Dense Chromatin Research Articles

Heterochromatin protein 1 alpha (HP1α) undergoes a monomer to dimer transition that opens and compacts live cell genome architecture.

Our understanding of heterochromatin nanostructure and its capacity to mediate gene silencing in a living cell has been prevented by the diffraction limit of optical microscopy. Thus, here to overcome this technical hurdle, and directly measure the nucleosome arrangement that underpins this dense chromatin state, we coupled fluorescence lifetime imaging microscopy (FLIM) of Förster resonance energy transfer (FRET) between histones core to the nucleosome, with molecular editing of heterochromatin protein 1 alpha (HP1α). Intriguingly, this super-resolved readout of nanoscale chromatin structure, alongside fluorescence fluctuation spectroscopy (FFS) and FLIM-FRET analysis of HP1α protein-protein interaction, revealed nucleosome arrangement to be differentially regulated by HP1α oligomeric state. Specifically, we found HP1α monomers to impart a previously undescribed global nucleosome spacing throughout genome architecture that is mediated by trimethylation on lysine 9 of histone H3 (H3K9me3) and locally reduced upon HP1α dimerisation. Collectively, these results demonstrate HP1α to impart a dual action on chromatin that increases the dynamic range of nucleosome proximity. We anticipate that this finding will have important implications for our understanding of how live cell heterochromatin structure regulates genome function.

Chromatin phase separation and nuclear shape fluctuations are correlated in a polymer model of the nucleus

ABSTRACT Abnormal cell nuclear shapes are hallmarks of diseases, including progeria, muscular dystrophy, and many cancers. Experiments have shown that disruption of heterochromatin and increases in euchromatin lead to nuclear deformations, such as blebs and ruptures. However, the physical mechanisms through which chromatin governs nuclear shape are poorly understood. To investigate how heterochromatin and euchromatin might govern nuclear morphology, we studied chromatin microphase separation in a composite coarse-grained polymer and elastic shell simulation model. By varying chromatin density, heterochromatin composition, and heterochromatin-lamina interactions, we show how the chromatin phase organization may perturb nuclear shape. Increasing chromatin density stabilizes the lamina against large fluctuations. However, increasing heterochromatin levels or heterochromatin-lamina interactions enhances nuclear shape fluctuations by a “wetting”-like interaction. In contrast, fluctuations are insensitive to heterochromatin’s internal structure. Our simulations suggest that peripheral heterochromatin accumulation could perturb nuclear morphology, while nuclear shape stabilization likely occurs through mechanisms other than chromatin microphase organization.

Correlative single molecule lattice light sheet imaging reveals the dynamic relationship between nucleosomes and the local chromatin environment

In the nucleus, biological processes are driven by proteins that diffuse through and bind to a meshwork of nucleic acid polymers. To better understand this interplay, we present an imaging platform to simultaneously visualize single protein dynamics together with the local chromatin environment in live cells. Together with super-resolution imaging, new fluorescent probes, and biophysical modeling, we demonstrate that nucleosomes display differential diffusion and packing arrangements as chromatin density increases whereas the viscoelastic properties and accessibility of the interchromatin space remain constant. Perturbing nuclear functions impacts nucleosome diffusive properties in a manner that is dependent both on local chromatin density and on relative location within the nucleus. Our results support a model wherein transcription locally stabilizes nucleosomes while simultaneously allowing for the free exchange of nuclear proteins. Additionally, they reveal that nuclear heterogeneity arises from both active and passive processes and highlight the need to account for different organizational principles when modeling different chromatin environments.

Probe-free optical chromatin deformation and measurement of differential mechanical properties in the nucleus.

The nucleus is highly organized to facilitate coordinated gene transcription. Measuring the rheological properties of the nucleus and its sub-compartments will be crucial to understand the principles underlying nuclear organization. Here, we show that strongly localized temperature gradients (approaching 1°C/µm) can lead to substantial intra-nuclear chromatin displacements (>1 µm), while nuclear area and lamina shape remain unaffected. Using particle image velocimetry (PIV), intra-nuclear displacement fields can be calculated and converted into spatio-temporally resolved maps of various strain components. Using this approach, we show that chromatin displacements are highly reversible, indicating that elastic contributions are dominant in maintaining nuclear organization on the time scale of seconds. In genetically inverted nuclei, centrally compacted heterochromatin displays high resistance to deformation, giving a rigid, solid-like appearance. Correlating spatially resolved strain maps with fluorescent reporters in conventional interphase nuclei reveals that various nuclear compartments possess distinct mechanical identities. Surprisingly, both densely and loosely packed chromatin showed high resistance to deformation, compared to medium dense chromatin. Equally, nucleoli display particularly high resistance and strong local anchoring to heterochromatin. Our results establish how localized temperature gradients can be used to drive nuclear compartments out of mechanical equilibrium to obtain spatial maps of their material responses.

