Local Chromatin Compaction Research Articles

Evaluating the role of the nuclear microenvironment in gene function by population-based modeling.

The nuclear folding of chromosomes relative to nuclear bodies is an integral part of gene function. Here, we demonstrate that population-based modeling-from ensemble Hi-C data-provides a detailed description of the nuclear microenvironment of genes and its role in gene function. We define the microenvironment by the subnuclear positions of genomic regions with respect to nuclear bodies, local chromatin compaction, and preferences in chromatin compartmentalization. These structural descriptors are determined in single-cell models, thereby revealing the structural variability between cells. We demonstrate that the microenvironment of a genomic region is linked to its functional potential in gene transcription, replication, and chromatin compartmentalization. Some chromatin regions feature a strong preference for a single microenvironment, due to association with specific nuclear bodies in most cells. Other chromatin shows high structural variability, which is a strong indicator of functional heterogeneity. Moreover, we identify specialized nuclear microenvironments, which distinguish chromatin in different functional states and reveal a key role of nuclear speckles in chromosome organization. We demonstrate that our method produces highly predictive three-dimensional genome structures, which accurately reproduce data from a variety of orthogonal experiments, thus considerably expanding the range of Hi-C data analysis.

Dispersal of PRC1 condensates disrupts polycomb chromatin domains and loops.

Polycomb repressive complex 1 (PRC1) strongly influences 3D genome organization, mediating local chromatin compaction and clustering of target loci. Several PRC1 subunits have the capacity to form biomolecular condensates through liquid-liquid phase separation in vitro and when tagged and over-expressed in cells. Here, we use 1,6-hexanediol, which can disrupt liquid-like condensates, to examine the role of endogenous PRC1 biomolecular condensates on local and chromosome-wide clustering of PRC1-bound loci. Using imaging and chromatin immunoprecipitation, we show that PRC1-mediated chromatin compaction and clustering of targeted genomic loci-at different length scales-can be reversibly disrupted by the addition and subsequent removal of 1,6-hexanediol to mouse embryonic stem cells. Decompaction and dispersal of polycomb domains and clusters cannot be solely attributable to reduced PRC1 occupancy detected by chromatin immunoprecipitation following 1,6-hexanediol treatment as the addition of 2,5-hexanediol has similar effects on binding despite this alcohol not perturbing PRC1-mediated 3D clustering, at least at the sub-megabase and megabase scales. These results suggest that weak hydrophobic interactions between PRC1 molecules may have a role in polycomb-mediated genome organization.

Denoising Autoencoder Trained on Simulation-Derived Structures for Noise Reduction in Chromatin Scanning Transmission Electron Microscopy.

Scanning transmission electron microscopy tomography with ChromEM staining (ChromSTEM), has allowed for the three-dimensional study of genome organization. By leveraging convolutional neural networks and molecular dynamics simulations, we have developed a denoising autoencoder (DAE) capable of postprocessing experimental ChromSTEM images to provide nucleosome-level resolution. Our DAE is trained on synthetic images generated from simulations of the chromatin fiber using the 1-cylinder per nucleosome (1CPN) model of chromatin. We find that our DAE is capable of removing noise commonly found in high-angle annular dark field (HAADF) STEM experiments and is able to learn structural features driven by the physics of chromatin folding. The DAE outperforms other well-known denoising algorithms without degradation of structural features and permits the resolution of α-tetrahedron tetranucleosome motifs that induce local chromatin compaction and mediate DNA accessibility. Notably, we find no evidence for the 30 nm fiber, which has been suggested to serve as the higher-order structure of the chromatin fiber. This approach provides high-resolution STEM images that allow for the resolution of single nucleosomes and organized domains within chromatin dense regions comprising of folding motifs that modulate the accessibility of DNA to external biological machinery.

FRET-FISH probes chromatin compaction at individual genomic loci in single cells

Chromatin compaction is a key biophysical property that influences multiple DNA transactions. Lack of chromatin accessibility is frequently used as proxy for chromatin compaction. However, we currently lack tools for directly probing chromatin compaction at individual genomic loci. To fill this gap, here we present FRET-FISH, a method combining fluorescence resonance energy transfer (FRET) with DNA fluorescence in situ hybridization (FISH) to probe chromatin compaction at select loci in single cells. We first validate FRET-FISH by comparing it with ATAC-seq, demonstrating that local compaction and accessibility are strongly correlated. FRET-FISH also detects expected differences in compaction upon treatment with drugs perturbing global chromatin condensation. We then leverage FRET-FISH to study local chromatin compaction on the active and inactive X chromosome, along the nuclear radius, in different cell cycle phases, and during increasing passage number. FRET-FISH is a robust tool for probing local chromatin compaction in single cells.

