Large-scale Chromatin Organization Research Articles

DiffGR: Detecting Differentially Interacting Genomic Regions from Hi-C Contact Maps

Abstract Recent advances in high-throughput chromosome conformation capture (Hi-C) techniques have allowed us to map genome-wide chromatin interactions and uncover higher-order chromatin structures, thereby shedding light on the principles of genome architecture and functions. However, statistical methods for detecting changes in large-scale chromatin organization such as topologically associating domains (TADs) are still lacking. We proposed a new statistical method, DiffGR, for detecting differentially interacting genomic regions at the TAD level between Hi-C contact maps. We utilized the stratum-adjusted correlation coefficient to measure similarity of local TAD regions. We then developed a nonparametric approach to identify statistically significant changes of genomic interacting regions. Through simulation studies, we demonstrated that DiffGR can robustly and effectively discover differential genomic regions under various conditions. Furthermore, we successfully revealed cell type-specific changes in genomic interacting regions in both human and mouse Hi-C datasets, and illustrated that DiffGR yielded consistent and advantageous results compared with state-of-the-art differential TAD detection methods. The DiffGR R package is published under the GNU General Public License (GPL) ≥ 2 license and is publicly available at https://github.com/wmalab/DiffGR.

Time-of-flight spin-echo small-angle neutron scattering applied to biological cell nuclei

The organization of chromatin in the nuclei of rat lymphocyte was studied by time-of-flight spin-echo small-angle neutron scattering (ToF-SESANS). The procedures of the measurements in ToF and monochromatic SESANS modes were compared. It is shown that the sensitivity of the ToF method is significantly higher than that of the monochromatic mode. As a result, the SESANS correlation function can always be extracted from the ToF measurement of polarization, while this is not always the case with the monochromatic mode. The applicability of SESANS to fractal objects at the micrometre scale is demonstrated. The SESANS correlation function is well fitted by the exponential decay G(z) = exp(−z/ξ) with the correlation length ξ = 3.7 ± 0.1 µm. The exponential decay of the SESANS correlation function can be connected to the logarithmic correlation function γ(r) = ln(r/ξ) and the cubic law of the scattering intensity I(Q) = Q −3, which is in agreement with the concept of scattering from fractal objects. This finding is interpreted as evidence of a very specific logarithmic fractal structure of the large-scale organization of chromatin. The model of the logarithmic fractal is visualized as a hierarchical object obeying the volume-conservation principle at different scales.

The hierarchical packing of euchromatin domains can be described as multiplicative cascades.

The genome is packed into the cell nucleus in the form of chromatin. Biochemical approaches have revealed that chromatin is packed within domains, which group into larger domains, and so forth. Such hierarchical packing is equally visible in super-resolution microscopy images of large-scale chromatin organization. While previous work has suggested that chromatin is partitioned into distinct domains via microphase separation, it is unclear how these domains organize into this hierarchical packing. A particular challenge is to find an image analysis approach that fully incorporates such hierarchical packing, so that hypothetical governing mechanisms of euchromatin packing can be compared against the results of such an analysis. Here, we obtain 3D STED super-resolution images from pluripotent zebrafish embryos labeled with improved DNA fluorescence stains, and demonstrate how the hierarchical packing of euchromatin in these images can be described as multiplicative cascades. Multiplicative cascades are an established theoretical concept to describe the placement of ever-smaller structures within bigger structures. Importantly, these cascades can generate artificial image data by applying a single rule again and again, and can be fully specified using only four parameters. Here, we show how the typical patterns of euchromatin organization are reflected in the values of these four parameters. Specifically, we can pinpoint the values required to mimic a microphase-separated state of euchromatin. We suggest that the concept of multiplicative cascades can also be applied to images of other types of chromatin. Here, cascade parameters could serve as test quantities to assess whether microphase separation or other theoretical models accurately reproduce the hierarchical packing of chromatin.

Retrotransposons as Drivers of Mammalian Brain Evolution

Retrotransposons, a large and diverse class of transposable elements that are still active in humans, represent a remarkable force of genomic innovation underlying mammalian evolution. Among the features distinguishing mammals from all other vertebrates, the presence of a neocortex with a peculiar neuronal organization, composition and connectivity is perhaps the one that, by affecting the cognitive abilities of mammals, contributed mostly to their evolutionary success. Among mammals, hominids and especially humans display an extraordinarily expanded cortical volume, an enrichment of the repertoire of neural cell types and more elaborate patterns of neuronal connectivity. Retrotransposon-derived sequences have recently been implicated in multiple layers of gene regulation in the brain, from transcriptional and post-transcriptional control to both local and large-scale three-dimensional chromatin organization. Accordingly, an increasing variety of neurodevelopmental and neurodegenerative conditions are being recognized to be associated with retrotransposon dysregulation. We review here a large body of recent studies lending support to the idea that retrotransposon-dependent evolutionary novelties were crucial for the emergence of mammalian, primate and human peculiarities of brain morphology and function.

