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Local Chromatin Motion and Transcription
Journal Pre-proof Local chromatin motion and transcription Michael Babokhov, Kayo Hibino, Yuji Itoh, Kazuhiro Maeshima PII:
S0022-2836(19)30614-X
DOI:
https://doi.org/10.1016/j.jmb.2019.10.018
Reference:
YJMBI 66304
To appear in:
Journal of Molecular Biology
Received Date: 7 May 2019 Revised Date:
7 October 2019
Accepted Date: 24 October 2019
Please cite this article as: M. Babokhov, K. Hibino, Y. Itoh, K. Maeshima, Local chromatin motion and transcription, Journal of Molecular Biology, https://doi.org/10.1016/j.jmb.2019.10.018. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier Ltd. All rights reserved.
Local chromatin motion and transcription
Michael Babokhov1#, Kayo Hibino1,2#, Yuji Itoh1, Kazuhiro Maeshima1,2*
1, Genome Dynamics Laboratory, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan. 2, Department of Genetics, School of Life Science, Sokendai (Graduate University for Advanced Studies), Mishima, Shizuoka 411-8540, Japan. *Correspondence: e-mail: [email protected] Tel: +81-55-981-6864 # These authors contributed equally to this paper.
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Abstract Eukaryotic chromatin is a complex of nucleic acids and proteins that is central to interpreting the information coded in the genome. Chromatin is rather irregularly folded inside the nucleus in a fluid-like state that exhibits dynamic local movement. The highly dynamic nature of chromatin has become increasingly appreciated, particularly in DNA-templated processes including transcription since this dynamic property ensures a degree of DNA accessibility, even in compacted chromatin. Many proteins globally constrain local chromatin movements, which seem to be driven essentially by thermal fluctuation in living cells. For instance, loss of the cohesin complex, which can capture chromatin fibers, leads to an increase in chromatin motion. Another constraining factor of chromatin motion is the transcription machinery. While the previously held view is that transcription requires open and highly dynamic chromatin, a number of studies are now pointing to a more nuanced role of transcription in constraining chromatin movement: dynamic clustering of active RNA polymerase II and other transcription factors can serve as a glue that transiently bridges active DNA regions to be transcribed, thereby loosely networking chromatin and constraining chromatin motion. In contrast, outside heterochromatin, the transcriptionally less active regions might be less constrained, more dynamic and accessible, implying a high competency state for rapid and efficient recruitment of protein factors. This new view on the interplay of local chromatin motion and transcription reflects traditional models of the transcription factories and, more recently, liquid-like hubs/droplets of transcription factors, providing new insight into chromatin function.
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Keywords nucleosome, chromatin, cohesin, single nucleosome imaging, transcription, RNA polymerase II, liquid droplet.
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Introduction All essential DNA-templated processes (e.g. RNA transcription, DNA replication, repair/recombination) in eukaryotic cells occur in the context of chromatin [1-4]. The fundamental unit of chromatin is the nucleosome, comprised of genomic DNA wrapped around an octamer of core histone proteins that regulate the access of DNA-templated processes to genetic information[5]. For higher order chromatin folding, based on initial in-vitro observations, the nucleosome fiber was predicted to helically fold upon itself to form a highly ordered “30-nm fiber”[6, 7] and further large regular fibers. However, further studies found the regular 30-nm fibers only under a few rare conditions, for instance under low salt conditions [8], or very partially and transiently [9]. Instead, chromatin was mainly found to consist of more irregular and variable nucleosome fibers [9-17]. Chromatin exists in a fluid-like state in the living cell. In this perspective we define the fluid-like chromatin state as one with diffusive movement, as opposed to vibration around a fixed position found in amorphous solids [18, 19]. Note that this state is contrasted with the static state based on the regular 30-nm fibers that has long been proposed [20, 21]. The biophysical properties of this dynamic chromatin also fit well with parameters like bendability obtained from chromatin conformation capture (3C) and related experiments[22-24]. Taken together, extensive advances in the past 10 years have highlighted the dynamic organization of chromatin in the nucleus and its significance in regulating genomic processes. In this perspective, we will discuss the dynamic aspect of chromatin, especially its interplay with transcription.
