The dynamics of the nuclear environment and their impact on gene function

Abstract

In the last decades, it has become increasingly clear how the modulation of spatial organization of chromatin over time and through the cell cycle is closely connected to gene function regulation. Different physicochemical stimuli contribute to the realization of specific transcriptional programs and finally to a specific cellular phenotype. In this review, we aim to describe the current knowledge about the dynamics regulating the movements and the interactions of molecules within the nucleus and their impact on gene functions. In particular, taking into account that these forces exert their effect in a nuclear environment characterized by a high concentration of molecules, we will discuss the role of proteins and structures that regulate these movements and transduce physicochemical signals acting on the cell to the nucleus.

The peculiar distribution of chromatin inside the nucleus determines specific contacts between DNA sequences, molecular complexes and nuclear structures, such as nucleoli and nuclear envelope, forming distinct functional domains like the Nucleolus associated domains (NADs) and the lamina-associated domains (LADs). During the different cell cycle phases, upon cellular differentiation or under conditions that perturb cell homoeostasis, nuclear architecture undergo structural rearrangements that influence gene expression and cell functions (1). These dynamics, regulated in space and time and influenced by different physicochemical signals acting on the cells, take place in a highly molecular crowded nucleoplasm that causes physical aggregation and separation of molecules into membrane-less compartment (2).

How nuclear motor and structural proteins influence gene function

The dynamic protein network, operationally defined as a nucleoskeleton, is composed of a series of nuclear motor and structural proteins such as actin, nuclear myosins (NMs), lamins and lamina-binding proteins, nuclear pore-linked filaments, nuclear mitotic apparatus, spectrins, titin and kinesins that shape the nucleus and regulate its mechanical properties and genome functionality. Many important nuclear functions, among which gene expression, are influenced by nucleoskeletal components (3).

Gene transcription is a highly regulated process that relies on a concerted interplay of proteins and regulatory DNA elements. Transcription requires extensive chromatin remodelling, to allow the access of the polymerases and transcription factors to the DNA regulatory elements and to expose the gene sequence. Proteins in transcriptional complexes have to move with respect to each other and along the DNA as transcription proceeds. Actin and myosin are able to convert, by ATP hydrolysis, chemical energy into mechanical work useful in transcription and in other nuclear processes. Nuclear Myosin 1 (NM1) is a monomeric, single-headed myosin localized in the cell nucleus. NM1 and actin have a role in pre-initiation complex assembly and in transcription elongation, acting more in general in chromatin remodelling (4, 5). NM1 and β-actin in fact co-localize and co-immunoprecipitate with RNA polymerase I and II (Pol I and Pol II) complexes (4, 5). Moreover, some studies have shown that decreasing NM1 levels causes transcription inhibition by both polymerases (6) and similarly, a decrease of monomeric nuclear actin, by inducing its polymerization, prevents RNA Pol II transcription (7). When NMI binds DNA and β-actin associates to the polymerase, they have been suggested to power the sliding of polymerase complexes relative to DNA (8).

Considering Pol I transcription machinery, NM1 and actin are required for transcription activation (9) and additionally NM1 plays a role during the elongation phase, interacting with the chromatin remodelling complex Williams syndrome transcription factor (WSTF)-SNF2h to promote Pol I transcription on chromatin (10). Finally, NMI and actin participate in the maturation of small ribosomal subunits (SSU), accompanying them to the nuclear pore, where they interact with actin-rich pore-linked filaments. Specifically, Cisterna et al. (11) demonstrated the spatial association of NMI and actin with the SSU from the nucleolar periphery to the nuclear pore. The export of SSU may occur through active mechanisms of transport, mediated by myosin and actin, under specific circumstances, particularly when cell metabolic activities require quickly new ribosome availability (11). However, generally, protein-coding and non-coding RNAs of different size seems to travel from the nucleus to the cytoplasm, mainly through a diffusion-based mechanism, exploiting export receptors and adaptor proteins (12). Mechanisms of gene tethering at NPCs can regulate gene activity either in a positive or negative manner depending on their interactions with different proteins (13). Export-competent messenger ribonucleoproteins mRNP complexes generated in the nucleoplasm reach NPCs passing through zones of heterochromatin exclusion (14). Then the translocation through NPCs requires assembly and disassembly of several mRNP-binding components to expose domains recognized by cytoplasmic translation adaptors and facilitate translation (14).

