scholarly journals Phase separation and nucleosome compaction are governed by the same domain of Polycomb Repressive Complex 1

2018 ◽  
Author(s):  
Aaron J. Plys ◽  
Christopher P. Davis ◽  
Jongmin Kim ◽  
Gizem Rizki ◽  
Madeline M. Keenen ◽  
...  
Keyword(s):  
Phase Separation ◽  
Point Mutations ◽  
Mammalian Development ◽  
Polycomb Repressive Complex ◽  
Axial Patterning ◽  
Core Proteins ◽  
Low Concentrations ◽  
Polycomb Bodies ◽  
Polycomb Repressive Complex 1

SummaryMammalian development requires effective mechanisms to repress genes whose expression would generate inappropriately specified cells. The Polycomb Repressive Complex 1 (PRC1) family complexes are central to maintaining this repression1. These include a set of canonical PRC1 complexes that each contain four core proteins, including one from the CBX family. These complexes have previously been shown to reside in membraneless organelles called Polycomb bodies, leading to speculation that canonical PRC1 might be found in a separate phase from the rest of the nucleus2,3. We show here that reconstituted PRC1 readily phase separates into droplets in vitro at low concentrations and physiological salt conditions. This behavior is driven by the CBX2 subunit. Point mutations in an internal domain of CBX2 eliminate phase separation. These same point mutations eliminate the formation of puncta in cells, and have previously been shown to eliminate nucleosome compaction in vitro4 and to generate axial patterning defects in mice5. Thus, a single domain in CBX2 is required for phase separation and nucleosome compaction, a finding that relates these functions to each other and to proper development.

scholarly journals Polycomb Cbx2 Condensates Assemble through Phase Separation

2018 ◽  
Author(s):  
Roubina Tatavosian ◽  
Samantha Kent ◽  
Kyle Brown ◽  
Tingting Yao ◽  
Huy Nguyen Duc ◽  
...  
Keyword(s):  
Phase Separation ◽  
Polycomb Group ◽  
Developmental Defects ◽  
Intrinsically Disordered ◽  
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.

scholarly journals Phase separation of Polycomb-repressive complex 1 is governed by a charged disordered region of CBX2

2019 ◽  
Vol 33 (13-14) ◽  
pp. 799-813 ◽  
Author(s):  
Aaron J. Plys ◽  
Christopher P. Davis ◽  
Jongmin Kim ◽  
Gizem Rizki ◽  
Madeline M. Keenen ◽  
...  
Keyword(s):  
Phase Separation ◽  
Polycomb Repressive Complex ◽  
Polycomb Repressive Complex 1

scholarly journals Selective sorting of microRNAs into exosomes by phase-separated YBX1 condensates

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Xiao-Man Liu ◽  
Liang Ma ◽  
Randy Schekman
Keyword(s):  
Phase Separation ◽  
Rna Binding ◽  
Cultured Cells ◽  
Rna Binding Proteins ◽  
Point Mutations ◽  
Cell Communication ◽  
Mediate Cell ◽  
Local Enrichment ◽  
In Cells

Exosomes may mediate cell-to-cell communication by transporting various proteins and nucleic acids to neighboring cells. Some protein and RNA cargoes are significantly enriched in exosomes. How cells efficiently and selectively sort them into exosomes remains incompletely explored. Previously we reported that YBX1 is required in sorting of miR-223 into exosomes. Here we show that YBX1 undergoes liquid-liquid phase separation (LLPS) in vitro and in cells. YBX1 condensates selectively recruit miR-223 in vitro and into exosomes secreted by cultured cells. Point mutations that inhibit YBX1 phase separation impair the incorporation of YBX1 protein into biomolecular condensates formed in cells, and perturb miR-233 sorting into exosomes. We propose that phase separation-mediated local enrichment of cytosolic RNA binding proteins and their cognate RNAs enables their targeting and packaging by vesicles that bud into multivesicular bodies. This provides a possible mechanism for efficient and selective engulfment of cytosolic proteins and RNAs into intraluminal vesicles which are then secreted as exosomes from cells.

scholarly journals Selective sorting of microRNAs into exosomes by phase-separated YBX1 condensates

2021 ◽  
Author(s):  
Xiao-Man Liu ◽  
Liang Ma ◽  
Randy Schekman
Keyword(s):  
Phase Separation ◽  
Rna Binding ◽  
Cultured Cells ◽  
Rna Binding Proteins ◽  
Point Mutations ◽  
Cell Communication ◽  
Mediate Cell ◽  
Local Enrichment ◽  
In Cells