Local chromatin motion
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Dynamic movements of chromatin in live cell imaging studies have long been revealed using LacO/LacI-GFP [25-29] and a related system[30, 31], CRISPR/dCas9-based strategies [32-34] and single nucleosome imaging [35, 36]. Genome-wide chromatin dynamics in a whole nucleus were also investigated using fluorescently labeled chromatin[37-39]. This dynamic property ensures a degree of DNA accessibility, even in compacted chromatin [35, 40], which was also supported by recent finding of genome-wide MNase accessibility [41]. This property may have several advantages in template-directed biological processes. For instance, in transcriptional regulation, the dynamic movement of chromatin would help with the targeting of transcription factors (TFs): the local chromatin fluctuation can facilitate movements of small TFs such as pioneering factors within the compact chromatin domain [35]. As another possibility, the movements may help to transfer the target sequence to the surface of compact chromatin domains that limit access of large TFs [40]. Furthermore, dynamically folding chromatin can more easily form loops, facilitating interaction between distant promoter and enhancer sequences[42, 43] or other genomic elements [31].
The widespread prevalence of chromatin motion raises the important question regarding the main driving force behind these movements. Local chromatin motion seems to be primarily driven by thermal fluctuation [19, 28, 36, 44]. Variability in this motion can be created by constraints from physical or geometrical factors [19]. Artificially decondensed chromatin (extended nucleosome fibers) is increasingly mobile because fewer local constraints are available to restrict thermal fluctuating motion (Figure 1A and left, 1B). In human cells, chromatin treated with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA)[13, 45] to decondense chromatin displayed increased chromatin movements (left, Figure 1B)[19, 36]. It is known that TSA treatment
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upregulates acetylation of the histone H3 and H4 tails, which can weaken the histone tail binding to the neighboring nucleosome and subsequent nucleosome-nucleosome interactions [46]. In addition, some ATP-dependent mechanisms have also been proposed e.g.[47]. Budding yeast studies showed that the INO80 remodeler enhances chromatin movements in response to DNA damage, mainly through nucleosome eviction and subsequent chromatin decompaction [48], which might facilitate repair processes of damaged DNA regions (Discussed later).
Many chromatin proteins can constrain local chromatin motion, which might contribute to various genome functions and also retention of each chromosome in its own “chromosome territory” [49] in the nuclear space. For instance, chromatin movements are constrained by the cohesin complex (center, Figure 1B) [19, 36, 50], which can capture chromatin fibers with its ring structure and thereby enable formation of chromatin loops as well as sister chromatid cohesion [51-53]. Fewer chromatin constraints by cohesin loss lead to an increase in local chromatin motion (Figure 1A and right, 1B)[19, 36, 50].
Consistently, heterochromatin-rich regions at the nuclear periphery, which are mainly laminaassociated chromatin domains LADs [54], showed less movement than the nucleoplasmic region [19, 36, 38, 39, 55], likely due to the chromatin tethering to inner nuclear membrane structures such as lamins [54]. Such chromatin constraining is true of the pericentric heterochromatin regions in mouse cells [36]. These regions are enriched with the heterochromatin marker trimethylation of histone H3 Lysine 9 and HP1 proteins, which can crosslink nucleosomes [56]and constrain chromatin motion [36].
