Nucleus

The spatiotemporal organisation of chromatin in the eukaryotic nucleus is fundamental for genome regulation. Chromatin undergoes rapid remodelling and rearrangements within minutes, altering its diffusion properties. Considering the tight coupling between genome function and nuclear architecture, a key question is how chromatin dynamics adapt to or promote nuclear processes. To elucidate the underlying physical principles, we employed High-resolution Diffusion mapping (Hi-D) to track chromatin motion throughout interphase in live human cells. Our analysis, that considers both diffusive motion and drift generated by active forces, revealed that chromatin dynamics are heterogeneous, with distinct behaviours in different subnuclear zones. Notably, both diffusive and processive contributions to chromatin motion progressively decrease from G1 to G2 phase, with this reduction occurring uniformly across all subzones. This suggests a global mechanism driving the observed decrease in chromatin mobility during cell cycle progression. By combining genetic knockout experiments and polymer modelling, we demonstrate that the doubling of DNA content, rather than cohesin-mediated sister chromatid entrapment, is responsible for the gradual decrease in chromatin motion during the cell cycle in human nuclei. These findings provide new insights into the physical and functional organisation of chromatin and its regulation during cellular proliferation. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/712877v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@75c654org.highwire.dtl.DTLVardef@2fbd3dorg.highwire.dtl.DTLVardef@31025aorg.highwire.dtl.DTLVardef@191808e_HPS_FORMAT_FIGEXP M_FIG C_FIG

This article has been withdrawn by bioRxiv owing to a technical error that created a duplicate posting of this manuscript. Please see doi: https://doi.org/10.1101/2020.06.24.168757 to access the preprint.

Transcription is a fundamental cellular process, and the first step of gene expression. In human cells, it depends on the binding to chromatin of various proteins, including RNA polymerases and numerous transcription factors (TFs). Observations indicate that these proteins tend to form macromolecular clusters, known as transcription factories, whose morphology and composition is still debated. While some microscopy experiments have revealed the presence of specialised factories, composed of similar TFs transcribing families of related genes, sequencing experiments suggest instead that mixed clusters may be prevalent, as a panoply of different TFs binds promiscuously the same chromatin region. The mechanisms underlying the formation of specialised or mixed factories remain elusive. With the aim of finding such mechanisms, here we develop a chromatin polymer model mimicking the chromatin binding-unbinding dynamics of different types of complexes of TFs. Surprisingly, both specialised (i.e., demixed) and mixed clusters spontaneously emerge, and which of the two types forms depends mainly on cluster size. The mechanism promoting mixing is the presence of non-specific interactions between chromatin and proteins, which become increasingly important as clusters become larger. This result, that we observe both in simple polymer models and more realistic ones for human chromosomes, reconciles the apparently contrasting experimental results obtained. Additionally, we show how the introduction of different types of TFs strongly affects the emergence of transcriptional networks, providing a pathway to investigate transcriptional changes following gene editing or naturally occurring mutations.

Underlying higher order chromatin organization are Structural Maintenance of Chromosomes (SMC) complexes, large protein rings that entrap DNA. The molecular mechanism by which SMC complexes organize chromatin is as yet incompletely understood. Two prominent models posit that SMC complexes actively extrude DNA loops (loop extrusion), or that they sequentially entrap two DNAs that come into proximity by Brownian motion (diffusion capture). To explore the implications of these two mechanisms, we perform biophysical simulations of a 3.76 Mb-long chromatin chain, the size of the long S. pombe chromosome I left arm. On it, the SMC complex condensin is modeled to perform loop extrusion or diffusion capture. We then compare computational to experimental observations of mitotic chromosome formation. Both loop extrusion and diffusion capture can result in native-like contact probability distributions. In addition, the diffusion capture model more readily recapitulates mitotic chromosome axis shortening and chromatin density enrichment. Diffusion capture can also explain why mitotic chromatin shows reduced, as well as more anisotropic, movements, features that lack support from loop extrusion. The condensin distribution within mitotic chromosomes, visualized by stochastic optical reconstruction microscopy (STORM), shows clustering predicted from diffusion capture. Our results inform the evaluation of current models of mitotic chromosome formation.

