Liquid-liquid phase separation (LLPS) contributes to the spatial and functional segregation of molecular processes within the cell nucleus. However, the role played by LLPS in chromatin folding in ...living cells remains unclear. Here, using stochastic optical reconstruction microscopy (STORM) and Hi-C techniques, we studied the effects of 1,6-hexanediol (1,6-HD)-mediated LLPS disruption/modulation on higher-order chromatin organization in living cells. We found that 1,6-HD treatment caused the enlargement of nucleosome clutches and their more uniform distribution in the nuclear space. At a megabase-scale, chromatin underwent moderate but irreversible perturbations that resulted in the partial mixing of A and B compartments. The removal of 1,6-HD from the culture medium did not allow chromatin to acquire initial configurations, and resulted in more compact repressed chromatin than in untreated cells. 1,6-HD treatment also weakened enhancer-promoter interactions and TAD insulation but did not considerably affect CTCF-dependent loops. Our results suggest that 1,6-HD-sensitive LLPS plays a limited role in chromatin spatial organization by constraining its folding patterns and facilitating compartmentalization at different levels.
Mammalian and Drosophila genomes are partitioned into topologically associating domains (TADs). Although this partitioning has been reported to be functionally relevant, it is unclear whether TADs ...represent true physical units located at the same genomic positions in each cell nucleus or emerge as an average of numerous alternative chromatin folding patterns in a cell population. Here, we use a single-nucleus Hi-C technique to construct high-resolution Hi-C maps in individual Drosophila genomes. These maps demonstrate chromatin compartmentalization at the megabase scale and partitioning of the genome into non-hierarchical TADs at the scale of 100 kb, which closely resembles the TAD profile in the bulk in situ Hi-C data. Over 40% of TAD boundaries are conserved between individual nuclei and possess a high level of active epigenetic marks. Polymer simulations demonstrate that chromatin folding is best described by the random walk model within TADs and is most suitably approximated by a crumpled globule build of Gaussian blobs at longer distances. We observe prominent cell-to-cell variability in the long-range contacts between either active genome loci or between Polycomb-bound regions, suggesting an important contribution of stochastic processes to the formation of the Drosophila 3D genome.
How the nuclear lamina (NL) impacts on global chromatin architecture is poorly understood. Here, we show that NL disruption in Drosophila S2 cells leads to chromatin compaction and repositioning from ...the nuclear envelope. This increases the chromatin density in a fraction of topologically-associating domains (TADs) enriched in active chromatin and enhances interactions between active and inactive chromatin. Importantly, upon NL disruption the NL-associated TADs become more acetylated at histone H3 and less compact, while background transcription is derepressed. Two-colour FISH confirms that a TAD becomes less compact following its release from the NL. Finally, polymer simulations show that chromatin binding to the NL can per se compact attached TADs. Collectively, our findings demonstrate a dual function of the NL in shaping the 3D genome. Attachment of TADs to the NL makes them more condensed but decreases the overall chromatin density in the nucleus by stretching interphase chromosomes.
We used dissipative particle dynamics simulations to study the copolymerization process in the presence of spatial heterogeneities caused by incompatibility between polymerizing monomers. We ...performed the model verification with the available literature data on styrene–acrylic acid copolymerization in the bulk, and a very good agreement between experimental and simulated data for both chain average composition and triad fractions was observed. Next, we studied the system properties for five model AB reaction processes with different sets of reactivity ratios at different compositions and Flory–Huggins parameters χ. We found that the system average copolymer composition does not change upon increasing χ value if the monomers have equal reactivity ratios r A = r B but changes toward higher A-monomer content if r A > r B. The local monomer fraction around growing chain ends was studied, and evidence of the presence of the bootstrap effect was found at nonzero values of χ. Next, significant changes in the copolymer sequences caused by the partial monomer incompatibility were observed: for all studied sets of reactivity ratios the resulting sequences demonstrated increased blockiness compared to the case of zero χ. Finally, we found that the reaction process in the presence of the monomer incompatibility cannot be described by a single set of terminal unit model reactivity ratios, as the “observable” reactivity ratios r′ extracted from the chain sequences depend strongly on the monomer feed composition.
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In this work we developed a RAFT polymerization model taking into account the main reactions of the experimental RAFT process and implemented that model in dissipative particle dynamics (DPD). With ...the help of a kinetic model based on the same reaction routine, we investigated the question of how to simulate realistic reactions using such models. We showed that a simultaneous
M
-fold increase of the initiation probability
p
i
and an
M
-fold decrease of the termination probability
p
t
does not result in significant changes in the chain length distribution. If the RAFT/initiator ratio is large, a simplified model with no termination and immediate radical formation can be used with good enough accuracy. After that we directly compared the reaction behavior within the kinetic model and DPD. We showed that steric restrictions, which were not present in the kinetic model, can introduce noticeable changes in the system behavior. Finally, we studied the influence of the incompatibility on the RAFT polymerization process on an example of classical implementation of polymerization-induced self-assembly (PISA). We showed that in systems with incompatible species the number of activation-deactivation cycles does not always reflect the dispersity of the resulting chain ensemble. Moreover, we demonstrated that specifically the incompatibility between the RAFT end group and other species can have a large effect on the polymerization results.
RAFT polymerization model for dissipative particle dynamics is developed. The question of how to choose parameters for the model is discussed. The incompatibility between the species is shown to have a dramatic effect on the polymerization results.
