Regulation of chromatin microphase separation by binding of protein complexes
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This fundamental work substantially advances our understanding of polymer physics underpinnings of genome folding, organization, and regulation. The conclusions are supported by both convincing computer simulations and analytical theory. The work will be of significant interest to the genome folding community.
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Abstract
We show evidence of the association of RNA polymerase II (RNAP) with chromatin in a core-shell organization, reminiscent of microphase separation where the cores comprise dense chromatin and the shell, RNAP and chromatin with low density. These observations motivate our physical model for the regulation of core-shell chromatin organization. Here, we model chromatin as a multiblock copolymer, comprising active and inactive regions (blocks) that are both in poor solvent and tend to be condensed in the absence of binding proteins. However, we show that the solvent quality for the active regions of chromatin can be regulated by the binding of protein complexes (e.g., RNAP and transcription factors). Using the theory of polymer brushes, we find that such binding leads to swelling of the active chromatin regions which in turn modifies the spatial organization of the inactive regions. In addition, we use simulations to study spherical chromatin micelles, whose cores comprise inactive regions and shells comprise active regions and bound protein complexes. In spherical micelles the swelling increases the number of inactive cores and controls their size. Thus, genetic modifications affecting the binding strength of chromatin-binding protein complexes may modulate the solvent quality experienced by chromatin and regulate the physical organization of the genome.
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Author Response
Reviewer #1 (Public Review):
The authors provide evidence for chromatin, which in Drosophila muscle cells is peripherally localized in the nucleus, whereas the central region is depleted of chromatin, and is organised such that RNA polymerase II (RNAp) is surrounding dense regions of chromatin. The authors theoretically study the formation of these regions by describing chromatin as a multi-block copolymer, where the blocks correspond to active and inactive chromatin regions. These regions are assumed to phase separately and to have different solvability. The solvability of the active region is regulated by binding RNAp. The authors study the core-shell organization in a layered geometry by analyzing the various contributions to free energy. In this way, they in particular obtain the dependence of the shell-layer …
Author Response
Reviewer #1 (Public Review):
The authors provide evidence for chromatin, which in Drosophila muscle cells is peripherally localized in the nucleus, whereas the central region is depleted of chromatin, and is organised such that RNA polymerase II (RNAp) is surrounding dense regions of chromatin. The authors theoretically study the formation of these regions by describing chromatin as a multi-block copolymer, where the blocks correspond to active and inactive chromatin regions. These regions are assumed to phase separately and to have different solvability. The solvability of the active region is regulated by binding RNAp. The authors study the core-shell organization in a layered geometry by analyzing the various contributions to free energy. In this way, they in particular obtain the dependence of the shell-layer thickness, which is described as a polymer brush. From these results, they infer chromatin organization in spherical coreshell chromatin domains and compare these results to Brownian dynamics simulations.
The work is well done and even though it uses standard methods for studying block copolymers and polymer brushes obtains interesting information about local chromatin organization. These findings should be of great interest to researchers in the field of chromatin organization and in general to everybody interested in understanding the physical principles of biological organization.
The work has two main weaknesses: The experimental evidence for RNAp and chromatin microorganization is weak as only one example is shown. It remains unclear whether the observed organization pattern is common or not. Also, no data is shown concerning the dependence of the extensions of the active and inactive phases on parameters, for example, solvent properties or transcriptional activity. Second, some parts could prove difficult for biologists to assess. For example, the expression for the brush-free energy should be explained in more detail and notions like that of 'mushrooms' need to be introduced. As a second example, biologists might benefit from a better explanation of the concept of a theta solvent and its relevance.
We thank Reviewer #1 for the positive review and critical feedback. Below we answer the points raised in the last paragraph of its review.