Another fundamental nuclear structure: the nuclear lamina (NL), a dense meshwork of intermediate filaments and associated proteins, which constitute the inner side scaffold of the nuclear envelope, exerts a role in regulating gene expression. A large portion of the heterochromatin in the nucleus is associated with the NL and referred as LADs. LADs are nuclear domains characterized by repressive histone marks, where genes are silenced or expressed at low levels (15). Nevertheless, lamin A/C can interact also with euchromatin, affecting epigenetic pathways and gene expression, as determined by experiments of chromatin immunoprecipitation of euchromatin- and heterochromatin-enriched samples. On the contrary, lamin B1 has been only associated with heterochromatin (16). Moreover, the NL influences various signalling pathways by sequestering transcription factors at the nuclear periphery preventing them from reaching their targets, or by acting as a scaffold for activation of signalling molecules (17). Over the last decade, different studies tried to clarify the influence of this structure on gene expression, exploiting artificial tethering of genomic reporter loci to the NL. Mixed results were obtained regarding transcriptional outcomes. Specifically, transcription of the reporter varies upon different localization to the NL and seems related to intrinsic differences in promoter sensitivity to the local LAD chromatin environment and the chromatin local features (18).

Physicochemical signals are transduced to the nucleus to modulate gene expression programmes

Cells of epithelial and connective tissues experience a series of tensional forces, like stretching or compression and are exposed to a variety of chemical signals that influence cell homoeostasis equilibrium. They are able to transduce and integrate these physicochemical stimuli from the local microenvironment into specific transcriptional programmes and genome organizations (19).

The cytoskeleton has a great influence on nuclear morphology, particularly during cell differentiation, when cell and nucleus acquire specific shapes. These morphological changes in turn affect chromatin dynamics. Specifically, mechanical and biochemical stimuli sensed by specialized membrane proteins are transduced to the nucleus biochemically (directly or through secondary messengers) and/or physically, thanks to the cytoskeleton networks that connect the cell membrane and the nucleus through the linker of nucleoskeleton and cytoskeleton complex (20). Stretch-activated receptors, binding to extracellular matrix proteins, are able to sense the local rigidity at the focal adhesion sites and transduce this parameter to the nucleus (21). Stem cells integrate physical and chemical signals coming from their niche, leading to differentiation into specific cell types (22, 23). They have a highly plastic cell nucleus due to a reduced cytoplasmic–nuclear ratio, whereas cell differentiation determines the dynamic reorganization of the cytoskeleton, which shape nuclear membranes leading to a stiffer nucleus in lineage-committed cells (24). Nuclear stiffness, which seems to be related mainly to the levels of lamin A/C proteins, strongly affect the possibility of nuclear deformation (25). The interplay among these mechanical forces can alter chromatin structure from a distance and influence chromatin-remodelling enzymes by switching them either in the bound or unbound forms, which in turn regulate gene expression and it could also modulate the opening and closing of nuclear pores (20).

Moreover, in case of strong mechanical deformations, cells may alter gene expression programmes trying to counterbalance such stress, but in extreme conditions this stimuli could cause cell transdifferentiation. Indeed, defects in cellular mechanosensory processes have been associated with various types of cancer (26).

Finally, chromatin exerts an outward entropic pressure onto the nuclear envelope, consequently, the degree of chromatin condensation affects importantly, not only gene expression but also the nuclear mechanical properties by modulating this pressure (27).