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Transcription as a regulator of chromatin motion Recently it was shown that active RNA polymerase II (RNAPII) has a constraining role for chromatin motion in the cell [57]. RNAPII is a multi-subunit complex that is responsible for the transcription of all protein coding mRNAs and many additional non-coding RNAs [58, 59]. That RNAPII normally constricts chromatin movement was a surprising finding since it was previously assumed that transcription would open up chromatin structure and increase local chromatin motion. However, it was demonstrated through the effects of RNAPII rapid depletion as well as RNAPII inhibitors 5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) and αamanitin, all of which evicted RNAPII from chromatin and increased chromatin movements (Figure 2A)[57]. Note that the transcription inhibition did not significantly change local chromatin structures or chromatin domain organization [36, 60]. Furthermore, treatment with actinomycin D, which stalled RNAPII on the chromatin template [61], decreased chromatin motion genome-wide (Figure 2A)[57]. Interestingly these effects were correlated with active RNAPII marked with Serine 5 phosphorylation on the C-terminal domain (RNAPII-Ser5P), suggesting that the initiating form of the RNAPII is involved in the chromatin constraining process. These findings also held up in physiological states of transcriptional activity such as in quiescent cells or UV-irradiated cells, which are transcriptionally less active [57]. Consistently, some specific genomic loci in human breast cancer, fly embryos, and mouse embryonic stem cells became less dynamic when actively transcribed [30, 62, 63]. On the other hand, Gu et al.[33] reported that movement of a transcriptional regulatory element increased upon transcription activation. Although further studies are required to reconcile these incompatible results, taken together, the genome-wide results suggest that transcription constrains genome DNA motion under a range of cellular conditions.
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The unexpected role of active RNAPII in constraining chromatin raises the question as to the mechanism behind this activity. A possible model is that active RNAPII and other transcription complexes can serve as a hub that transiently bridges DNA regions, thereby forming a loose network of connections constraining chromatin motion (center, Figure 2B): less transcription leads to an increase in chromatin motion (right, Figure 2B)[57]. Indeed, recent chromatin interactome analyses by Hi-C reported genome-wide stable promoter-enhancer interactions maintained by RNAPII [64, 65].
This model suggests that transcription may occur around or between clusters of chromatin domains (center, Figure 2B), which is in a good agreement with a recent imaging work by Miron et al. [66] that transcription does not occur where chromatin is the most decondensed, but more at intermediate level of chromatin density, especially in so-called “inter-chromatin compartment” between chromatin domains [4].
Consistent with the above model, RNAPII and other transcription proteins have been long proposed to gather into “transcriptional factories”, which have been defined as clusters of RNAPII and other transcription factors that immobilize genome chromatin for efficient transcription [67]. Recent studies have uncovered dynamic liquid-droplet/cluster formations of RNAPII, Mediator, and other factors like P-TEFb complex as one potential mechanism to gather transcriptional complexes, possibly by phase separation processes [68-72]. Although a measured lifetime of RNPII clusters is ~ 5 s on average, a number of the transient RNAPII clusters were observed throughout the nucleus at a particular time point [73, 74]. If each cluster can constrain
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the local chromatin for 5 sec, local chromatin motion at the whole nucleus level would be considerably constrained. Indeed, a computational modeling suggested that such dynamic clusters of RNAPII can constrain chromatin genome-wide [57]. Furthermore, knockdown (KD) of CDK9, a component of P-TEFb, was found to increase chromatin motion in a manner similar to removal of RNAPII [57], suggesting that constraint of chromatin movement is achieved through dynamic clusters/droplets of several different transcription factors, presumably to increase the efficiency of the transcriptional process. It is intriguing to note that chromatin motion is also constrained in the pericentromeric heterochromatin[36], where HP1 was suggested to form droplets by a phase separation [75, 76].
Constraint of chromatin by RNAPII also implies that transcriptionally less active regions might be less constrained and more dynamic. Since it was suggested that an increase in local chromatin motion can facilitate chromatin accessibility of transcription factors and other proteins [35], the more dynamic (and repressive) chromatin may be in a high competency state for rapid and efficient recruitment of transcription factors to turn on certain genes in response to extracellular signals. A similar mechanism might be at work in the UV-irradiated cells to recruit DNA repair machinery to damage sites. Further work will be required to understand the physiological role(s) of constraining chromatin motion during transcriptional processes.