The cell nucleus is a compartment in which essential processes such as gene transcription and DNA replication occur. While the large amount of chromatin confined in the finite nuclear space could install the picture of a particularly dense organelle surrounded by less dense cytoplasm, recent studies have begun to report the opposite. However, the generality of this newly emerging, opposite picture has so far not been tested. Here, we used combined optical diffraction tomography (ODT) and epi-fluorescence microscopy to systematically quantify the mass densities of cytoplasm, nucleoplasm, and nucleoli of human cell lines, challenged by various perturbations. We found that the nucleoplasm maintains a lower mass density than cytoplasm during cell cycle progression by scaling its volume to match the increase of dry mass during cell growth. At the same time, nucleoli exhibited a significantly higher mass density than the cytoplasm. Moreover, actin and microtubule depolymerization and changing chromatin condensation altered volume, shape, and dry mass of those compartments, while the relative distribution of mass densities was generally unchanged. Our findings suggest that the relative mass densities across membrane-bound and membraneless compartments are robustly conserved, likely by different as of yet unknown mechanisms, which hints at an underlying functional relevance. This surprising robustness of mass densities contributes to an increasing recognition of the importance of physico-chemical properties in determining cellular characteristics and compartments.

Linker histones (LH) have been shown to preferentially bind to AT-rich DNA, particularly A-tracts, contiguous stretches of adenines. Using spFRET (single pair Forster/Fluorescence Resonance Energy Transfer), we recently found that the globular domain (gH) of Xenopus laevis H1.0b LH orients towards A-tracts on the linker-DNA (L-DNA) while binding on-dyad in LH:mononucleosome complexes. Here, we investigate the impact of this A-tract-mediated orientation of the gH on the compaction of higher-order structures by studying trinucleosomes as minimal models for chromatin. Two 600 bp DNA sequences were constructed, each containing three consecutive Widom 601 core sequences connected by about 40 bp L-DNA but differing in the positioning of A-tracts on either the outer or the inner L-DNAs flanking the first and third Widom 601 sequences. The two inner L-DNAs were fluorescently labelled at their midpoints. Trinucleosomes were reconstituted using the doubly labelled DNA, core histone octamers and H1.0b. SpFRET was performed for a range of NaCl concentrations to measure the compaction and whether gH orientations affected the stability of the trinucleosomes to salt-induced dissociation. While the LH compacted the trinucleosomes, the extent of compaction and the stability were similar for the two DNA sequences. Modeling constrained by the measured FRET efficiency suggests that the structures adopted by the trinucleosomes correspond to the standard zig-zagged two-helical start arrangement with the first and third nucleosomes stacked on top of each other. In this arrangement, the first and third LHs are insufficiently close to interact and affect compaction. Thus, despite differences in the positioning of the A-tracts in the sequences studied, LH binding compacts the corresponding trinucleosomes similarly. Why it mattersThe compaction and three-dimensional structure of chromatin affect the exposure of the DNA and thus regulate gene expression. Linker histone proteins bind to nucleosomes and thereby contribute to chromatin compaction. We here investigated whether the DNA A-tract-mediated orientation of a linker histone globular domain affects chromatin structure by using a trinucleosome as a minimal model for chromatin. Our observations suggest that the trinucleosome structure and compaction are robust against differences in linker histone globular domain orientations. eTOC blurbWe investigate whether DNA sequences, such as adenine-tracts, and sequence-induced linker histone reorientation affect chromatin structure. Using trinucleosomes as model systems for chromatin, we demonstrate that the chromatin structure and compaction are robust to the studied DNA sequence differences and sequence-induced linker histone orientation.