In the original version of the manuscript we only showed a representative image of nuclei of muscle cells in an intact, live Drosophila larvae. Notably, this organization is representative of many nuclei analyzed in muscle tissue. In the revised version we show that in a distinct tissue, e.g. salivary gland epithelium of live Drosophila larvae, RNA Pol II distribution is similarly facing the nucleoplasm, although chromatin condensation differs due to higher DNA ploidy. The new images were added as Supplement information (Fig A1). Since these representative images are the main motivation behind our theoretical analysis, we think that including them will help the reader in understanding the relevance of our minimal model. The effect of different biological perturbations, such as changes in the repressive marks and how these change the core-shell structure require extensive experiments that are outside the scope of the present paper. We also note, that in live organisms (not just live cells) such as those studied here, one can only reliably use genetic perturbations; solvent quality is regulated by the organism and cannot be controlled as in synthetic polymer experiments. Our main focus in the present paper is to highlight an area that has been relatively unexplored by the chromatin organization community, which is how changes in concentrations binding-partners of chromatin may have a strong effect in nuclear architecture.
We have also improved the explanation of the physical concepts for biologists. We added a more thorough explanation of the concept of a polymer brush and explained more clearly what the concept of theta solvent in terms of the scaling properties of a polymer in solution. We quote these revisions below.
Reviewer #2 (Public Review):
This work formulates a detailed theoretical polymer physics model intended to explain the observed morphology of chromatin in the Drosophila cell nucleus. The model is examined in detail by both analytical calculation and computer simulation. The central premise of the suggested theory is that it is again based on equilibrium statistical mechanics. Within this paradigm, authors explore the model that views chromatin fiber as a block copolymer and, most importantly, describes the role of RNA polymerase as it interacts with one of the copolymer blocks and regulates its effective solvent quality. Blocks are assumed to be fixed on the time scale of interest by, e.g., different levels of acetylation or methylation. RNA polymerase is supposed to interact only with one of the chromatin blocks, called active, and assumed interaction is quite peculiar. Namely, RNA polymerase complex may absorb on chromatin fiber and, the model assumes, the fiber decorated with absorbed RNA polymerase molecules is less sticky to itself, or more repulsive than the fiber itself. This peculiar assumption allows authors to make interesting predictions about how proteins can regulate the genome folding architecture.
We thank the reviewer for the positive and critical feedback. We agree that our assumption of changes in the effective solvent stemming from protein complexes binding to chromatin is at the core of our analysis and we justify it further below.
STRENGTH
The work includes a rather detailed theoretical description of the model and its equilibrium statistical mechanics. As both analytical theory and accompanying simulation indicate, the assumptions put forward in formulating the model do indeed produce the desired morphology, with isolated regions ("micelles") of core inactive chromatin surrounded by the less dense shell region in which RNA polymerization may potentially take place. Having such a detailed theory is potentially beneficial for the field and opens up avenues for further exploration.
We thank the referee for appreciating the potential benefit of our minimal theory of solvent-quality regulation by binding processes.
WEAKNESS
The underlying assumption about the interaction of RNA polymerase complex with the fiber, although important and organic for the model, does not seem easy to justify from a molecular standpoint, especially thinking of the charges and electrostatic interactions.
We visualize that the binding of RNA Pol II (mediated by different transcription factors) to chromatin is also associated with larger protein complexes that may contain hydrophobic and hydrophilic components, such as pre-initiation complexes. Some regions of these complexes might associate directly with chromatin due to positive charges on the surface of the Pol II complex , whereas the hydrophilic negative regions may be directed towards the solvent. Our theory is typical of the approach used in polymer physics where coarse-grained interactions are considered. While the origin of hydrophilic interactions lies in electrostatics, such interactions are highly screened in cells (typically 200 mM concentration of salts) and can be considered as short-ranged and competitive with hydrophobic interactions. Chromatin in solution is known to condense (see Gibson, et. al., Cell 2019 and Strickfaden, et. al., Cell 2020) and even phase separate from the nucleoplasm (see Amiad-Pavlov, et. al., Science Advances, 2021); this can arise either from hydrophobic interactions of the histone tails or from opposite charge attraction of the histones and linker DNA. In our model, this competes with the binding of protein complexes which then disrupt the self-attraction of chromatin. Previous work has shown that RNA Pol II associating with chromatin (in the absence of transcription) prevents the coarsening of dense chromatin domains (see Hilbert, et. al. Nat. Comm. 2021), which agrees with our modeling of protein complexes that bind to chromatin and interfere with its condensation; in addition, the binding of Pol-II and all its binding partners that form the pre-initiation complex (see Hahn, Nat. Struct. & Mol. Biol. 2004, 11) will result in effective, steric repulsion between different active and Pol II bound chromatin domains. Another interesting observation is that most of the surface of RNA Polymerase II is negatively charged with a few positively charged patches with which it specifically interacts with DNA while others serve as exit paths of RNA (see Cramer, et. al., Science, 2001.). We agree that a more thorough analysis of the molecular interactions between what we name protein complexes and chromatin is interesting, but it is out of the scope of our paper that uses a coarsegrained, polymer physics approach. This approach also allows our model to be to be predictive as to the physical organization and growth of the domains, independent of those molecular details that are as yet unknown.