Mammalian DNA Replication Timing.

Immediately following the discovery of the structure of DNA and the semi-conservative replication of the parental DNA sequence into two new DNA strands, it became apparent that DNA replication is organized in a temporal and spatial fashion during the S phase of the cell cycle, correlated with the large-scale organization of chromatin in the nucleus. After many decades of limited progress, technological advances in genomics, genome engineering, and imaging have finally positioned the field to tackle mechanisms underpinning the temporal and spatial regulation of DNA replication and the causal relationships between DNA replication and other features of large-scale chromosome structure and function. In this review, we discuss these major recent discoveries as well as expectations for the coming decade.

Phase separation by the polyhomeotic sterile alpha motif compartmentalizes Polycomb Group proteins and enhances their activity

Polycomb Group (PcG) proteins organize chromatin at multiple scales to regulate gene expression. A conserved Sterile Alpha Motif (SAM) in the Polycomb Repressive Complex 1 (PRC1) subunit Polyhomeotic (Ph) has been shown to play an important role in chromatin compaction and large-scale chromatin organization. Ph SAM forms helical head to tail polymers, and SAM-SAM interactions between chromatin-bound Ph/PRC1 are believed to compact chromatin and mediate long-range interactions. To understand the underlying mechanism, here we analyze the effects of Ph SAM on chromatin in vitro. We find that incubation of chromatin or DNA with a truncated Ph protein containing the SAM results in formation of concentrated, phase-separated condensates. Ph SAM-dependent condensates can recruit PRC1 from extracts and enhance PRC1 ubiquitin ligase activity towards histone H2A. We show that overexpression of Ph with an intact SAM increases ubiquitylated H2A in cells. Thus, SAM-induced phase separation, in the context of Ph, can mediate large-scale compaction of chromatin into biochemical compartments that facilitate histone modification.

Chromatin as an active polymeric material.

The patterns of the large-scale spatial organization of chromatin in interphase human somatic cells are not random. Such patterns include the radial separation of euchromatin and heterochromatin, the territorial organization of individual chromosomes, the non-random locations of chromosome territories and the differential positioning of the two X chromosomes in female cells. These features of large-scale nuclear architecture follow naturally from the hypothesis that ATP-consuming non-equilibrium processes associated with highly transcribed regions of chromosomes are a source of 'active' forces. These forces are in excess of those that arise from Brownian motion. Simulations of model chromosomes that incorporate such activity recapitulate these features. In addition, they reproduce many other aspects of the spatial organization of chromatin at large scales that are known from experiments. Our results, reviewed here, suggest that the distribution of transcriptional activity across chromosomes underlies many aspects of large-scale nuclear architecture that were hitherto believed to be unrelated.

Emergence of chromatin hierarchical loops from protein disorder and nucleosome asymmetry

Protein flexibility and disorder is emerging as a crucial modulator of chromatin structure. Histone tail disorder enables transient binding of different molecules to the nucleosomes, thereby promoting heterogeneous and dynamic internucleosome interactions and making possible recruitment of a wide-range of regulatory and remodeling proteins. On the basis of extensive multiscale modeling we reveal the importance of linker histone H1 protein disorder for chromatin hierarchical looping. Our multiscale approach bridges microsecond-long bias-exchange metadynamics molecular dynamics simulations of atomistic 211-bp nucleosomes with coarse-grained Monte Carlo simulations of 100-nucleosome systems. We show that the long C-terminal domain (CTD) of H1-a ubiquitous nucleosome-binding protein-remains disordered when bound to the nucleosome. Notably, such CTD disorder leads to an asymmetric and dynamical nucleosome conformation that promotes chromatin structural flexibility and establishes long-range hierarchical loops. Furthermore, the degree of condensation and flexibility of H1 can be fine-tuned, explaining chromosomal differences of interphase versus metaphase states that correspond to partial and hyperphosphorylated H1, respectively. This important role of H1 protein disorder in large-scale chromatin organization has a wide range of biological implications.

