2 in plant shoot apical meristem (SAM) triggers reversible protein phase separation of TERMINATING FLOWER (TMF), a transcription factor that times flowering transition in the tomato by repressing pre-maturation of SAM. Cysteine residues within TMF sense cellular redox to form disulfide bonds that concatenate multiple TMF molecules and elevate the amount of intrinsically disordered regions to drive phase separation. Oxidation triggered phase separation enables TMF to bind and sequester the promoter of a floral identity gene ANANTHA to repress its expression. The reversible transcriptional condensation via redox-regulated phase separation endows aerobic organisms with the flexibility of gene control in dealing with developmental cues.


Assuntos
Flores/genética , Regulação da Expressão Gênica de Plantas , Lycopersicon esculentum/genética , Proteínas de Plantas/genética , RNA de Plantas/genética , Espécies Reativas de Oxigênio/metabolismo , Agrobacterium/genética , Agrobacterium/metabolismo , Flores/crescimento & desenvolvimento , Flores/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hidroponia/métodos , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Lycopersicon esculentum/crescimento & desenvolvimento , Lycopersicon esculentum/metabolismo , Meristema/genética , Meristema/crescimento & desenvolvimento , Meristema/metabolismo , Oxirredução , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Protoplastos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , Espécies Reativas de Oxigênio/uso terapêutico , S-Adenosilmetionina/metabolismo , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transformação Genética
9.
Cell Res ; 31(6): 613-630, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33514913

RESUMO

Organization of the genome into euchromatin and heterochromatin appears to be evolutionarily conserved and relatively stable during lineage differentiation. In an effort to unravel the basic principle underlying genome folding, here we focus on the genome itself and report a fundamental role for L1 (LINE1 or LINE-1) and B1/Alu retrotransposons, the most abundant subclasses of repetitive sequences, in chromatin compartmentalization. We find that homotypic clustering of L1 and B1/Alu demarcates the genome into grossly exclusive domains, and characterizes and predicts Hi-C compartments. Spatial segregation of L1-rich sequences in the nuclear and nucleolar peripheries and B1/Alu-rich sequences in the nuclear interior is conserved in mouse and human cells and occurs dynamically during the cell cycle. In addition, de novo establishment of L1 and B1 nuclear segregation is coincident with the formation of higher-order chromatin structures during early embryogenesis and appears to be critically regulated by L1 and B1 transcripts. Importantly, depletion of L1 transcripts in embryonic stem cells drastically weakens homotypic repeat contacts and compartmental strength, and disrupts the nuclear segregation of L1- or B1-rich chromosomal sequences at genome-wide and individual sites. Mechanistically, nuclear co-localization and liquid droplet formation of L1 repeat DNA and RNA with heterochromatin protein HP1α suggest a phase-separation mechanism by which L1 promotes heterochromatin compartmentalization. Taken together, we propose a genetically encoded model in which L1 and B1/Alu repeats blueprint chromatin macrostructure. Our model explains the robustness of genome folding into a common conserved core, on which dynamic gene regulation is overlaid across cells.

10.
Anal Chem ; 93(5): 2988-2995, 2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33512148

RESUMO

The formation of biomolecular condensates is driven by liquid-liquid phase separation, which is prevalent in cells to govern crucial cellular functions. However, understanding the properties of phase-separated condensates remains very challenging for the lack of suitable techniques. Here, we report a photoluminescence lifetime imaging method for real-time monitoring of phase-separated condensates, both in vitro and in living cells, using a microsecond-scale photoluminescence lifetime probe based on iridium complex. The probe has a large Stokes shift, excellent cell permeability, and minimal cell autofluorescence interference. With this method, the dynamic process of phase separation of fused in sarcoma protein has been well explored, showing high spatiotemporal resolution and high throughput. Beginning with initial formation, the protein droplets get bigger and more viscous, and then a final maturation to solidified aggregates has been characterized. This study paves the path for a deeper understanding of the properties of phase-separated biomolecular condensates.