Testing the Effect of Histone Acetyltransferases on Local Chromatin Compaction.

Experiments determining the chromatin association of histone acetylases (HATs) and deacetylases (HDACs) at the genome-wide level provide precise maps of locus occupancy, but do not allow conclusions on the functional consequences of this locus-specific enrichment. Here we describe a protocol that allows tethering of HATs or HDACs to specific genomic loci upon fusion with a fluorescent protein and a DNA-binding protein such as the E. coli Lac repressor (LacI), which binds to genomically inserted lac operon sequences (lacO) via DNA/protein interactions. Integration of these lacO sequences into a genomic region of interest allows to monitor the functional consequences of HAT/HDAC targeting on chromatin (de)compaction, histone modification, and interaction with other proteins by quantitative light microscopy, as described here. As DNA-binding of LacI can be tightly controlled by the addition of galactose-derivatives, this method also allows to monitor the effects of locus-specific recruitment in a time-resolved manner.

Integrative genome modeling platform reveals essentiality of rare contact events in 3D genome organizations

A multitude of sequencing-based and microscopy technologies provide the means to unravel the relationship between the three-dimensional organization of genomes and key regulatory processes of genome function. Here, we develop a multimodal data integration approach to produce populations of single-cell genome structures that are highly predictive for nuclear locations of genes and nuclear bodies, local chromatin compaction and spatial segregation of functionally related chromatin. We demonstrate that multimodal data integration can compensate for systematic errors in some of the data and can greatly increase accuracy and coverage of genome structure models. We also show that alternative combinations of different orthogonal data sources can converge to models with similar predictive power. Moreover, our study reveals the key contributions of low-frequency (‘rare’) interchromosomal contacts to accurately predicting the global nuclear architecture, including the positioning of genes and chromosomes. Overall, our results highlight the benefits of multimodal data integration for genome structure analysis, available through the Integrative Genome Modeling software package.

HP1α is a chromatin crosslinker that controls nuclear and mitotic chromosome mechanics.

Chromatin, which consists of DNA and associated proteins, contains genetic information and is a mechanical component of the nucleus. Heterochromatic histone methylation controls nucleus and chromosome stiffness, but the contribution of heterochromatin protein HP1α (CBX5) is unknown. We used a novel HP1α auxin-inducible degron human cell line to rapidly degrade HP1α. Degradation did not alter transcription, local chromatin compaction, or histone methylation, but did decrease chromatin stiffness. Single-nucleus micromanipulation reveals that HP1α is essential to chromatin-based mechanics and maintains nuclear morphology, separate from histone methylation. Further experiments with dimerization-deficient HP1αI165E indicate that chromatin crosslinking via HP1α dimerization is critical, while polymer simulations demonstrate the importance of chromatin-chromatin crosslinkers in mechanics. In mitotic chromosomes, HP1α similarly bolsters stiffness while aiding in mitotic alignment and faithful segregation. HP1α is therefore a critical chromatin-crosslinking protein that provides mechanical strength to chromosomes and the nucleus throughout the cell cycle and supports cellular functions.

Tetranucleosome Interactions Drive Chromatin Folding.

The multiscale organizational structure of chromatin in eukaryotic cells is instrumental to DNA transcription, replication, and repair. At mesoscopic length scales, nucleosomes pack in a manner that serves to regulate gene expression through condensation and expansion of the genome. The particular structures that arise and their respective thermodynamic stabilities, however, have yet to be fully resolved. In this study, we combine molecular modeling using the 1CPN mesoscale model of chromatin with nonlinear manifold learning to identify and characterize the structure and free energy of metastable states of short chromatin segments comprising between 4- and 16-nucleosomes. Our results reveal the formation of two previously characterized tetranucleosomal conformations, the “α-tetrahedron” and the “β-rhombus”, which have been suggested to play an important role in the accessibility of DNA and, respectively, induce local chromatin compaction or elongation. The spontaneous formation of these motifs is potentially responsible for the slow nucleosome dynamics observed in experimental studies. Increases of the nucleosome repeat length are accompanied by more pronounced structural irregularity and flexibility and, ultimately, a dynamic liquid-like behavior that allows for frequent structural reorganization. Our findings indicate that tetranucleosome motifs are intrinsically stable structural states, driven by local internucleosomal interactions, and support a mechanistic picture of chromatin packing, dynamics, and accessibility that is strongly influenced by emergent local mesoscale structure.