Nuclear crowding: between transcription and chromatin architecture

Nuclear elements are not randomly distributed in the cell nucleus, but their spatial organization rather suggests a form of compartmentalization (1). These compartments lack the usual lipid membranes as boundary elements, as commonly found in cell–cell or cell–organelles separation. The reasons for this rely in the leading force of this pattern formation: macromolecular crowding (28). The crowded nuclear environment favours biomolecules assembly, which is also essential for nucleolar stability and integrity (29). Moreover, Richter et al. investigated the effects of molecular crowding alteration on nuclear architecture. They highlighted that crowding directly affects chromatin compaction. Consequently, an important role has been proposed for nuclear crowding, since it could work as a stabilization parameter for several nuclear processes (30). Transcription resides among them. As proteins and non-coding chromatin occupy some 20–40% of the nuclear volume, transcription reactions are kinetically influenced by the surrounding elements that can deeply affect thermodynamics and equilibria of these events (29). For instance, under crowding conditions, nuclear molecules diffusion rates are reduced, thus favouring interaction with DNA (31). By increasing the time proteins can interact with promoters, the transcriptional noise is reduced, since less unstable gene expression intermediates are formed. Moreover, the size of crowding agents is reported to reflect the noise decrease: larger molecules reduce the diffusion of transcription factors with higher efficiency compared to smaller ones, thus promoting protein–DNA interaction (32). Matsuda and colleagues highlighted a direct link between nuclear crowding and gene expression regulation, hence it is no surprise that some evidence has been found suggesting an evolutionary conservation: multiple mammalian cell lines share similar nuclear crowding levels. In contrast, dysregulations in crowding phenomena have been reported to be associated to defective gene transcription, which results in pathologies, cancer and other severe conditions (33). For instance, crowded environments promote protein misfolding, that is associated to several consequences regarding health (34). Nuclear crowding can therefore be considered an environmental complexity that regulates thermodynamics and kinetics of several reactions, nuclear architecture and chromatin mobility and, importantly, gene expression: position inside the nucleus, time-depending crowding, nature of the crowding agents and much more must be kept in mind while assessing the complicated transcription events (30).

Phase separation drives nuclear compartmentalization

Another main contributor to nuclear microcompartmentalization, formation of membrane-less sectors, is a process known as liquid–liquid phase separation (LLPS) (35). LLPS could be directly correlated to the phenomenon of nuclear crowding. Indeed, phase-separated compartments are formed as a consequence of interaction between different elements that reside in the nucleus: proteins, RNA and DNA (2). The driving force that leads to phase separation is the strength of interaction, that must be strong enough to allow association, but at the same time it must be sufficiently weak to permit the internal dynamicity typical of every liquid state (36). The compartments formed in this way show permeability for specific biomolecules, hence determining a partition without the need of lipid layers: molecules can be either inside the compartment or excluded, according to their different nature and chemical characteristics (37). As long as a minimum in free energy is maintained, LLPS stays preserved on a wide scale, too. Among these cases, chromatin compartments held a primary place, since they are formed and maintained stable thanks to LLPS. It seems that heterochromatin domains are organized via phase separation driven by specific proteins, such as the heterochromatin protein 1 (HP1α), which is immobilized by dimerization and interactions with different binding partners (2). Differences in diffusion rates of heterochromatin protein 1 inside these heterochromatic domains have been pointed out: slower rates close to boundaries, higher ones inside. This reflects the typical properties of liquid compartments. HP1α summarizes the properties proteins must share to drive LLPS: the presence of oligomerization domains (in this case, dimerization), an intrinsically disordered region and a substrate-binding domain (that of HP1α binds methylated chromatin). These aspects allow the organization of molecules in membrane-less domains, de-mixed from the surrounding solutions and elements (38, 39). Moreover, because in various cases the substrate-binding domains recognize epigenetic modifications, as it happens for HP1α, the division in compartments due to LLPS is important in the field of gene expression regulation—in this specific case, by inducing chromatin compaction, thus a condition that limits DNA accessibility to transcription factors (39).

In the light of LLPS function in the cell nucleus, deepening the level of knowledge about this topic may elucidate several points which are still quite unclear, for instance the role of all the proteins and molecules that are at the base of this phenomenon. The same molecules could indeed be targets of new drugs. In the case of chromatin compartmentalization, the possibility to directly alter heterochromatin formation could have many outcomes regarding gene expression (40).

Investigating chromatin dynamics: from chromosome painting to 4D analysis

As described so far, chromatin conformation gives to each cell specificity and identity. Because of this, studying how chromatin architecture impacts on gene function has become crucial for decoding and predicting specific genetic information. Here, we discuss some methods for this purpose.