Perspectives In recent years there has been a growing appreciation for the highly variable and dynamic nature of chromatin organization and how these properties can contribute to regulating cellular processes including RNA transcription, DNA replication, and repair/recombination. This
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progress raises a concern that detailed structural determination of chromatin might not be meaningful. As noted by Dubochet [77], chemical fixation with compounds such as formaldehyde and glutaraldehyde, which are commonly used for cell biology analyses, can have artifactual effects on chromatin structures. Studies of chromatin dynamics, which presumably reflect chromatin organization in living cells, become more and more important to understand chromatin organization for multiple DNA-templated processes, especially with a combination of new technologies e.g. [66, 78].
Transcription has been shown to play an active role in regulating local chromatin motion on a genome-wide scale: a loose network consisting of clusters of transcriptionally active RNAPIIs and other transcription factors that serve as transient network hubs (Figure 2B). It would be intriguing to see whether chromatin around the transcription hub is constrained. Recent findings point to a role for transcription as not just a “reader” of genome chromatin, but also a “modulator” – a process that can actively shape the dynamic chromatin movements and organization to globally enable more efficient transcription and transcription-related processes.
ACKNOWLEDGMENTS We are grateful to the collaborators in the Nagashima et al. (2019) for their contribution, Ms. Sachiko Tamura for the Figure preparation. We thank Dr. M. Sasai and Dr. S. Ide for critical reading of this manuscript, and Dr. Bystricky and Maeshima lab members for helpful discussions, and the anonymous reviews for their valuable comments to improve this paper. We must apologize that we could not mention many important works and related papers on chromatin dynamics due to space limitations. This work was supported by JSPS grant
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(16H04746, 16H06279(PAGS) and 19H05273), Takeda Science Foundation, a JST CREST grant (JPMJCR15G2).
COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.
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Figure 1. Fewer chromatin constraints lead to an increase in chromatin motion. (A) Mean square displacement (MSD) plots (± standard deviation [SD] among cells) of control, TSAtreated, and RAD21-KD HeLa cells. MSD of TSA-treated and RAD21-KD cells were significantly greater than the control (Kolmogorov–Smirnov, p < 10–14 at all time points in control vs. TSA and control vs. RAD21-KD, error bars ± SD). For each condition, n = 25–75 cells. Data was reproduced from [36] with permission. (B) (left) Decondensed chromatin in cells treated with HDAC inhibitor TSA[13, 45] showed increased chromatin movements because of weakening nucleosome-nucleosome interactions and subsequent less local chromatin constraining. (center) Usual state of chromatin: Chromatin domain is organized by local nucleosome-nucleosome interactions and global holding by cohesin. (right) Cohesin loss lead to less constraining and a resultant increase in chromatin motion [36].
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Figure 2. Formation of a loose spatial genome chromatin network via transcription machinery. (A) MSD plots (±SD among cells) of nucleosomes in the human RPE-1 cells treated with RNAPII inhibitors, α-amanitin (pink), actinomycin D (brown) and control DMSO (gray). For each condition, n = 20 cells. The inhibition of RNAPII increased the chromatin motion, except for actinomycin D. Note that RPE-1 cells have slightly higher MSD value in control than HeLa cells, which were originated from a cervical cancer and have higher transcriptional activity than RPE-1 cells. ***, P < 0.0001 by the Kolmogorov– Smirnov test for control versus α-amanitin (P = 1.0 × 10 8) and for control versus actinomycin D (P = 9.5 × 10 6). Data was reproduced from [57]. (B) (left and center) Dynamic cluster/droplet of active RNAPII (red sphere with tails) and transcription factors (Mediator, activators, other factors; blue sphere) can work as a transient hub (pink sphere) to weakly connect multiple chromatin domains into a loose spatial genome network. RNAPII-Ser5P is a glue that bridges active DNA regions to be transcribed. This transcription hub can constrain chromatin motion. (right) RNAPII inhibition or its rapid depletion released the chromatin constraints and increased chromatin motion.
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Highlights
Chromatin is predominantly folded irregularly inside the nucleus in a fluid-like state.
Local chromatin movements, driven principally by thermal fluctuation, are constrained by multiple different proteins.