Reviewer #3 (Public Review):
This theoretical study provides a theoretical explanation for a puzzling question arising from recent experiments: How can chromosomes behave like polymers collapsed in a poor solvent but also contain "open" active chromatin sections? The authors propose that the binding of proteins (e.g. RNAP's) to the active sections can effectively change the solvent quality for these sections and thus open them. They suggest further that chromosomes show micellar structures with inactive blocks forming the cores of the micelles. Protein binding causes swelling of the micellar shells which affects the whole chromosome structure by changing the total number of micelles. This theory fits well to live imaging data of chromatin in Drosophila larvae, like the one shown in the striking Figure 1.
The manuscript is written very clearly.
My only suggestion is that the authors, in both the theory and simulation parts, are more explicit about how the interactions between the various components are modeled. From what I could see, in the theory part, one needs to look closely at Eq. 5 to understand how the influence of the binding of proteins affects the interaction between active monomers, and in the simulation part, one needs to go to the appendix to learn that interaction strengths between monomers within the active blocks and monomers within the inactive blocks have different values. The latter is crucial to understand the micellar structure shown at the top of Fig. 5A.
We thank the reviewer for his positive response. We have explained Eq. 5 more carefully now and included other explanatory remarks throughout the text. We also explained more clearly the interactions considered in the simulations. Below we answer point by point and add quotes from the revised manuscript.
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eLife assessment
This fundamental work substantially advances our understanding of polymer physics underpinnings of genome folding, organization, and regulation. The conclusions are supported by both convincing computer simulations and analytical theory. The work will be of significant interest to the genome folding community.
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Reviewer #1 (Public Review):
The authors provide evidence for chromatin, which in Drosophila muscle cells is peripherally localized in the nucleus, whereas the central region is depleted of chromatin, and is organised such that RNA polymerase II (RNAp) is surrounding dense regions of chromatin. The authors theoretically study the formation of these regions by describing chromatin as a multi-block copolymer, where the blocks correspond to active and inactive chromatin regions. These regions are assumed to phase separately and to have different solvability. The solvability of the active region is regulated by binding RNAp. The authors study the core-shell organization in a layered geometry by analyzing the various contributions to free energy. In this way, they in particular obtain the dependence of the shell-layer thickness, which is …
Reviewer #1 (Public Review):
The authors provide evidence for chromatin, which in Drosophila muscle cells is peripherally localized in the nucleus, whereas the central region is depleted of chromatin, and is organised such that RNA polymerase II (RNAp) is surrounding dense regions of chromatin. The authors theoretically study the formation of these regions by describing chromatin as a multi-block copolymer, where the blocks correspond to active and inactive chromatin regions. These regions are assumed to phase separately and to have different solvability. The solvability of the active region is regulated by binding RNAp. The authors study the core-shell organization in a layered geometry by analyzing the various contributions to free energy. In this way, they in particular obtain the dependence of the shell-layer thickness, which is described as a polymer brush. From these results, they infer chromatin organization in spherical core-shell chromatin domains and compare these results to Brownian dynamics simulations.
The work is well done and even though it uses standard methods for studying block copolymers and polymer brushes obtains interesting information about local chromatin organization. These findings should be of great interest to researchers in the field of chromatin organization and in general to everybody interested in understanding the physical principles of biological organization.
The work has two main weaknesses: The experimental evidence for RNAp and chromatin micro-organization is weak as only one example is shown. It remains unclear whether the observed organization pattern is common or not. Also, no data is shown concerning the dependence of the extensions of the active and inactive phases on parameters, for example, solvent properties or transcriptional activity. Second, some parts could prove difficult for biologists to assess. For example, the expression for the brush-free energy should be explained in more detail and notions like that of 'mushrooms' need to be introduced. As a second example, biologists might benefit from a better explanation of the concept of a theta solvent and its relevance.