H1 histones control the epigenetic landscape by local chromatin compaction.

H1 linker histones are the most abundant chromatin-binding proteins1. In vitro studies indicate that their association with chromatin determines nucleosome spacing and enables arrays of nucleosomes to fold into more compact chromatin structures. However, the in vivo roles of H1 are poorly understood2. Here we show that the local density of H1 controls the balance of repressive and active chromatin domains by promoting genomic compaction. We generated a conditional triple-H1-knockout mouse strain and depleted H1 in haematopoietic cells. H1 depletion in T cells leads to de-repression of T cell activation genes, a process that mimics normal T cell activation. Comparison of chromatin structure in normal and H1-depleted CD8+ T cells reveals that H1-mediated chromatin compaction occurs primarily in regions of the genome containing higher than average levels of H1: the chromosome conformation capture (Hi-C) B compartment and regions of the Hi-C A compartment marked by PRC2. Reduction of H1 stoichiometry leads to decreased H3K27 methylation, increased H3K36 methylation, B-to-A-compartment shifting and an increase in interaction frequency between compartments. In vitro, H1 promotes PRC2-mediated H3K27 methylation and inhibits NSD2-mediated H3K36 methylation. Mechanistically, H1 mediates these opposite effects by promoting physical compaction of the chromatin substrate. Our results establish H1 as a critical regulator of gene silencing through localized control of chromatin compaction, 3D genome organization and the epigenetic landscape.

Analysis of muntjac deer genome and chromatin architecture reveals rapid karyotype evolution

Closely related muntjac deer show striking karyotype differences. Here we describe chromosome-scale genome assemblies for Chinese and Indian muntjacs, Muntiacus reevesi (2n = 46) and Muntiacus muntjak vaginalis (2n = 6/7), and analyze their evolution and architecture. The genomes show extensive collinearity with each other and with other deer and cattle. We identified numerous fusion events unique to and shared by muntjacs relative to the cervid ancestor, confirming many cytogenetic observations with genome sequence. One of these M. muntjak fusions reversed an earlier fission in the cervid lineage. Comparative Hi-C analysis showed that the chromosome fusions on the M. muntjak lineage altered long-range, three-dimensional chromosome organization relative to M. reevesi in interphase nuclei including A/B compartment structure. This reshaping of multi-megabase contacts occurred without notable change in local chromatin compaction, even near fusion sites. A few genes involved in chromosome maintenance show evidence for rapid evolution, possibly associated with the dramatic changes in karyotype.

Structural and Dynamical Signatures of Local DNA Damage in Live Cells

The dynamic organization of chromatin inside the cell nucleus plays a key role in gene regulation and genome replication, as well as maintaining genome integrity. Although the static folded state of the genome has been extensively studied, dynamical signatures of processes such as transcription or DNA repair remain an open question. Here, we investigate the interphase chromatin dynamics in human cells in response to local DNA damage, specifically, DNA double-strand breaks (DSBs). Using simultaneous two-color spinning-disk confocal microscopy, we monitor the DSB dynamics and the compaction of the surrounding chromatin, visualized by fluorescently labeled 53BP1 and histone H2B, respectively. Our study reveals a surprising difference between the mobility of DSBs located in the nuclear interior versus periphery (less than 1 μm from the nuclear envelope), with the interior DSBs being almost twice as mobile as the periphery DSBs. Remarkably, we find that the DSB sites possess a robust structural signature in a form of a unique chromatin compaction profile. Moreover, our data show that the DSB motion is subdiffusive and ATP-dependent and exhibits unique dynamical signatures, different from those of undamaged chromatin. Our findings reveal that the DSB mobility follows a universal relationship defined solely by the physical parameters describing the DSBs and their local environment, such as the DSB focus size (represented by the local accumulation of 53BP1), DSB density, and the local chromatin compaction. This suggests that the DSB-related repair processes are robust and likely deterministic because the observed dynamical signatures (DSB mobility) can be explained solely by their structural features (DSB focus size, local chromatin compaction). Such knowledge might help in detecting local DNA damage in live cells, as well as in aiding our biophysical understanding of genome integrity in health and disease.

Fluorescence fluctuation spectroscopy: an invaluable microscopy tool for uncovering the biophysical rules for navigating the nuclear landscape.