Assuntos
Irídio , Proteínas
11.
Nat Cell Biol ; 23(1): 32-39, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33288888

RESUMO

MicroRNA (miRNA) production entails the step-wise processing of primary miRNAs (pri-miRNAs) into precursor miRNAs (pre-miRNAs) and miRNA/* duplexes by Dicing complexes containing DCL1, HYL1 and SE, which are localized in nuclear dicing bodies (D-bodies)1,2. Here, we show that D-bodies are phase-separated condensates. SE forms droplets and drives DCL1, HYL1 and pri/pre-miRNAs into the droplets in vitro, and mutation of SE abrogates the formation of D-bodies in vivo, which indicates that D-bodies arise through SE-mediated phase separation. Disruption of SE phase separation greatly reduces its activity in promoting miRNA processing both in vitro and in vivo. We further show that pre-miRNAs are processed into miRNA/* duplexes in the droplets and, after processing, miRNA/* duplexes are bound by HYL1 and released from the droplets. Our findings provide evidence that efficient miRNA processing depends on the SE-phase-separation-mediated formation of D-bodies and suggest a paradigm that the products made in phase-separated condensates can be shipped out for subsequent processes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Núcleo Celular , MicroRNAs/genética , Transição de Fase , Proteínas de Ligação a RNA/genética
12.
Proc Natl Acad Sci U S A ; 117(44): 27124-27131, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33087563

RESUMO

Liquid-liquid phase separation, driven by multivalent macromolecular interactions, causes formation of membraneless compartments, which are biomolecular condensates containing concentrated macromolecules. These condensates are essential in diverse cellular processes. Formation and dynamics of micrometer-scale phase-separated condensates are examined routinely. However, limited by commonly used methods which cannot capture small-sized free-diffusing condensates, the transition process from miscible individual molecules to micrometer-scale condensates is mostly unknown. Herein, with a dual-color fluorescence cross-correlation spectroscopy (dcFCCS) method, we captured formation of nanoscale condensates beyond the detection limit of conventional fluorescence microscopy. In addition, dcFCCS is able to quantify size and growth rate of condensates as well as molecular stoichiometry and binding affinity of client molecules within condensates. The critical concentration to form nanoscale condensates, identified by our experimental measurements and Monte Carlo simulations, is at least several fold lower than the detection limit of conventional fluorescence microscopy. Our results emphasize that, in addition to micrometer-scale condensates, nanoscale condensates are likely to play important roles in various cellular processes and dcFCCS is a simple and powerful quantitative tool to examine them in detail.

13.
Sci China Life Sci ; 63(7): 953-985, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32548680

RESUMO

Cells are compartmentalized by numerous membrane-enclosed organelles and membraneless compartments to ensure that a wide variety of cellular activities occur in a spatially and temporally controlled manner. The molecular mechanisms underlying the dynamics of membrane-bound organelles, such as their fusion and fission, vesicle-mediated trafficking and membrane contactmediated inter-organelle interactions, have been extensively characterized. However, the molecular details of the assembly and functions of membraneless compartments remain elusive. Mounting evidence has emerged recently that a large number of membraneless compartments, collectively called biomacromolecular condensates, are assembled via liquid-liquid phase separation (LLPS). Phase-separated condensates participate in various biological activities, including higher-order chromatin organization, gene expression, triage of misfolded or unwanted proteins for autophagic degradation, assembly of signaling clusters and actin- and microtubule-based cytoskeletal networks, asymmetric segregations of cell fate determinants and formation of pre- and post-synaptic density signaling assemblies. Biomacromolecular condensates can transition into different material states such as gel-like structures and solid aggregates. The material properties of condensates are crucial for fulfilment of their distinct functions, such as biochemical reaction centers, signaling hubs and supporting architectures. Cells have evolved multiple mechanisms to ensure that biomacromolecular condensates are assembled and disassembled in a tightly controlled manner. Aberrant phase separation and transition are causatively associated with a variety of human diseases such as neurodegenerative diseases and cancers. This review summarizes recent major progress in elucidating the roles of LLPS in various biological pathways and diseases.