Each proliferating cell replicates its DNA and internal components before distributing this material evenly to its daughters. Although the regulation of cyclin-dependent kinases (Cdks) that dictate orderly cell cycle progression is well characterized, how the subcellular localization of the cell cycle machinery contributes to timing is not well understood. We investigated the influence of the nucleus by reconstituting cell cycle oscillations in droplets of frog egg extract in the absence or presence of a nuclear compartment and monitoring dynamics by time-lapse microscopy. We found that the cell cycle time increased in the presence of nuclei, which grew larger with each cell cycle. The correlation between increasing nuclear volume and a longer cell cycle period was maintained across extracts and nuclei from various Xenopus species and persisted upon inhibition of DNA replication or transcription. However, inhibition of nuclear import or the kinase Wee1 impacted the relationship between the nuclear-cytoplasmic ratio and the cell cycle period. To conceptually capture these experimental observations, we developed a computational model that incorporates cell cycle oscillations, nuclear-cytoplasmic compartmentalization, and periodic nuclear envelope breakdown and reformation. Altogether, our results support the major role of the nuclear compartment in setting the pace of the cell cycle and provide an explanation for the increase in cell cycle length observed at the midblastula transition when cells become smaller and the nuclear-cytoplasmic ratio increases.

Liquid-liquid phase separation (LLPS) is driven by weak multi-valent interactions. Such interactions can result in the formation of puncta in cells and droplets in vitro. The heterochromatin protein HP1 forms droplets with chromatin in vitro and is found in puncta in cells. A common approach to visualize the dynamics of HP1 in cells is to genetically encode fluorescent tags on the protein. HP1 modified with tags such as GFP has been shown to localize to dynamic puncta in vivo. However, whether tagged HP1 retains its intrinsic phase separation properties has not been systematically studied. Here, using different C-terminal tags (AID-sfGFP, mEGFP, and UnaG), we assessed how tag size and linker length affected the phase separation ability of HP1 with DNA in vitro. We found that the AID-sfGFP tag (52 kDa) promoted HP1 phase-separation, possibly driven by the highly disordered AID degron. The mEGFP tag (27 kDa) inhibited phase-separation by HP1, whereas an UnaG tag (13 kDa) with a 16 amino acid linker showed minimal perturbation. The UnaG tag can thus be used in cellular studies of HP1 to better correlate in vitro and in vivo studies. To test if cellular crowding overcomes the negative effects of large tags in vivo, we used polyethylene glycol (PEG) to mimic crowding in vitro. We found that addition of 10% PEG8000 or PEG4000 enables phase separation by GFP-tagged HP1 at comparable concentrations as untagged HP1. However, these crowding agents also substantially dampened the differences in phase-separation between wild-type and mutant HP1 proteins. PEG further drove phase-separation of Maltose Binding Protein (MBP), a tag often used to solubilize other proteins. These results suggest that phase-separation of biological macromolecules with PEG should be interpreted with caution as PEG-based crowding agents may change the types of interactions made within the phases.

The three-dimensional organization of chromatin is influenced by DNA-binding proteins, through specific and non-specific interactions. However, the role of DNA sequence and interaction between binding proteins in influencing chromatin structure is not yet fully understood. By employing a simple polymer-based model of chromatin, that explicitly considers sequence-dependent binding of proteins to DNA and protein-protein interactions, we elucidate a mechanism for chromatin organization. We find that: (1) Tuning of protein-protein interaction and protein concentration is sufficient to either promote or inhibit the compartmentalization of chromatin. (2) The presence of chromatin acts as a nucleating site for the condensation of the proteins at a density lower than in isolated protein systems. (3) The exponents describing the spatial distance between the different parts of the chromatin, and their contact probabilities are strongly influenced by both sequence and the protein-protein attraction. Our findings have the potential application of re-interpreting data obtained from various chromosome conformation capture technologies, thereby laying the groundwork for advancing our understanding of chromatin organization.