Active RNA Polymerase II globally constrains chromatin motion, suggesting the existence of loose genome chromatin networks via transcriptional machinery.
S0022-2836(19)30614-X
DOI:
https://doi.org/10.1016/j.jmb.2019.10.018
Reference:
YJMBI 66304
To appear in:
Journal of Molecular Biology
Received Date: 7 May 2019 Revised Date:
7 October 2019
Accepted Date: 24 October 2019
Please cite this article as: M. Babokhov, K. Hibino, Y. Itoh, K. Maeshima, Local chromatin motion and transcription, Journal of Molecular Biology, https://doi.org/10.1016/j.jmb.2019.10.018. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier Ltd. All rights reserved.
Local chromatin motion and transcription
Michael Babokhov1#, Kayo Hibino1,2#, Yuji Itoh1, Kazuhiro Maeshima1,2*
1, Genome Dynamics Laboratory, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan. 2, Department of Genetics, School of Life Science, Sokendai (Graduate University for Advanced Studies), Mishima, Shizuoka 411-8540, Japan. *Correspondence: e-mail: [email protected] Tel: +81-55-981-6864 # These authors contributed equally to this paper.
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Abstract Eukaryotic chromatin is a complex of nucleic acids and proteins that is central to interpreting the information coded in the genome. Chromatin is rather irregularly folded inside the nucleus in a fluid-like state that exhibits dynamic local movement. The highly dynamic nature of chromatin has become increasingly appreciated, particularly in DNA-templated processes including transcription since this dynamic property ensures a degree of DNA accessibility, even in compacted chromatin. Many proteins globally constrain local chromatin movements, which seem to be driven essentially by thermal fluctuation in living cells. For instance, loss of the cohesin complex, which can capture chromatin fibers, leads to an increase in chromatin motion. Another constraining factor of chromatin motion is the transcription machinery. While the previously held view is that transcription requires open and highly dynamic chromatin, a number of studies are now pointing to a more nuanced role of transcription in constraining chromatin movement: dynamic clustering of active RNA polymerase II and other transcription factors can serve as a glue that transiently bridges active DNA regions to be transcribed, thereby loosely networking chromatin and constraining chromatin motion. In contrast, outside heterochromatin, the transcriptionally less active regions might be less constrained, more dynamic and accessible, implying a high competency state for rapid and efficient recruitment of protein factors. This new view on the interplay of local chromatin motion and transcription reflects traditional models of the transcription factories and, more recently, liquid-like hubs/droplets of transcription factors, providing new insight into chromatin function.
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Keywords nucleosome, chromatin, cohesin, single nucleosome imaging, transcription, RNA polymerase II, liquid droplet.
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Introduction All essential DNA-templated processes (e.g. RNA transcription, DNA replication, repair/recombination) in eukaryotic cells occur in the context of chromatin [1-4]. The fundamental unit of chromatin is the nucleosome, comprised of genomic DNA wrapped around an octamer of core histone proteins that regulate the access of DNA-templated processes to genetic information[5]. For higher order chromatin folding, based on initial in-vitro observations, the nucleosome fiber was predicted to helically fold upon itself to form a highly ordered “30-nm fiber”[6, 7] and further large regular fibers. However, further studies found the regular 30-nm fibers only under a few rare conditions, for instance under low salt conditions [8], or very partially and transiently [9]. Instead, chromatin was mainly found to consist of more irregular and variable nucleosome fibers [9-17]. Chromatin exists in a fluid-like state in the living cell. In this perspective we define the fluid-like chromatin state as one with diffusive movement, as opposed to vibration around a fixed position found in amorphous solids [18, 19]. Note that this state is contrasted with the static state based on the regular 30-nm fibers that has long been proposed [20, 21]. The biophysical properties of this dynamic chromatin also fit well with parameters like bendability obtained from chromatin conformation capture (3C) and related experiments[22-24]. Taken together, extensive advances in the past 10 years have highlighted the dynamic organization of chromatin in the nucleus and its significance in regulating genomic processes. In this perspective, we will discuss the dynamic aspect of chromatin, especially its interplay with transcription.