Anomalous coarsening of coalescing nucleoli in human cells

Coarsening is a ubiquitous phenomenon in droplet systems near thermodynamic equilibrium—as an increase in droplet size lowers the system’s free energy—however, coarsening of droplets in nonequilibrium systems, such as the cell nucleus, is far from understood. Liquid condensates in the cell nucleus, like nucleoli, form by liquid-liquid phase separation and play a key role in the nuclear organization. In human cells, nucleolar droplets are nucleated at the beginning of the cell cycle and coarsen with time by coalescing with each other. Upon coarsening, human nucleoli exhibit an anomalous volume distribution P(V)∼V−1, which cannot be explained by any existing theory. In this work, we investigate physical mechanisms behind the anomalous coarsening of human nucleoli. Using spinning disk confocal microscopy, we simultaneously record dynamic behavior of nucleoli and their surrounding chromatin before their coalescence in live human cells. We find that nucleolar anomalous coarsening persists during the entire cell cycle. We measure chromatin flows and density between and around nucleoli, as well as relative motion of two nucleoli before they coalesce. We find that, before nucleolar coalescence, chromatin concentration decreases in the space between nucleoli and the nucleoli move faster toward each other, resembling an effective depletion attraction between the coalescing nucleoli. Indeed, our computational simulations of nucleolar dynamics show that short-ranged attraction is sufficient to explain the observed anomalous volume distribution of human nucleoli. Overall, our results reveal a potential physical mechanism contributing to coarsening of human nucleoli. Such knowledge expands our picture of the physical behavior of liquid condensates inside the cell nucleus and our understanding of the dynamic nuclear organization.

Histone FRET reports the spatial heterogeneity in nanoscale chromatin architecture that is imparted by the epigenetic landscape at the level of single foci in an intact cell nucleus

Genome sequencing has identified hundreds of histone post-translational modifications (PTMs) that define an open or compact chromatin nanostructure at the level of nucleosome proximity, and therefore serve as activators or repressors of gene expression. Direct observation of this epigenetic mode of transcriptional regulation in an intact single nucleus, is however, a complex task. This is because despite the development of fluorescent probes that enable observation of specific histone PTMs and chromatin density, the changes in nucleosome proximity regulating gene expression occur on a spatial scale well below the diffraction limit of optical microscopy. In recent work, to address this research gap, we demonstrated that the phasor approach to fluorescence lifetime imaging microscopy (FLIM) of Förster resonance energy transfer (FRET) between fluorescently labelled histones core to the nucleosome, is a readout of chromatin nanostructure that can be multiplexed with immunofluorescence (IF) against specific histone PTMs. Here from application of this methodology to gold standard gene activators (H3K4Me3 and H3K9Ac) versus repressors (e.g., H3K9Me3 and H3K27Me), we find that while on average these histone marks do impart an open versus compact chromatin nanostructure, at the level of single chromatin foci, there is significant spatial heterogeneity. Collectively this study illustrates the importance of studying the epigenetic landscape as a function of space within intact nuclear architecture and opens the door for the study of chromatin foci sub-populations defined by combinations of histone marks, as is seen in the context of bivalent chromatin.

Primary gastric rhabdomyosarcoma. A rare cause of an upper gastrointestinal bleeding.

Adenocarcinoma accounts for 90% of malignant gastric tumours. Lymphomas, gastrointestinal stromal tumours (GIST) and other less frequent tumours account for 10%. However, rhabdomyosarcoma (RMS) remains a rare cause within the broad differential diagnosis of gastric lesions. Most cases of gastrointestinal RMS in adults are metastatic disease so primary RMS is extremely rare. A few cases reports of esophageal and gastric RMS have been published. This is a 76-year-old patient with a history of oncological oncology of poorly differentiated lung adenocarcinoma T2N1M0 in 2008 treated with surgery with no signs of recurrence. She presented to emergency with occasional episodes of melena and iron deficiency anemia for the last 3 months. The laboratory findings showed moderated anemia. Gastroscopy was performed and a prominent submucosal and ulcerated bulge was observed at the posterior wall of the gastric body and fundus. A histologic examination reveled tissue fragments composed of small round spindle-shaped cells. Tumor cells had atypical nuclei showing dense chromatin ad pale to eosinophilic cytoplasm. Immunohistochemically, the tumor was positive for AE1/3, desmin, myogenin and CD56. Patient was diagnosed with primary gastric RMS. Thoraco-abdominopelvic CT showed a locally advanced stage (T2 N1M0).

Near-Infrared Light-Driven Nanoparticles for Cancer Photoimmunotherapy by Synergizing Immune Cell Death and Epigenetic Regulation.