The authors have withdrawn this manuscript after detecting issues with two of the figures in the manuscript. Therefore, the authors do not wish this work to be cited as a reference. If you have any questions, please contact the corresponding authors.

Methodological advances in conformation capture techniques have fundamentally changed our understanding of chromatin architecture. However, the nanoscale organization of chromatin and its cell-to-cell variance are less studied. By using a combination of high throughput super-resolution microscopy and coarse-grained modelling we investigated properties of active and inactive chromatin in interphase nuclei. Using DNase I hypersensitivity as a criterion, we have selected prototypic active and inactive regions from ENCODE data that are representative for K-562 and more than 150 other cell types. By using oligoFISH and automated STED microscopy we systematically measured physical distances of the endpoints of 5kb DNA segments in these regions. These measurements result in high-resolution distance distributions which are right-tailed and range from very compact to almost elongated configurations of more than 200 nm length for both the active and inactive regions. Coarse-grained modeling of the respective DNA segments suggests that in regions with high DNase I hypersensitivity cell-to-cell differences in nucleosome occupancy determine the histogram shape. Simulations of the inactive region cannot sufficiently describe the compaction measured by microscopy, although internucleosomal interactions were elevated and the linker histone H1 was included in the model. These findings hint at further organizational mechanisms while the microscopy-based distance distribution indicates high cell-to-cell differences also in inactive chromatin regions. The analysis of the distance distributions suggests that direct enhancer-promoter contacts, which most models of enhancer action assume, happen for proximal regulatory elements in a probabilistic manner due to chromatin flexibility.

The nuclei of undifferentiated cells show uniform decompacted chromatin while during development nuclei decrease in size and foci of condensed chromatin appear, reminiscent of phase separation. This study is motivated by recent experiments that suggest that the unbinding of enzymes that chemically modify (acetylate) histone tails causes decompaction of condensed chromatin. Here we take into account the enzymatic reactions of histone modifications to predict the phase separation of chromatin in a model system, the chromatin brush, which mimics chromatin at the proximity of a nuclear membrane. The model contains activators and silencers, which change the state of the nucleosomes to (transcriptionally) active or inactive via the Michaelis-Menten kinetics. Our theory predicts that the chromatin brush will phase separate when the brush height is reduced below a threshold height. The phase separation is driven by an anti-correlation: Activators change the state of nucleosomes to the active state suppressing the binding of silencers to these nucleosomes and vice versa.

Molecular crowding causes the compaction of chromatin fibers, contributing to the formation of the nuclear architecture. However, the molecular mechanism of compaction under crowded conditions is not yet fully understood. In this study, we employed the single-molecule optical tweezer method to investigate the effect of molecular crowding on chromatin structure. Force-extension experiments on a 12-mer polynucleosome in the presence of different sizes and concentrations of polyethylene glycol (PEG) as a crowding agent showed that at low concentrations of low-molecular-weight (MW) PEG, the compaction of the polynucleosome was not significant. In this respect, nucleosomes predominantly remained separated, while DNA-histone interactions within individual nucleosomes were slightly stabilized. In contrast, high concentrations of high-MW PEG significantly promote internucleosomal interactions, leading to highly compact polynucleosome conformations. Under these conditions, approximately 30 pN of force was required to disrupt the internucleosomal interactions and release DNA; this force was 36% higher than that required for DNA unwrapping in the absence of PEG. These findings suggest that molecular crowding impacts cellular processes by mechanically regulating chromatin accessibility for regulatory proteins and the passage of motor molecules such as RNA polymerase. Significance StatementChromatin condensation is closely related to biological processes such as transcription and replication. Molecular crowding has recently attracted attention as a factor regulating chromatin condensation. In this study, we used the optical tweezer method to analyze the molecular mechanisms underlying chromatin condensation. We found that high-molecular weight and high-concentration crowders (polyethylene glycol) induced significant compaction, which involved internucleosomal interactions that markedly reduced DNA accessibility. Our results suggest that molecular crowding not only alters the condensation state, but also mechanically regulates chromatin accessibility.