Small-angle neutron scattering (SANS) and spin-echo SANS measurements reveal the logarithmic fractal structure of the large-scale chromatin organization in HeLa nuclei

This paper reports on the two-scale fractal structure of chromatin organization in the nucleus of the HeLa cell. Two neutron scattering methods, small-angle neutron scattering (SANS) and spin-echo SANS, are used to unambiguously identify the large-scale structure as being a logarithmic fractal with the correlation function γ(r) ∼ ln(r/ξ). The smaller-scale structural level is shown to be a volume fractal with dimension D F = 2.41. By definition, the volume fractal is self-similar at different scales, while the logarithmic fractal is hierarchically changed upon scaling. As a result, the logarithmic fractal is more compact than the volume fractal but still has a rather high surface area, which provides accessibility at all length scales. Apparently such bi-fractal chromatin organization is the result of an evolutionary process of optimizing the compactness and accessibility of gene packing. As they are in a water solution, the HeLa nuclei tend to agglomerate over time. The large-scale logarithmic fractal structure of chromatin provides the HeLa nucleus with the possibility of penetrating deeply into the adjacent nucleus during the agglomeration process. The interpenetration phenomenon of the HeLa nuclei shows that the chromatin-free space of one nucleus is not negligible but is as large as the volume occupied by chromatin itself. It is speculated that it is the logarithmic fractal architecture of chromatin that provides a comfortable compartment for this most important function of the cell.

Interplay of lamin A and lamin B LADs on the radial positioning of chromatin

ABSTRACTImmunosuppressive drugs such as cyclosporin A (CsA) can elicit hepatotoxicity by affecting gene expression. Here, we address the link between CsA and large-scale chromatin organization in HepG2 hepatocarcinoma cells. We show the existence of lamina-associated domains (LADs) interacting with lamin A, lamin B, or both. These ‘A-B’, ‘A-only’ and ‘B-only’ LADs display distinct fates after CsA treatment: A-B LADs remain constitutive or lose A, A-only LADs mainly lose A or switch to B, and B-only LADs remain B-only or acquire A. LAD rearrangement is overall uncoupled from changes in gene expression. Three-dimensional (3D) genome modeling predicts changes in radial positioning of LADs as LADs switch identities, which are corroborated by fluorescence in situ hybridization. Our results reveal interplay between A- and B-type lamins on radial locus positioning, suggesting complementary contributions to large-scale genome architecture. The data also unveil a hitherto unsuspected impact of cytotoxic drugs on genome conformation.Abbreviations: ChIP-seq: chromatin immunoprecipitation sequencing; CsA: cyclosporin A; FISH; fluorescence in situ hybridization; ICMT: isoprenylcysteine methyltransferase; LAD: lamina-associated domain; TAD: topologically-associated domain

Characterizing the nuclease accessibility of DNA in human cells to map higher order structures of chromatin.

Packaging of DNA into chromatin regulates DNA accessibility and consequently all DNA-dependent processes. The nucleosome is the basic packaging unit of DNA forming arrays that are suggested, by biochemical studies, to fold hierarchically into ordered higher-order structures of chromatin. This organization has been recently questioned using microscopy techniques, proposing an irregular structure. To address the principles of chromatin organization, we applied an in situ differential MNase-seq strategy and analyzed in silico the results of complete and partial digestions of human chromatin. We investigated whether different levels of chromatin packaging exist in the cell. We assessed the accessibility of chromatin within distinct domains of kb to Mb genomic regions, performed statistical analyses and computer modelling. We found no difference in MNase accessibility, suggesting no difference in fiber folding between domains of euchromatin and heterochromatin or between other sequence and epigenomic features of chromatin. Thus, our data suggests the absence of differentially organized domains of higher-order structures of chromatin. Moreover, we identified only local structural changes, with individual hyper-accessible nucleosomes surrounding regulatory elements, such as enhancers and transcription start sites. The regulatory sites per se are occupied with structurally altered nucleosomes, exhibiting increased MNase sensitivity. Our findings provide biochemical evidence that supports an irregular model of large-scale chromatin organization.