A large family of P,S-bidentate diamidophosphite ligands were readily synthesized from accessible hydroxyl-thioether compounds. One type of Pd(II) cationic allylic complex with these ...diamidophosphites fulfilling a P-monodentate function, and three types, where the ligands act as P,S-bridging ligands (coordination polymer and head-to-head and head-to-tail dimers), were obtained. In addition, neutral Pd(II) halide complexes were generated in situ as common intermediates for both groups of cationic dimers. The structures of the ligands and complexes were elucidated by means of 2D-NMR and were confirmed by powder X-ray diffraction, as well as by DFT calculations. Asymmetric inducers of this type exhibited up to 94% ee in the Pd-mediated allylic substitution of (E)-1,3-diphenylallyl acetate with various C- and N-nucleophiles. Ee values of up to 80% were obtained in the Pd-catalyzed allylic alkylation of cinnamyl acetate with β-ketoesters. In addition, up to 61% ee was achieved in the asymmetric amination of 2-(diethoxyphosphoryl)-1-phenylallyl acetate with aniline. The effects of the diamidophosphite and thioether moieties on the catalytic activity and enantioselectivity were investigated.
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In this work we presented a novel computational model of precipitation polymerization allowing one to obtain core-shell microgels via a realistic cross-linking process based on the ...experimental procedure. We showed that the cross-linker–monomer reactivity ratios r are responsible for the microgel internal structure. Values of r lower than 1 correspond to the case when alternating sequences occur at the early reaction stages; this leads to the formation of microgels with pronounced core-shell structure. The distribution of dangling ends for small values of r becomes bimodal with two well-distinguished peaks, which correspond to the core (short dangling ends) and corona (long dangling ends) regions. The density profiles confirm the existence of two distinct regions for small r: a densely cross-linked core and a loose corona entirely consisting of dangling ends with no cross-linker. The consumption of the cross-linker in the course in the microgel formation was found to be in a perfect agreement with the predictions of Monte Carlo (MC) model in the sequence space.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
In the current work, atom transfer radical polymerization-induced self-assembly (ATRP PISA) phase diagrams were obtained by the means of dissipative particle dynamics simulations. A fast algorithm ...for determining the equilibrium morphology of block copolymer aggregates was developed. Our goal was to assess how the chemical nature of ATRP affects the self-assembly of diblock copolymers in the course of PISA. We discovered that the chain growth termination via recombination played a key role in determining the ATRP PISA phase diagrams. In particular, ATRP with turned off recombination yielded a PISA phase diagram very similar to that obtained for a simple ideal living polymerization process. However, an increase in the recombination probability led to a significant change of the phase diagram: the transition between cylindrical micelles and vesicles was strongly shifted, and a dependence of the aggregate morphology on the concentration was observed. We speculate that this effect occurred due to the simultaneous action of two factors: the triblock copolymer architecture of the terminated chains and the dispersity of the solvophobic blocks. We showed that these two factors affected the phase diagram weakly if they acted separately; however, their combination, which naturally occurs during ATRP, affected the ATRP PISA phase diagram strongly. We suggest that the recombination reaction is a key factor leading to the complexity of experimental PISA phase diagrams.
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In this work, we studied the question of whether it is possible to develop a one-step approach for the creation of microphase-separated materials with long-range order with the help of spontaneous ...gradient copolymers, i.e., formed during controlled copolymerization solely due to the large difference in the reactivity ratios. To that end, we studied the polymerization-induced microphase separation in bulk on the example of a monomer pair with realistic parameters based on styrene (S) and vinylpirrolydone (VP) by means of computer simulation. We showed that for experimentally reasonable chain lengths, the structures with long-range order start to appear at the conversion degree as low as 76%; a full phase diagram in coordinates (fraction of VP-conversion degree) was constructed. Rather rich phase behavior was obtained; moreover, at some VP fractions, order-order transitions were observed. Finally, we studied how the conversion degree at which the order-disorder transition occurs changes upon varying the maximum average chain length in the system.
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Two groups of modular chiral diamidophosphite ligands were easily synthesised from accessible
N
-Boc-amino alcohols and pseudodipeptides. The reaction of these compounds with Pd(allyl)Cl
2
in the ...presence of AgBF
4
yielded complexes Pd(allyl)(L)
2
BF
4
. In addition, metallochelates Pd(allyl)(L)BF
4
with (
S
)-methioninol-based
P
,
S
-bidentate ligands were prepared. The structures of the novel ligands and complexes were elucidated by means of 2D-NMR and were confirmed by single-crystal X-ray diffraction, as well as by DFT calculations. Asymmetric inducers of this type exhibited high enantioselectivities in the Pd-mediated allylic substitution of (
E
)-1,3-diphenylallyl ethyl carbonate with CH
2
(CO
2
Me)
2
(up to 98% ee) and (CH
2
)
4
NH (up to 92% ee). Ee values of up to 86% and 73% were obtained in the Pd-catalyzed allylic alkylation of cinnamyl acetate with ethyl 2-oxocyclohexane-1-carboxylate and ethyl 2-oxocyclopentane-1-carboxylate, respectively. The effects of the structural modules, such as the nature of the phosphorus-containing ring or exocyclic substituent, on the catalytic activity and enantioselectivity were investigated.
Novel diamidophosphites based on β-hydroxyamides were prepared, and their individual and
in situ
formed complexes were tested in Pd-mediated allylations.
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