A layer of dense heterochromatin is found at the periphery of the nucleus. Because this peripheral heterochromatin functions as a repressive phase, mechanisms that relocate genes to the periphery play an important role in regulating transcription. Using Monte-Carlo simulations, we show that an interaction between chromatin and the nuclear boundary need not be specific to heterochromatin in order to preferentially locate heterochromatin to the nuclear periphery. This observation considerably broadens the class of possible interactions that result in peripheral positioning to include boundary interactions that either weakly attract all chromatin or strongly bind to a randomly chosen small subset of loci. The key distinguishing feature of heterochromatin is its high chromatin density with respect to euchromatin. In our model this densification is caused by HP1s preferential binding to H3K9me3 marked histone tails. We conclude that factors that are themselves unrelated to the nuclear periphery can determine which genomic regions condense to form heterochromatin and thereby control which regions are relocated to the periphery.

The six PCGF proteins (PCGF1-6) define the biochemical identity of Polycomb Repressor Complex 1 (PRC1) subcomplexes. While structural and functional studies of PRC1 subcomplexes have revealed specialized roles in distinct aspects of epigenetic regulation, our understanding of variation in protein interaction networks between the PCGF subunits is incomplete. We carried out an affinity purification mass spectrometry (AP-MS) screen of subunits PCGF1 (NSPC1), PCGF2 (MEL18), and PCGF4 (BMI1), using an immunoprecipitation approach that replicated endogenous cellular conditions in a cell line capable of differentiation programs. Over 200 interactions were found, including 83 that had not been described previously. Bioinformatic analysis found that these interacting proteins covered a range of functional pathways, often focused on cell biology and chromatin regulation. We found evidence of mutual regulation (at mRNA and protein level) between distinct PCGF subunits. Furthermore, we confirmed that disruption of each subunit using shRNA results in reduced proliferation ability. Overall, our work adds to understanding of the role of PCGF proteins within the wider cellular network.

The chromatin associated with the nuclear lamina (NL) is referred to as Lamina-Associated Domains (LADs). While mapping of this feature has been done using various technologies, technical limitations exist for each of the methods. Here, we present an adaptation of the Tyramide-Signal Amplification sequencing (TSA-seq) protocol, which we call chromatin pull down-based TSA-seq (cTSA-seq), that can be used to map chromatin regions at or near the NL from as little as 50,000 cells without using carriers. The cTSA-seq mapped regions are composed of LADs and smaller chromatin regions that fall within the chromatin B-compartment known to be enriched for heterochromatin and be present at the nuclear periphery. As a proof of principle, we used cTSA-seq to map chromatin at or near the assembling NL as cells exit mitosis and progress through early and later G1. Consistent with previous reports, lamin-B1 based cTSA-seq revealed that regions toward the distal ends of chromosomes are near or at the reassembling NL during early G1. The cTSA-seq mapping and analyses revealed similarity between the early G1 chromatin and oncogene-induced senescent cell populations. The cTSA-seq reported here represents a useful method for analyzing chromatin at or near the NL from small numbers of cells.

Eukaryotic nuclear DNA is wrapped around histone proteins to form nucleosomes, which further assemble to package and regulate the genome. Understanding of the physical mechanisms that contribute to higher order chromatin organization is limited. Previously, we reported the intrinsic capacity of chromatin to undergo phase separation and form dynamic liquid-like condensates, which can be regulated by cellular factors. Recent work from Hansen, Hendzel, and colleagues suggested these intrinsic chromatin condensates are solid in all but a specific set of conditions. Here we show that intrinsic chromatin condensates are fluid in diverse solutions, without need for specific buffering components. Exploring experimental differences in sample preparation and imaging between these two studies, we suggest what may have led Hansen, Hendzel, and colleagues to mischaracterize the innate properties of chromatin condensates. We also describe how liquid-like in vitro behaviors can translate to the locally dynamic but globally constrained movement of chromatin in cells.