Assuntos
Membrana Celular/metabolismo , Organelas/metabolismo , Animais , Autofagia , Fenômenos Fisiológicos Celulares , Cromatina/metabolismo , Expressão Gênica , Humanos , Cinética , Neoplasias/metabolismo , Doenças Neurodegenerativas/metabolismo , Transição de Fase , Dobramento de Proteína , Proteínas , Proteólise , Propriedades de Superfície
14.
Trends Biochem Sci ; 45(6): 457-458, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32413321

RESUMO

Chromatin readers are important intermediaries linking epigenetic information and biological phenotypes. Many diseases are caused by mutations in epigenetic readers. Recently, a study by Wan et al. uncovered that cancer-associated mutations promote self-association of eleven-nineteen-leukemia protein (ENL), leading to abnormal condensates, elevated gene expression, and impaired cell fate determination.


Assuntos
Cromatina , Mutação com Ganho de Função , Diferenciação Celular , Mutação
15.
J Biol Chem ; 295(33): 11420-11434, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32461254

RESUMO

Modification-dependent and -independent biomolecular interactions, including protein-protein, protein-DNA/RNA, protein-sugar, and protein-lipid interactions, play crucial roles in all cellular processes. Dysregulation of these biomolecular interactions or malfunction of the associated enzymes results in various diseases; therefore, these interactions and enzymes are attractive targets for therapies. High-throughput screening can greatly facilitate the discovery of drugs for these targets. Here, we describe a biomolecular interaction detection method, called phase-separated condensate-aided enrichment of biomolecular interactions in test tubes (CEBIT). The readout of CEBIT is the selective recruitment of biomolecules into phase-separated condensates harboring their cognate binding partners. We tailored CEBIT to detect various biomolecular interactions and activities of biomolecule-modifying enzymes. Using CEBIT-based high-throughput screening assays, we identified known inhibitors of the p53/MDM2 (MDM2) interaction and of the histone methyltransferase, suppressor of variegation 3-9 homolog 1 (SUV39H1), from a compound library. CEBIT is simple and versatile, and is likely to become a powerful tool for drug discovery and basic biomedical research.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Mapeamento de Interação de Proteínas/métodos , Descoberta de Drogas/métodos , Humanos , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Transição de Fase , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo
16.
Cell Res ; 30(5): 393-407, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32111972

RESUMO

Rett syndrome (RTT), a severe postnatal neurodevelopmental disorder, is caused by mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2). MeCP2 is a chromatin organizer regulating gene expression. RTT-causing mutations have been shown to affect this function. However, the mechanism by which MeCP2 organizes chromatin is unclear. In this study, we found that MeCP2 can induce compaction and liquid-liquid phase separation of nucleosomal arrays in vitro, and DNA methylation further enhances formation of chromatin condensates by MeCP2. Interestingly, RTT-causing mutations compromise MeCP2-mediated chromatin phase separation, while benign variants have little effect on this process. Moreover, MeCP2 competes with linker histone H1 to form mutually exclusive chromatin condensates in vitro and distinct heterochromatin foci in vivo. RTT-causing mutations reduce or even abolish the ability of MeCP2 to compete with histone H1 and to form chromatin condensates. Together, our results identify a novel mechanism by which phase separation underlies MeCP2-mediated heterochromatin formation and reveal the potential link between this process and the pathology of RTT.