-
Reviewer #2 (Public Review):
This work formulates a detailed theoretical polymer physics model intended to explain the observed morphology of chromatin in the Drosophila cell nucleus. The model is examined in detail by both analytical calculation and computer simulation. The central premise of the suggested theory is that it is based on equilibrium statistical mechanics. Within this paradigm, authors explore the model that views chromatin fiber as a block copolymer and, most importantly, describes the role of RNA polymerase as it interacts with one of the copolymer blocks and regulates its effective solvent quality. Blocks are assumed to be fixed on the time scale of interest by, e.g., different levels of acetylation or methylation. RNA polymerase is supposed to interact only with one of the chromatin blocks, called active, and assumed …
Reviewer #2 (Public Review):
This work formulates a detailed theoretical polymer physics model intended to explain the observed morphology of chromatin in the Drosophila cell nucleus. The model is examined in detail by both analytical calculation and computer simulation. The central premise of the suggested theory is that it is based on equilibrium statistical mechanics. Within this paradigm, authors explore the model that views chromatin fiber as a block copolymer and, most importantly, describes the role of RNA polymerase as it interacts with one of the copolymer blocks and regulates its effective solvent quality. Blocks are assumed to be fixed on the time scale of interest by, e.g., different levels of acetylation or methylation. RNA polymerase is supposed to interact only with one of the chromatin blocks, called active, and assumed interaction is quite peculiar. Namely, RNA polymerase complex may absorb on chromatin fiber and, the model assumes, the fiber decorated with absorbed RNA polymerase molecules is less sticky to itself, or more repulsive than the fiber itself. This peculiar assumption allows authors to make interesting predictions about how proteins can regulate the genome folding architecture.
STRENGTH
The work includes a rather detailed theoretical description of the model and its equilibrium statistical mechanics. As both analytical theory and accompanying simulation indicate, the assumptions put forward in formulating the model do indeed produce the desired morphology, with isolated regions ("micells") of core inactive chromatin surrounded by the less dense shell region in which RNA polymerization may potentially take place. Having such a detailed theory is potentially beneficial for the field and opens up avenues for further exploration.
WEAKNESS
The underlying assumption about the interaction of RNA polymerase complex with the fiber, although important and organic for the model, does not seem easy to justify from a molecular standpoint, especially thinking of the charges and electrostatic interactions.
-
Reviewer #3 (Public Review):
This theoretical study provides a theoretical explanation for a puzzling question arising from recent experiments: How can chromosomes behave like polymers collapsed in a poor solvent but also contain "open" active chromatin sections? The authors propose that the binding of proteins (e.g. RNAP's) to the active sections can effectively change the solvent quality for these sections and thus open them. They suggest further that chromosomes show micellar structures with inactive blocks forming the cores of the micelles. Protein binding causes swelling of the micellar shells which affects the whole chromosome structure by changing the total number of micelles. This theory fits well to live imaging data of chromatin in Drosophila larvae, like the one shown in the striking Figure 1.
The manuscript is written very …
Reviewer #3 (Public Review):
This theoretical study provides a theoretical explanation for a puzzling question arising from recent experiments: How can chromosomes behave like polymers collapsed in a poor solvent but also contain "open" active chromatin sections? The authors propose that the binding of proteins (e.g. RNAP's) to the active sections can effectively change the solvent quality for these sections and thus open them. They suggest further that chromosomes show micellar structures with inactive blocks forming the cores of the micelles. Protein binding causes swelling of the micellar shells which affects the whole chromosome structure by changing the total number of micelles. This theory fits well to live imaging data of chromatin in Drosophila larvae, like the one shown in the striking Figure 1.
The manuscript is written very clearly.
My only suggestion is that the authors, in both the theory and simulation parts, are more explicit about how the interactions between the various components are modeled. From what I could see, in the theory part, one needs to look closely at Eq. 5 to understand how the influence of the binding of proteins affects the interaction between active monomers, and in the simulation part, one needs to go to the appendix to learn that interaction strengths between monomers within the active blocks and monomers within the inactive blocks have different values. The latter is crucial to understand the micellar structure shown at the top of Fig. 5A.
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