Exosomes may mediate cell-to-cell communication by transporting various proteins and nucleic acids to neighboring cells. Some protein and RNA cargoes are significantly enriched in exosomes. How cells efficiently and selectively sort them into exosomes remains incompletely explored. Previously we reported that YBX1 is required in sorting of miR-223 into exosomes. Here we show that YBX1 undergoes liquid-liquid phase separation (LLPS) in vitro and in cells. YBX1 condensates selectively recruit miR-223 in vitro and into exosomes secreted by cultured cells. Point mutations that inhibit YBX1 phase separation impair the incorporation of YBX1 protein into biomolecular condensates formed in cells, and perturb miR-233 sorting into exosomes. We propose that phase separation-mediated local enrichment of cytosolic RNA binding proteins and their cognate RNAs enables their targeting and packaging by vesicles that bud into multivesicular bodies. This provides a possible mechanism for efficient and selective engulfment of cytosolic proteins and RNAs into intraluminal vesicles which are then secreted as exosomes from cells.

scholarly journals SALL1 Modulates CBX4 Stability, Nuclear Bodies, and Regulation of Target Genes

2021 ◽  
Vol 9 ◽  
Author(s):  
Immacolata Giordano ◽  
Lucia Pirone ◽  
Veronica Muratore ◽  
Eukene Landaluze ◽  
Coralia Pérez ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Cell Differentiation ◽  
Limb Development ◽  
Target Genes ◽  
Nuclear Bodies ◽  
Polycomb Repressive Complex ◽  
Complex Interplay ◽  
Polycomb Bodies ◽  
Polycomb Repressive Complex 1 ◽  
The Relationship

Development is orchestrated through a complex interplay of multiple transcription factors. The comprehension of this interplay will help us to understand developmental processes. Here we analyze the relationship between two key transcription factors: CBX4, a member of the Polycomb Repressive Complex 1 (PRC1), and SALL1, a member of the Spalt-like family with important roles in embryogenesis and limb development. Both proteins localize to nuclear bodies and are modified by the small ubiquitin-like modifier (SUMO). Our results show that CBX4 and SALL1 interact in the nucleoplasm and that increased SALL1 expression reduces ubiquitination of CBX4, enhancing its stability. This is accompanied by an increase in the number and size of CBX4-containing Polycomb bodies, and by a greater repression of CBX4 target genes. Thus, our findings uncover a new way of SALL1-mediated regulation of Polycomb bodies through modulation of CBX4 stability, with consequences in the regulation of its target genes, which could have an impact in cell differentiation and development.

scholarly journals H2A ubiquitination is essential for Polycomb Repressive Complex 1-mediated gene regulation in Marchantia polymorpha

2021 ◽  
Author(s):  
Shujing Liu ◽  
Minerva S. Trejo-Arellano ◽  
Yichun Qiu ◽  
D. Magnus Eklund ◽  
Claudia Köhler ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Transcriptional Repression ◽  
Point Mutations ◽  
Marchantia Polymorpha ◽  
Transcriptional Level ◽  
Polycomb Repressive Complex ◽  
Direct Role ◽  
Chromatin Regulators ◽  
Intergenic Regions ◽  
Polycomb Repressive Complex 1

AbstractPolycomb repressive complex 1 (PRC1) and PRC2 are chromatin regulators maintaining transcriptional repression. The deposition of H3 lysine 27 tri-methylation (H3K27me3) by PRC2 is known to be required for transcriptional repression, whereas the contribution of H2A ubiquitination (H2Aub) in the Polycomb repressive system remains unclear in plants. We directly tested the requirement of H2Aub for gene regulation in Marchantia polymorpha by generating point mutations in H2A that prevent ubiquitination by PRC1. These mutants show reduced H3K27me3 levels on the same target sites as mutants defective in PRC1 subunits MpBMI1 and the homolog MpBMI1L, revealing that PRC1-catalyzed H2Aub is essential for Polycomb system function. Furthermore, by comparing transcriptome data between mutants in MpH2A and MpBMI1/1L, we demonstrate that H2Aub contributes to the PRC1-mediated transcriptional level of genes and transposable elements. Together, our data demonstrate that H2Aub plays a direct role in H3K27me3 deposition and is required for PRC1-mediated transcriptional changes in both genic and intergenic regions in Marchantia.

scholarly journals H2A ubiquitination is essential for Polycomb Repressive Complex 1-mediated gene regulation in Marchantia polymorpha

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Shujing Liu ◽  
Minerva S. Trejo-Arellano ◽  
Yichun Qiu ◽  
D. Magnus Eklund ◽  
Claudia Köhler ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Transposable Elements ◽  
Transcriptional Repression ◽  
Point Mutations ◽  
Marchantia Polymorpha ◽  
Transcriptional Level ◽  
Polycomb Repressive Complex ◽  
Direct Role ◽  
Chromatin Regulators ◽  
Polycomb Repressive Complex 1