Local chromatin motion
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Dynamic movements of chromatin in live cell imaging studies have long been revealed using LacO/LacI-GFP [25-29] and a related system[30, 31], CRISPR/dCas9-based strategies [32-34] and single nucleosome imaging [35, 36]. Genome-wide chromatin dynamics in a whole nucleus were also investigated using fluorescently labeled chromatin[37-39]. This dynamic property ensures a degree of DNA accessibility, even in compacted chromatin [35, 40], which was also supported by recent finding of genome-wide MNase accessibility [41]. This property may have several advantages in template-directed biological processes. For instance, in transcriptional regulation, the dynamic movement of chromatin would help with the targeting of transcription factors (TFs): the local chromatin fluctuation can facilitate movements of small TFs such as pioneering factors within the compact chromatin domain [35]. As another possibility, the movements may help to transfer the target sequence to the surface of compact chromatin domains that limit access of large TFs [40]. Furthermore, dynamically folding chromatin can more easily form loops, facilitating interaction between distant promoter and enhancer sequences[42, 43] or other genomic elements [31].
The widespread prevalence of chromatin motion raises the important question regarding the main driving force behind these movements. Local chromatin motion seems to be primarily driven by thermal fluctuation [19, 28, 36, 44]. Variability in this motion can be created by constraints from physical or geometrical factors [19]. Artificially decondensed chromatin (extended nucleosome fibers) is increasingly mobile because fewer local constraints are available to restrict thermal fluctuating motion (Figure 1A and left, 1B). In human cells, chromatin treated with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA)[13, 45] to decondense chromatin displayed increased chromatin movements (left, Figure 1B)[19, 36]. It is known that TSA treatment
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upregulates acetylation of the histone H3 and H4 tails, which can weaken the histone tail binding to the neighboring nucleosome and subsequent nucleosome-nucleosome interactions [46]. In addition, some ATP-dependent mechanisms have also been proposed e.g.[47]. Budding yeast studies showed that the INO80 remodeler enhances chromatin movements in response to DNA damage, mainly through nucleosome eviction and subsequent chromatin decompaction [48], which might facilitate repair processes of damaged DNA regions (Discussed later).
Many chromatin proteins can constrain local chromatin motion, which might contribute to various genome functions and also retention of each chromosome in its own “chromosome territory” [49] in the nuclear space. For instance, chromatin movements are constrained by the cohesin complex (center, Figure 1B) [19, 36, 50], which can capture chromatin fibers with its ring structure and thereby enable formation of chromatin loops as well as sister chromatid cohesion [51-53]. Fewer chromatin constraints by cohesin loss lead to an increase in local chromatin motion (Figure 1A and right, 1B)[19, 36, 50].
Consistently, heterochromatin-rich regions at the nuclear periphery, which are mainly laminaassociated chromatin domains LADs [54], showed less movement than the nucleoplasmic region [19, 36, 38, 39, 55], likely due to the chromatin tethering to inner nuclear membrane structures such as lamins [54]. Such chromatin constraining is true of the pericentric heterochromatin regions in mouse cells [36]. These regions are enriched with the heterochromatin marker trimethylation of histone H3 Lysine 9 and HP1 proteins, which can crosslink nucleosomes [56]and constrain chromatin motion [36].