Since genome sequencing is not a sufficient approach to evaluate gene expression, the scientific community has provided several molecular and imaging technologies in order to study chromatin organization and molecular crowding.

From the 70s onwards, fluorescence in situ hybridization and chromosome painting, showed how specific chromatin portions occupy specific regions of the nucleus (41).

Recently, we also reviewed how light and electron microscopy were able to prove that the majority of biological processes require to localize in a specific compartment of the nucleus: the perichromatin region (42).

The advent of high-throughput, together with, genome manipulation technologies give new resources for the exploration of this field.

Chromosome conformation capture techniques (3C, 4C, 5C, Hi-C) allow to study higher order chromatin structures, such as topologically associating domains and to define the chromatin organization in chromosome territories (43). Later on, chromosome organization was observed dynamically in vivo through the utilization of super resolution technologies such as the combination of photoactivated localization microscopy and single-nucleosome tracking (44).

As mentioned before, chromatin by definition is matter of interaction between proteins and DNA; moreover, chromatin dynamics cannot occur without the influence of nucleoskeletal components as well as external stimuli. The study of this web of interactions is another brick in structure to function comprehension.

A common and easy way to study molecular interactions is live cell imaging through the usage of endogenous fluorescence molecules. A cutting-edge procedure is the usage of CRISPR-mediated fluorescence tagging, which allows to follow precisely interactions between specific DNA sequences (45).

Additionally, mimicking biological stimuli with desired temporal dynamics in vitro can be very difficult through the usage of conventional tools. Recently, a microfluidic device has been developed. This ‘chamber’ can be used under an inverted fluorescence microscope and thanks to an automatic analysis makes possible the study of both gene expression and signalling pathway activity (46, 47). On top of that, other tools such as nuclear magnetic resonance or Förster resonance energy transfer are basic but essential in the studying of molecular interactions. These latter are only few examples of the required integration of bioengineering, biophysics and chemistry for this research topic.

As described in one of the above paragraphs, mechanical forces play critical roles in the function of living cells. This type of biological forces may be applied even in vitro, for instance, with the usage of three-dimensional magnetic twisting cytometry. With this type of technique is possible to apply a stressing force on the nuclear membrane and follow specific tagged protein transcription related to this force, as described in 2016 by Tajik et al. (48).

Finally, when it comes to phase separation phenomena, multiple approaches are generally combined to describe it: from basic sedimentation assays (49) to the more complicated usage of atomic force microscopy, from which the soft material parameters can be quantitatively extracted (50).

Based on these up-to-date technologies, we can highlight how the three-dimensional analysis is getting on, transforming in a more composite scenario in which parameters as time and external stimuli must be comprehended.

Future developments integrating different approaches together with bioinformatic tools and models offer further promises for the knowledge of nucleus choreography.

Conclusions

We have overviewed nuclear dynamics and nuclear characteristics affecting gene expression. This complex chain of events is discussed with a particular focus on the nuclear mechanical properties and on the potential roles of LLPS in the genesis and maintenance of chromatin domains. The events and potential causes described here are complex, and further knowledge in this field could be useful in basic research as well as in clinical diagnostics.

Funding

This research was supported by the Italian Ministry of Education, University and Research (MIUR): Dipartimenti di Eccellenza Program (2018–2022)—Department of Biology and Biotechnology ‘L. Spallanzani,’ University of Pavia (to M.B.). supported by the Italian Ministry of Education, University and Research (MIUR): Dipartimenti di Eccellenza Program (2018–2022)—Department of Biology and Biotechnology ‘L. Spallanzani,’ University of Pavia (to M.B.).

Conflict of Interest

None declared.

References

Abbreviations

 
• HP1α

  heterochromatin protein 1

 
• LADs

  lamina-associated domains

 
• LLPS

  liquid–liquid phase separation

 
• NADs

  nucleolus-associated domains

 
• NL

  nuclear lamina

 
• NM1

  nuclear myosin 1

 
• Pol

  polymerase

 
• SSU

  small ribosomal subunits

 
• WSTF

  Williams syndrome transcription factor.

Author notes