Activation of the alpha-globin gene expression correlates with dramatic upregulation of nearby non-globin genes and changes in local and large-scale chromatin spatial structure

BackgroundIn homeotherms, the alpha-globin gene clusters are located within permanently open genome regions enriched in housekeeping genes. Terminal erythroid differentiation results in dramatic upregulation of alpha-globin genes making their expression comparable to the rRNA transcriptional output. Little is known about the influence of the erythroid-specific alpha-globin gene transcription outburst on adjacent, widely expressed genes and large-scale chromatin organization. Here, we have analyzed the total transcription output, the overall chromatin contact profile, and CTCF binding within the 2.7 Mb segment of chicken chromosome 14 harboring the alpha-globin gene cluster in cultured lymphoid cells and cultured erythroid cells before and after induction of terminal erythroid differentiation.ResultsWe found that, similarly to mammalian genome, the chicken genomes is organized in TADs and compartments. Full activation of the alpha-globin gene transcription in differentiated erythroid cells is correlated with upregulation of several adjacent housekeeping genes and the emergence of abundant intergenic transcription. An extended chromosome region encompassing the alpha-globin cluster becomes significantly decompacted in differentiated erythroid cells, and depleted in CTCF binding and CTCF-anchored chromatin loops, while the sub-TAD harboring alpha-globin gene cluster and the upstream major regulatory element (MRE) becomes highly enriched with chromatin interactions as compared to lymphoid and proliferating erythroid cells. The alpha-globin gene domain and the neighboring loci reside within the A-like chromatin compartment in both lymphoid and erythroid cells and become further segregated from the upstream gene desert upon terminal erythroid differentiation.ConclusionsOur findings demonstrate that the effects of tissue-specific transcription activation are not restricted to the host genomic locus but affect the overall chromatin structure and transcriptional output of the encompassing topologically associating domain.

PML protein organizes heterochromatin domains where it regulates histone H3.3 deposition by ATRX/DAXX.

Maintenance of chromatin homeostasis involves proper delivery of histone variants to the genome. The interplay between different chaperones regulating the supply of histone variants to distinct chromatin domains remains largely undeciphered. We report a role of promyelocytic leukemia (PML) protein in the routing of histone variant H3.3 to chromatin and in the organization of megabase-size heterochromatic PML-associated domains that we call PADs. Loss of PML alters the heterochromatic state of PADs by shifting the histone H3 methylation balance from K9me3 to K27me3. Loss of PML impairs deposition of H3.3 by ATRX and DAXX in PADs but preserves the H3.3 loading function of HIRA in these regions. Our results unveil an unappreciated role of PML in the large-scale organization of chromatin and demonstrate a PML-dependent role of ATRX/DAXX in the deposition of H3.3 in PADs. Our data suggest that H3.3 loading by HIRA and ATRX-dependent H3K27 trimethylation constitute mechanisms ensuring maintenance of heterochromatin when the integrity of these domains is compromised.

Cytology of DNA Replication Reveals Dynamic Plasticity of Large-Scale Chromatin Fibers

In higher eukaryotic interphase nuclei, the 100- to>1,000-fold linear compaction of chromatin is difficult to reconcile with its function as a template for transcription, replication, and repair. It is challenging to imagine how DNA and RNA polymerases with their associated molecular machinery would move along the DNA template without transient decondensation of observed large-scale chromatin "chromonema" fibers [1]. Transcription or "replication factory" models [2], in which polymerases remain fixed while DNA is reeled through, are similarly difficult to conceptualize without transient decondensation of these chromonema fibers. Here, we show how a dynamic plasticity of chromatin folding within large-scale chromatin fibers allows DNA replication to take place without significantchanges in the global large-scale chromatin compaction or shape of these large-scale chromatin fibers. Time-lapse imaging of lac-operator-tagged chromosome regions shows no major change inthe overall compaction of these chromosome regions during their DNA replication. Improved pulse-chase labeling of endogenous interphase chromosomes yields a model in which the global compaction and shape of large-Mbp chromatin domains remains largely invariant during DNA replication, with DNA within these domains undergoing significant movements and redistribution as they move into and then out of adjacent replication foci. In contrast to hierarchical folding models, this dynamic plasticity of large-scale chromatin organization explains how localized changes in DNA topology allow DNA replication to take place without an accompanying global unfolding of large-scale chromatin fibers while suggesting a possible mechanism for maintaining epigenetic programming of large-scale chromatin domains throughout DNA replication.

A negative loop within the nuclear pore complex controls global chromatin organization.

The nuclear pore complex (NPC) tethers chromatin to create an environment for gene regulation, but little is known about how this activity is regulated to avoid excessive tethering of the genome. Here we propose a negative regulatory loop within the NPC controlling the chromatin attachment state, in which Nup155 and Nup93 recruit Nup62 to suppress chromatin tethering by Nup155. Depletion of Nup62 severely disrupts chromatin distribution in the nuclei of female germlines and somatic cells, which can be reversed by codepleting Nup155. Thus, this universal regulatory system within the NPC is crucial to control large-scale chromatin organization in the nucleus.