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Transcription as a regulator of chromatin motion Recently it was shown that active RNA polymerase II (RNAPII) has a constraining role for chromatin motion in the cell [57]. RNAPII is a multi-subunit complex that is responsible for the transcription of all protein coding mRNAs and many additional non-coding RNAs [58, 59]. That RNAPII normally constricts chromatin movement was a surprising finding since it was previously assumed that transcription would open up chromatin structure and increase local chromatin motion. However, it was demonstrated through the effects of RNAPII rapid depletion as well as RNAPII inhibitors 5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) and αamanitin, all of which evicted RNAPII from chromatin and increased chromatin movements (Figure 2A)[57]. Note that the transcription inhibition did not significantly change local chromatin structures or chromatin domain organization [36, 60]. Furthermore, treatment with actinomycin D, which stalled RNAPII on the chromatin template [61], decreased chromatin motion genome-wide (Figure 2A)[57]. Interestingly these effects were correlated with active RNAPII marked with Serine 5 phosphorylation on the C-terminal domain (RNAPII-Ser5P), suggesting that the initiating form of the RNAPII is involved in the chromatin constraining process. These findings also held up in physiological states of transcriptional activity such as in quiescent cells or UV-irradiated cells, which are transcriptionally less active [57]. Consistently, some specific genomic loci in human breast cancer, fly embryos, and mouse embryonic stem cells became less dynamic when actively transcribed [30, 62, 63]. On the other hand, Gu et al.[33] reported that movement of a transcriptional regulatory element increased upon transcription activation. Although further studies are required to reconcile these incompatible results, taken together, the genome-wide results suggest that transcription constrains genome DNA motion under a range of cellular conditions.
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The unexpected role of active RNAPII in constraining chromatin raises the question as to the mechanism behind this activity. A possible model is that active RNAPII and other transcription complexes can serve as a hub that transiently bridges DNA regions, thereby forming a loose network of connections constraining chromatin motion (center, Figure 2B): less transcription leads to an increase in chromatin motion (right, Figure 2B)[57]. Indeed, recent chromatin interactome analyses by Hi-C reported genome-wide stable promoter-enhancer interactions maintained by RNAPII [64, 65].
This model suggests that transcription may occur around or between clusters of chromatin domains (center, Figure 2B), which is in a good agreement with a recent imaging work by Miron et al. [66] that transcription does not occur where chromatin is the most decondensed, but more at intermediate level of chromatin density, especially in so-called “inter-chromatin compartment” between chromatin domains [4].
Consistent with the above model, RNAPII and other transcription proteins have been long proposed to gather into “transcriptional factories”, which have been defined as clusters of RNAPII and other transcription factors that immobilize genome chromatin for efficient transcription [67]. Recent studies have uncovered dynamic liquid-droplet/cluster formations of RNAPII, Mediator, and other factors like P-TEFb complex as one potential mechanism to gather transcriptional complexes, possibly by phase separation processes [68-72]. Although a measured lifetime of RNPII clusters is ~ 5 s on average, a number of the transient RNAPII clusters were observed throughout the nucleus at a particular time point [73, 74]. If each cluster can constrain
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the local chromatin for 5 sec, local chromatin motion at the whole nucleus level would be considerably constrained. Indeed, a computational modeling suggested that such dynamic clusters of RNAPII can constrain chromatin genome-wide [57]. Furthermore, knockdown (KD) of CDK9, a component of P-TEFb, was found to increase chromatin motion in a manner similar to removal of RNAPII [57], suggesting that constraint of chromatin movement is achieved through dynamic clusters/droplets of several different transcription factors, presumably to increase the efficiency of the transcriptional process. It is intriguing to note that chromatin motion is also constrained in the pericentromeric heterochromatin[36], where HP1 was suggested to form droplets by a phase separation [75, 76].
Constraint of chromatin by RNAPII also implies that transcriptionally less active regions might be less constrained and more dynamic. Since it was suggested that an increase in local chromatin motion can facilitate chromatin accessibility of transcription factors and other proteins [35], the more dynamic (and repressive) chromatin may be in a high competency state for rapid and efficient recruitment of transcription factors to turn on certain genes in response to extracellular signals. A similar mechanism might be at work in the UV-irradiated cells to recruit DNA repair machinery to damage sites. Further work will be required to understand the physiological role(s) of constraining chromatin motion during transcriptional processes.