The transfer of regulatory information between distal loci on chromatin is thought to involve physical proximity, but key biophysical features of these contacts remain unclear. For instance, it is unknown how close and for how long two loci need to be in order to productively interact. The main challenge is that it is currently impossible to measure chromatin dynamics with high spatiotemporal resolution at scale. Polymer simulations provide an accessible and rigorous way to test biophysical models of chromatin regulation, yet there is a lack of simple and general methods for extracting the values of model parameters. Here we adapt the Nelder-Mead simplex optimization algorithm to select the best polymer model matching a given Hi-C dataset, using the MYC locus as an example. The models biophysical parameters predict a compartmental rearrangement of the MYC locus in leukemia, which we validate with single-cell measurements. Leveraging trajectories predicted by the model, we find that loci with similar Hi-C contact frequencies can exhibit widely different contact dynamics. Interestingly, the frequency of productive interactions between loci exhibits a non-linear relationship with their Hi-C contact frequency when we enforce a specific capture radius and contact duration. These observations are consistent with recent experimental observations and suggest that the dynamic ensemble of chromatin configurations, rather than average contact matrices, is required to fully predict productive long-range chromatin interactions.

Despite their importance, how linker histone H1s interact in chromatin and especially how the highly positively charged and intrinsically disordered H1 C-terminal domain (CTD) binds and stabilizes nucleosomes and higher-order chromatin structures remains unclear. Using single-molecule FRET we found that about half of the H1 CTDs in H1-nucleosome complexes exhibit well-defined FRET values indicative of distinct, static conformations, while the remainder of the population exhibits dynamically changing values, similar to that observed for H1 in the absence of nucleosomes. We also find that the first 30 residues of the CTD participate in relatively localized interactions with the first [~]20 bp of linker DNA, and that two separate regions in the CTD contribute to H1-dependent organization of linker DNA, consistent with some non-random CTD-linker DNA interactions. Finally, our data show that acetylation mimetics within the histone H3 tail induce decondensation and enhanced dynamics of the nucleosome-bound H1 CTD. (148 words)

When the effect of various posttranslational histone tail modifications (PTMs) on nucleosome stability was compared in an in situ assay involving agarose-embedded nuclei, the promoter proximal H3K4me3, H3K27ac and H4K8ac positive nucleosomes exhibited relative sensitivity to intercalators as compared to bulk H3-GFP or nucleosomes carrying any of the following marks: H3K27me1, H3K27me2, H3K27me3, H3K9me1, H3K9me2, H3K9me3, H3K36me3, H3K4me0, H3K4me1, H3K4me2, H3K9ac, and H3K14ac. Nickase or DNase I treatment of the nuclei, or bleomycin treatment of live cells, did not affect the stability of nucleosomes carrying H3K4me3 or H3K27ac, while those of the second group were all destabilized upon treatment with intercalators. These observations support the possibility that the promoter proximal marks specify dynamic nucleosomes accomodating relaxed DNA sequences due to DNA breaks generated in vivo. In line with this interpretation, endogeneous, 3OH nicks were mapped within the nucleosome free region of promoters controlling genes active in human mononuclear cells, a conclusion supported by superresolution colocalization studies. The +1 nucleosomes were stabilized and the incidence of nicks was decreased at the promoters upon KDM4a,b,c KO induction (Pedersen et al, EMBO J, 2016) in mouse embryonic stem cells (mES). While etoposide did not further destabilize +1 nucleosomes in control mES, their stabilized state in the KO state was reversed by the drug. A significant fraction of the DNA breaks comprises TOP2-generated nicks according to the results of molecular combing experiments. The chromatin regions harboring nicks are topologicaly separated from the domains containing superhelical chromatin. These observations lend support for a model where the role of DNA strand discontinuities in transcriptional regulation and in higher-order chromatin organization are integrated.