17.
J Mol Biol ; 432(1): 160-169, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31260696

RESUMO

The selective degradation of protein aggregates is called aggrephagy. Misfolded proteins are thought to form aggregates, which are then surrounded by selective autophagy receptors and targeted to autophagosomes for degradation. Recent studies of p62 bodies, PGL granules, and stress granules indicate that proteins targeted for aggrephagy are not simple protein aggregates but rather form liquid-like protein condensates through liquid-liquid phase separation. The liquid-like properties of the condensates and hardening to a gel-like state may be crucial in the initiation of aggrephagy. Dysregulation of phase separation may cause human diseases. Here we review the potential roles of liquid-liquid phase separation in the process of aggrephagy.


Assuntos
Macroautofagia , Agregados Proteicos , Animais , Autofagossomos/metabolismo , Humanos , Transição de Fase , Poliubiquitina/metabolismo , Proteólise , Proteína Sequestossoma-1/metabolismo
18.
Dev Cell ; 52(3): 277-293.e8, 2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-31866201

RESUMO

Compacted heterochromatin blocks are prevalent in differentiated cells and present a barrier to cellular reprogramming. It remains obscure how heterochromatin remodeling is orchestrated during cell differentiation. Here we find that the evolutionarily conserved homeodomain transcription factor Prospero (Pros)/Prox1 ensures neuronal differentiation by driving heterochromatin domain condensation and expansion. Intriguingly, in mitotically dividing Drosophila neural precursors, Pros is retained at H3K9me3+ pericentromeric heterochromatin regions of chromosomes via liquid-liquid phase separation (LLPS). During mitotic exit of neural precursors, mitotically retained Pros recruits and concentrates heterochromatin protein 1 (HP1) into phase-separated condensates and drives heterochromatin compaction. This establishes a transcriptionally repressive chromatin environment that guarantees cell-cycle exit and terminal neuronal differentiation. Importantly, mammalian Prox1 employs a similar "mitotic-implantation-ensured heterochromatin condensation" strategy to reinforce neuronal differentiation. Together, our results unveiled a new paradigm whereby mitotic implantation of a transcription factor via LLPS remodels H3K9me3+ heterochromatin and drives timely and irreversible terminal differentiation.


Assuntos
Diferenciação Celular , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Heterocromatina/metabolismo , Mitose , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Proteínas Nucleares/metabolismo , Transição de Fase , Fatores de Transcrição/metabolismo , Animais , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Regulação da Expressão Gênica , Heterocromatina/genética , Histonas/genética , Histonas/metabolismo , Extração Líquido-Líquido , Masculino , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Proteínas Nucleares/genética , Nucleossomos , Fatores de Transcrição/genética
19.
Mol Cell ; 76(4): 646-659.e6, 2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31543422

RESUMO

Eukaryotic chromosomes contain compartments of various functions, which are marked by and enriched with specific histone modifications. However, the molecular mechanisms by which these histone marks function in chromosome compartmentalization are poorly understood. Constitutive heterochromatin is a largely silent chromosome compartment characterized in part by H3K9me2 and 3. Here, we show that heterochromatin protein 1 (HP1), an H3K9me2 and 3 "reader," interacts with SUV39H1, an H3K9me2 and 3 "writer," and with TRIM28, an abundant HP1 scaffolding protein, to form complexes with increased multivalent engagement of H3K9me2 and 3-modified chromatin. H3K9me2 and 3-marked nucleosomal arrays and associated complexes undergo phase separation to form macromolecule-enriched liquid droplets. The droplets are reminiscent of heterochromatin as they are highly dense chromatin-containing structures that are resistant to DNase and exclude the general transcription factor TFIIB. Our data suggest a general mechanism by which histone marks regulate chromosome compartmentalization by promoting phase separation.


Assuntos
Montagem e Desmontagem da Cromatina , Heterocromatina/metabolismo , Histonas/metabolismo , Gotículas Lipídicas/metabolismo , Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Células HEK293 , Heterocromatina/genética , Humanos , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Complexos Multiproteicos , Nucleossomos/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Tempo , Proteína 28 com Motivo Tripartido/genética , Proteína 28 com Motivo Tripartido/metabolismo
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