Perspectives In recent years there has been a growing appreciation for the highly variable and dynamic nature of chromatin organization and how these properties can contribute to regulating cellular processes including RNA transcription, DNA replication, and repair/recombination. This
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progress raises a concern that detailed structural determination of chromatin might not be meaningful. As noted by Dubochet [77], chemical fixation with compounds such as formaldehyde and glutaraldehyde, which are commonly used for cell biology analyses, can have artifactual effects on chromatin structures. Studies of chromatin dynamics, which presumably reflect chromatin organization in living cells, become more and more important to understand chromatin organization for multiple DNA-templated processes, especially with a combination of new technologies e.g. [66, 78].
Transcription has been shown to play an active role in regulating local chromatin motion on a genome-wide scale: a loose network consisting of clusters of transcriptionally active RNAPIIs and other transcription factors that serve as transient network hubs (Figure 2B). It would be intriguing to see whether chromatin around the transcription hub is constrained. Recent findings point to a role for transcription as not just a “reader” of genome chromatin, but also a “modulator” – a process that can actively shape the dynamic chromatin movements and organization to globally enable more efficient transcription and transcription-related processes.
ACKNOWLEDGMENTS We are grateful to the collaborators in the Nagashima et al. (2019) for their contribution, Ms. Sachiko Tamura for the Figure preparation. We thank Dr. M. Sasai and Dr. S. Ide for critical reading of this manuscript, and Dr. Bystricky and Maeshima lab members for helpful discussions, and the anonymous reviews for their valuable comments to improve this paper. We must apologize that we could not mention many important works and related papers on chromatin dynamics due to space limitations. This work was supported by JSPS grant
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(16H04746, 16H06279(PAGS) and 19H05273), Takeda Science Foundation, a JST CREST grant (JPMJCR15G2).
COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.
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Figure 1. Fewer chromatin constraints lead to an increase in chromatin motion. (A) Mean square displacement (MSD) plots (± standard deviation [SD] among cells) of control, TSAtreated, and RAD21-KD HeLa cells. MSD of TSA-treated and RAD21-KD cells were significantly greater than the control (Kolmogorov–Smirnov, p < 10–14 at all time points in control vs. TSA and control vs. RAD21-KD, error bars ± SD). For each condition, n = 25–75 cells. Data was reproduced from [36] with permission. (B) (left) Decondensed chromatin in cells treated with HDAC inhibitor TSA[13, 45] showed increased chromatin movements because of weakening nucleosome-nucleosome interactions and subsequent less local chromatin constraining. (center) Usual state of chromatin: Chromatin domain is organized by local nucleosome-nucleosome interactions and global holding by cohesin. (right) Cohesin loss lead to less constraining and a resultant increase in chromatin motion [36].
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Figure 2. Formation of a loose spatial genome chromatin network via transcription machinery. (A) MSD plots (±SD among cells) of nucleosomes in the human RPE-1 cells treated with RNAPII inhibitors, α-amanitin (pink), actinomycin D (brown) and control DMSO (gray). For each condition, n = 20 cells. The inhibition of RNAPII increased the chromatin motion, except for actinomycin D. Note that RPE-1 cells have slightly higher MSD value in control than HeLa cells, which were originated from a cervical cancer and have higher transcriptional activity than RPE-1 cells. ***, P < 0.0001 by the Kolmogorov– Smirnov test for control versus α-amanitin (P = 1.0 × 10 8) and for control versus actinomycin D (P = 9.5 × 10 6). Data was reproduced from [57]. (B) (left and center) Dynamic cluster/droplet of active RNAPII (red sphere with tails) and transcription factors (Mediator, activators, other factors; blue sphere) can work as a transient hub (pink sphere) to weakly connect multiple chromatin domains into a loose spatial genome network. RNAPII-Ser5P is a glue that bridges active DNA regions to be transcribed. This transcription hub can constrain chromatin motion. (right) RNAPII inhibition or its rapid depletion released the chromatin constraints and increased chromatin motion.
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Highlights
Chromatin is predominantly folded irregularly inside the nucleus in a fluid-like state.
Local chromatin movements, driven principally by thermal fluctuation, are constrained by multiple different proteins.
Active RNA Polymerase II globally constrains chromatin motion, suggesting the existence of loose genome chromatin networks via transcriptional machinery.