Nuclear architecture is fundamental to the manner by which molecules traverse the nucleus. The nucleoplasm is a crowded environment where dynamic rearrangements in local chromatin compaction locally redefine the space accessible toward nuclear protein diffusion. Here, we review a suite of methods based on fluorescence fluctuation spectroscopy (FFS) and how they have been employed to interrogate chromatin organization, as well as the impact this structural framework has on nuclear protein target search. From first focusing on a set of studies that apply FFS to an inert fluorescent tracer diffusing inside the nucleus of a living cell, we demonstrate the capacity of this technology to measure the accessibility of the nucleoplasm. Then with a baseline understanding of the exploration volume available to nuclear proteins during target search, we review direct applications of FFS to fluorescently labeled transcription factors (TFs). FFS can detect changes in TF mobility due to DNA binding, as well as the formation of TF complexes via changes in brightness due to oligomerization. Collectively, we find that FFS-based methods can uncover how nuclear proteins in general navigate the nuclear landscape.

Dynamical Signatures of Local DNA Damage in Live Cells

The dynamic organization of chromatin inside the eukaryotic cell nucleus is essential to the regulation of the genome such as maintaining its integrity, gene expression and replication. However, the physical picture of chromatin structure and dynamics remains unclear and requires identifying the dynamical and structural signatures of specific biological processes. In this work, we study the chromatin dynamics in interphase, time between two cell divisions, in response to DNA damage. As shown previously, chromatin dynamics across the whole nucleus change dramatically after DNA damage; chromatin's coherent motion is eliminated, while its local mobility increases and its compaction decreases [1]. Here, we explore the local chromatin dynamics and compaction in response to local DNA damage in human cells. To do so, we induce double stranded DNA breaks (DSBs), which we visualize using 53BP1-mCherry, and observe the spatiotemporal evolution of their dynamics using single particle tracking. Simultaneously, we use histone H2B-GFP as a reporter on the chromatin compaction, to compare DSB dynamics inside the heterochromatin and euchromatin. To elucidate the dynamic signature of the local DNA damage response, we vary the number of DSBs as well as explore the ATP-dependence of these processes. Our preliminary results show the local subdiffusive dynamics of the DSBs changes with an increasing DSB count inside the cell nucleus. We also explore the relationship between the protein aggregation, diffusion coefficient and chromatin compaction at a DSB. [1] Zidovska A, Weitz DA & Mitchison TJ, Proc. Natl. Acad. Sci., 110(39), 15555-60, 2013.

Interphase Chromatin Dynamics in Response to Double Stranded DNA Breaks

The dynamic organization of chromatin inside the eukaryotic cell nucleus is essential to the regulation of the genome such as maintaining its integrity, gene expression and replication. However, the physical picture of chromatin structure and dynamics remains unclear and requires identifying the dynamical and structural signatures of specific biological processes. In this work, we study the chromatin dynamics in interphase, time between two cell divisions, in response to DNA damage. As shown previously, chromatin dynamics across the whole nucleus change dramatically after DNA damage; chromatin's coherent motion is eliminated, while its local mobility increases and its compaction decreases [1]. Here, we explore the local chromatin dynamics and compaction in response to local DNA damage in human cells. To do so, we induce double stranded DNA breaks (DSBs), which we visualize using 53BP1-mCherry, and observe the spatiotemporal evolution of their dynamics using single particle tracking. Simultaneously, we use histone H2B-GFP as a reporter on the chromatin compaction, to compare DSB dynamics inside the heterochromatin and euchromatin. To elucidate the dynamic signature of the local DNA damage response, we vary the number of DSBs as well as explore the ATP-dependence of these processes. Our preliminary results show that with an increasing DSB count the local subdiffusive dynamics of the DSBs remains unchanged. We also explore the relationship between the protein aggregation, diffusion coefficient and chromatin compaction at a DSB. [1] Zidovska A, Weitz DA, Mitchison TJ, Proc. Natl. Acad. Sci. USA, 110(39), 1555560, 2013.

Quantitative analysis of chromatin accessibility in mouse embryonic fibroblasts

Genomic DNA of eukaryotic cells is hierarchically packaged into chromatin by histones. The dynamic organization of chromatin fibers plays a critical role in the regulation of gene transcription and other DNA-associated biological processes. Recently, numerous approaches have been developed to map the chromatin organization by characterizing chromatin accessibilities in genome-wide. However, reliable methods to quantitatively map chromatin accessibility are not well-established, especially not on a genome-wide scale. Here, we developed a modified MNase-seq for mouse embryonic fibroblasts, wherein chromatin was partially digested at multiple digestion times using micrococcal nuclease (MNase), allowing quantitative analysis of local yet genome-wide chromatin compaction. Our results provide strong evidence that the chromatin accessibility at promoter regions are positively correlated with gene activity. In conclusion, our assay is an ideal tool for the quantitative study of gene regulation in the perspective of chromatin accessibility.