Abstract Background Polycomb repressive complex 1 (PRC1) and PRC2 are chromatin regulators maintaining transcriptional repression. The deposition of H3 lysine 27 tri-methylation (H3K27me3) by PRC2 is known to be required for transcriptional repression, whereas the contribution of H2A ubiquitination (H2Aub) in the Polycomb repressive system remains unclear in plants. Results We directly test the requirement of H2Aub for gene regulation in Marchantia polymorpha by generating point mutations in H2A that prevent ubiquitination by PRC1. These mutants show reduced H3K27me3 levels on the same target sites as mutants defective in PRC1 subunits MpBMI1 and the homolog MpBMI1L, revealing that PRC1-catalyzed H2Aub is essential for Polycomb system function. Furthermore, by comparing transcriptome data between mutants in MpH2A and MpBMI1/1L, we demonstrate that H2Aub contributes to the PRC1-mediated transcriptional level of genes and transposable elements. Conclusion Together, our data demonstrates that H2Aub plays a direct role in H3K27me3 deposition and is required for PRC1-mediated transcriptional changes in both genes and transposable elements in Marchantia.

Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

1977 ◽  
Vol 16 (04) ◽  
pp. 157-162 ◽  
Author(s):  
C. Schümichen ◽  
B. Mackenbrock ◽  
G. Hoffmann
Keyword(s):  
Normal Saline ◽  
Half Life ◽  
Compound A ◽  
Short Half Life ◽  
Low Concentrations ◽  
The Stability ◽  
Kinetics Of ◽  
In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

In Vitro Effects of Ethanol on Rabbit Platelet Aggregation, Secretion of Granule Contents, and Cyclic AMP Levels in the Presence of Prostacyclin

1989 ◽  
Vol 61 (02) ◽  
pp. 254-258 ◽  
Author(s):  
Margaret L Rand ◽  
Peter L Gross ◽  
Donna M Jakowec ◽  
Marian A Packham ◽  
J Fraser Mustard
Keyword(s):  
Cyclic Amp ◽  
Inhibitory Effect ◽  
Rabbit Platelet ◽  
Inhibitory Effects ◽  
Low Concentrations ◽  
Rabbit Platelets ◽  
High Concentrations ◽  
In Vitro Effects ◽  
Aggregation Of Platelets

SummaryEthanol, at physiologically tolerable concentrations, inhibits platelet responses to low concentrations of collagen or thrombin, but does not inhibit responses of washed rabbit platelets stimulated with high concentrations of ADP, collagen, or thrombin. However, when platelet responses to high concentrations of collagen or thrombin had been partially inhibited by prostacyclin (PGI2), ethanol had additional inhibitory effects on aggregation and secretion. These effects were also observed with aspirin- treated platelets stimulated with thrombin. Ethanol had no further inhibitory effect on aggregation of platelets stimulated with ADP, or the combination of ADP and epinephrine. Thus, the inhibitory effects of ethanol on platelet responses in the presence of PGI2 were very similar to its inhibitory effects in the absence of PGI2, when platelets were stimulated with lower concentrations of collagen or thrombin. Ethanol did not appear to exert its inhibitory effects by increasing cyclic AMP above basal levels and the additional inhibitory effects of ethanol in the presence of PGI2 did not appear to be brought about by further increases in platelet cyclic AMP levels.

Aggregation of Cat Platelets in Vitro

1970 ◽  
Vol 23 (03) ◽  
pp. 601-620 ◽  
Author(s):  
Th. B Tschopp
Keyword(s):  
Adenosine Monophosphate ◽  
Blood Collection ◽  
Base Line ◽  
Reversible Aggregation ◽  
Low Concentrations ◽  
Irreversible Aggregation ◽  
High Concentrations ◽  
Two Phases ◽  
Improved Technique

SummaryAggregation of cat platelets in the citrated plasma is examined by means of Born’s absorptiometer. A marked tendency of the platelets of this species to spontaneous aggregation necessitated first of all the development of an improved technique of blood collection.A hypothesis according to which 5-HT is released from the platelets, explains the absence of oscillations on the base line of the absorptiometer, the absence of platelet swelling, when ADP is added, and the effect of stirring on the aggregation curves in cat PRP. The average volume of cat platelets amounts to 10.46 μ3 when directly fixed in the blood, when fixed from PRP to 12.17 μ3, when fixed from stirred PRP to 13.51 μ3.In low concentrations (0.3-2 μM) ADP produce reversible aggregation; in narrowly restricted, individually dissimilar mean concentrations irreversible aggregation in two phases and in high concentrations, irreversible aggregation in one phase. Like ADP serotonin produces 2 phase irreversible aggregation in concentrations of 3-10 μM, but unlike ADP, the aggregation velocity decreases again with high 5-HT concentrations (>100 μM). Adrenaline does not produce aggregation and it is likely that adenosine and adenosine monophosphate inhibit the aggregation by serotonin but not by ADP. Species differences in the aggregation of human, rabbit and cat platelets are discussed.

Sign in / Sign up

Export Citation Format

Share Document