The oncogenic BRD4-NUT chromatin regulator drives aberrant transcription within large topological domains.

NUT midline carcinoma (NMC), a subtype of squamous cell cancer, is one of the most aggressive human solid malignancies known. NMC is driven by the creation of a translocation oncoprotein, BRD4-NUT, which blocks differentiation and drives growth of NMC cells. BRD4-NUT forms distinctive nuclear foci in patient tumors, which we found correlate with ∼100 unprecedented, hyperacetylated expanses of chromatin that reach up to 2 Mb in size. These "megadomains" appear to be the result of aberrant, feed-forward loops of acetylation and binding of acetylated histones that drive transcription of underlying DNA in NMC patient cells and naïve cells induced to express BRD4-NUT. Megadomain locations are typically cell lineage-specific; however, the cMYC and TP63 regions are targeted in all NMCs tested and play functional roles in tumor growth. Megadomains appear to originate from select pre-existing enhancers that progressively broaden but are ultimately delimited by topologically associating domain (TAD) boundaries. Therefore, our findings establish a basis for understanding the powerful role played by large-scale chromatin organization in normal and aberrant lineage-specific gene transcription.

CTCF protein recruits centromeric protein CENP‐E to the centromeric regions through unusual CTCF binding sites

Centromere‐associated protein E (CENP‐E) is an integral component of the kinetochore, and has been shown to be essential for chromosome congression during mitosis. However, the molecular mechanism by which CENP‐E is recruited to the kinetochore is poorly understood. In the present study, we report that CENP‐E interacts directly with CTCF, the insulator protein that is involved in large scale organization of chromatin and in insulation. We demonstrate that α‐satellite repeats containing both CENP‐B boxes­­­ and CTCF binding sites in HeLa cells that are arrested in G2/M or Mitosis are co‐occupied by CTCF and CENP‐E. CENP‐E localization appears to depend on CTCF, because deletion of CTCF by siRNA disrupts binding of CENP‐E to the centromeric regions. The CTCF binding sites in centromeric regions are unusual in that most of them carry only the ‘M2′ sequence motif, a 9‐bp long DNA fragment that was originally thought to appear together with the core CTCF binding motif (also called ‘M1′ motif). Furthermore, we provide evidence that the ‘M2′ motif engages only the C‐terminal zinc fingers of CTCF, and binds CTCF quite strongly. Based on these findings, we suggest that CTCF helps recruit CENP‐E to the kinetochore and that the CTCF‐CENP‐E complex may represent a novel structural feature of centromeres.

Topokaryotyping – a proposal for a novel approach to study nuclear organization

Gene positioning in the nucleus plays an important role in gene activity and genome stability, both in norm and pathology. We propose a new principle for the analysis of the large scale chromatin organization at the single-cell level, termed Topokaryotyping. It is based on coexpression in cells of (i) biotin ligase BirA targeted to a particular intranuclear domain (via its fusion with a protein-marker of this domain) together with (ii) BAP-histone fusion. The application of biotin pulse chase followed by (i) generation of mitotic spreads, (ii) detection of the biotin label and (iii) karyotyping technique to identify the chromosomes should provide information on the association of particular chromosome regions with the nuclear domain under study. We discuss potential advantages of the proposed approach as compared to other methods to study genome topology in the nucleus.

Background-Free Super-Resolution Microscopy of Subcellular Structures by Lifetime Tuning and Photons Separation

Background-Free Super-Resolution Microscopy of Subcellular Structures by Lifetime Tuning and Photons Separation

Large-scale organization of ribosomal DNA chromatin is regulated by Tip5

The DNase I accessibility and chromatin organization of genes within the nucleus do correlate to their transcriptional activity. Here, we show that both serum starvation and overexpression of Tip5, a key regulator of ribosomal RNA gene (rDNA) repression, dictate DNase I accessibility, facilitate the association of rDNA with the nuclear matrix and thus regulate large-scale rDNA chromatin organization. Tip5 contains four AT-hooks and a TAM (Tip5/ARBP/MBD) domain, which were proposed to bind matrix-attachment regions (MARs) of the genome. Remarkably, the TAM domain of Tip5 functions as nucleolar localization and nuclear matrix targeting module, whereas AT-hooks do not mediate association with the nuclear matrix, but they are required for nucleolar targeting. These findings suggest a dual role for Tip5’s AT-hooks and TAM domain, targeting the nucleolus and anchoring to the nuclear matrix, and suggest a function for Tip5 in the regulation of higher-order rDNA chromatin structure.