Dynamics of Passive and Active Particles in the Cell Nucleus

Inspite of being embedded in a dense meshwork of nuclear chromatin, gene loci and large nuclear components are highly dynamic at C. To understand this apparent unfettered movement in an overdense environment, we study the dynamics of a passive micron size bead in live cell nuclei at two different temperatures ( and C) with and without external force. In the absence of a force, the beads are caged over large time scales. On application of a threshold uniaxial force (about 10 pN), the passive beads appear to hop between cages; this large scale movement is absent upon ATP-depletion, inhibition of chromatin remodeling enzymes and RNAi of lamin B1 proteins. Our results suggest that the nucleus behaves like an active solid with a finite yield stress when probed at a micron scale. Spatial analysis of histone fluorescence anisotropy (a measure of local chromatin compaction, defined as the volume fraction of tightly bound chromatin) shows that the bead movement correlates with regions of low chromatin compaction. This suggests that the physical mechanism of the observed yielding is the active opening of free-volume in the nuclear solid via chromatin remodeling. Enriched transcription sites at C also show caging in the absence of the applied force and directed movement beyond a yield stress, in striking contrast with the large scale movement of transcription loci at C in the absence of a force. This suggests that at physiological temperatures, the loci behave as active particles which remodel the nuclear mesh and reduce the local yield stress.

Dense Chromatin Activates Polycomb Repressive Complex 2 to Regulate H3 Lysine 27 Methylation

Polycomb repressive complex 2 (PRC2)-mediated histone H3 lysine 27 (H3K27) methylation is vital for Polycomb gene silencing, a classic epigenetic phenomenon that maintains transcriptional silencing throughout cell divisions. We report that PRC2 activity is regulated by the density of its substrate nucleosome arrays. Neighboring nucleosomes activate the PRC2 complex with a fragment of their H3 histones (Ala(31) to Arg(42)). We also identified mutations on PRC2 subunit Su(z)12, which impair its binding and response to the activating peptide and its ability in establishing H3K27 trimethylation levels in vivo. In mouse embryonic stem cells, local chromatin compaction occurs before the formation of trimethylated H3K27 upon transcription cessation of the retinoic acid-regulated gene CYP26a1. We propose that PRC2 can sense the chromatin environment to exert its role in the maintenance of transcriptional states.

Changes in Chromatin Compaction During the Cell Cycle Revealed by Micrometer-Scale Measurement of Molecular Flow in the Nucleus

We present a quantitative fluctuation-based assay to measure the degree of local chromatin compaction and investigate how chromatin density regulates the diffusive path adopted by an inert protein in dividing cells. The assay uses CHO-K1 cells coexpressing untagged enhanced green fluorescent protein (EGFP) and histone H2B tagged mCherry. We measure at the single-cell level the EGFP localization and molecular flow patterns characteristic of each stage of chromatin compaction from mitosis through interphase by means of pair-correlation analysis. We find that the naturally occurring changes in chromatin organization impart a regulation on the spatial distribution and temporal dynamics of EGFP within the nucleus. Combined with the analysis of Ca2+ intracellular homeostasis during cell division, EGFP flow regulation can be interpreted as the result of controlled changes in chromatin compaction. For the first time, to our knowledge, we were able to probe chromatin compaction on the micrometer scale, where the regulation of molecular diffusion may become relevant for many cellular processes.

Histone H2B ubiquitylation disrupts local and higher-order chromatin compaction

Regulation of chromatin structure involves histone post-translational modifications which can modulate intrinsic properties of the chromatin fiber to change the chromatin state. We used chemically defined nucleosome arrays to demonstrate that H2B ubiquitylation (uH2B), a modification associated with transcription, interferes with chromatin compaction and leads to an open and biochemically accessible fiber conformation. Importantly, these effects were specific for ubiquitin, as compaction of chromatin modified with a similar ubiquitin-sized protein, Hub1, was only weakly affected. Applying a fluorescence based method we found that uH2B acts through a mechanism distinct from H4 tail acetylation (acH4), a modification known to disrupt chromatin folding. Finally, incorporation of both uH2B and acH4 in nucleosomes resulted in synergistic inhibition of higher order chromatin structure formation, possibly a result of their distinct mode of action.

Histone H2B ubiquitylation disrupts local and higher-order chromatin compaction

Histone H2B ubiquitylation disrupts local and higher-order chromatin compaction