Histone deacetylases (HDACs) are a class of epigenetic enzymes that are closely related to tumorigenesis and suppress the expression of tumor suppressor genes. Whereas the HDACs inhibitors can release DNA into the cytoplasm and trigger innate immunity. However, the high density of chromatin limits DNA damage and release. In this study, suitable nanosized CycNHOH NPs (150nm) and CypNHOH NPs (85nm) efficiently accumulate at the tumor site due to the enhanced permeability and retention (EPR) effect. In addition, robust single-linear oxygen generation and good photothermal conversion efficiency under NIR laser irradiation accelerated the DNA damage process. By effectively initiating immune cell death, CypNHOH NPs activated both innate and adaptive immunity by maturing dendritic cells, infiltrating tumors with natural killer cells, and activating cytotoxic T lymphocytes, which offer a fresh perspective for the development of photo-immunotherapy.

Dynamic changes characteristics of the spermatozoon during the reproductive process of mud crab (Scylla paramamosain): From spermatophore formation, transportation to dispersion

To enhance comprehension of the destiny of the spermatozoon throughout the reproductive process of mud crab (Scylla paramamosain) is important for achieving large-scale artificial breeding of Scylla. The reproductive systems of sexually mature male and female mud crabs were studied, utilizing the histomorphological method. The spermatozoon traveled from testicular lobules through the seminiferous duct, into the anterior vas deferens (AVD) via the collecting duct. The spermatophore wall formed at the end of the AVD. During copulation, spermatophores stored in the median vas deferens (MVD) were transferred to the seminal receptacle through the posterior vas deferens (PVD), ejaculatory duct, and gonopod, with seminal plasma secreted by both MVD and PVD together. After mating, the sperm plug was formed within the seminal receptacle's ventral zone. At ovary stage Ⅲ, the spermatophore wall ruptured, and the sperm plug disappeared. Bundled sperm packets congregated into a dense mass, while a few spermatozoa dispersed along the intermediate chamber to the dorsal zone. At ovary stage Ⅳ, the dorsal zone also exhibited a dense sperm mass due to increased dispersal. At ovary stage Ⅴ, merely a modest portion of the dense sperm mass remained in the ventral zone, and the rest dissociated and dispersed within the seminal receptacle. Following ovulation, sperm mass remained in the ventral zone, re-dissociating and re-dispersing once ovarian development reached stage Ⅲ again. The phased dispersion supply mode of spermatozoa behind multiple ovipositions was unveiled. Furthermore, we observed the ultrastructural changes in the spermatozoon during the process of the capacitation, including operculum widening, actin-containing substance accumulation, and the density of chromatin in the nucleus decreased. As presented herein, the elucidation of the dynamic changes of the spermatozoon, as well as the pattern of spermatozoa supply, contributes significantly to our enhanced comprehension of the reproductive biology of Scylla.

Chromatin-mediated silencing on the inactive X chromosome.

In mammals, the second X chromosome in females is silenced to enable dosage compensation between XX females and XY males. This essential process involves the formation of a dense chromatin state on the inactive X (Xi) chromosome. There is a wealth of information about the hallmarks of Xi chromatin and the contribution each makes to silencing, leaving the tantalising possibility of learning from this knowledge to potentially remove silencing to treat X-linked diseases in females. Here, we discuss the role of each chromatin feature in the establishment and maintenance of the silent state, which is of crucial relevance for such a goal.

A quantitative evaluation method utilizing the homology concept to assess the state of chromatin within the nucleus of lung cancer

Homology is a mathematical tool to quantify "the contact degree", which can be expressed in terms of Betti numbers. The Betti numbers used in this study consisted of two numbers, b0 (a zero-dimensional Betti number) and b1 (a one-dimensional Betti number). We developed a chromatin homology profile (CHP) method to quantify the chromatin contact degree based on this mathematical tool. Using the CHP method we analyzed the number of holes (surrounded areas = b1 value) formed by the chromatin contact and calculated the maximum value of b1 (b1MAX), the value of b1 exceeding 5 for the first time or Homology Value (HV), and the chromatin density (b1MAX/ns2). We attempted to detect differences in chromatin patterns and differentiate histological types of lung cancer from respiratory cytology using these three features. The HV of cancer cells was significantly lower than that of non-cancerous cells. Furthermore, b1MAX and b1MAX/ns2 showed significant differences between small cell and non-small cell carcinomas and between adenocarcinomas and squamous cell carcinomas, respectively. We quantitatively analyzed the chromatin patterns using homology and showed that the CHP method may be a useful tool for differentiating histological types of lung cancer in respiratory cytology.

Nuclear growth and import can be uncoupled.

What drives nuclear growth? Studying nuclei assembled in Xenopus egg extract and focusing on importin α/β-mediated nuclear import, we show that, while import is required for nuclear growth, nuclear growth and import can be uncoupled when chromatin structure is manipulated. Nuclei treated with micrococcal nuclease to fragment DNA grew slowly despite exhibiting little to no change in import rates. Nuclei assembled around axolotl chromatin with 20-fold more DNA than Xenopus grew larger but imported more slowly. Treating nuclei with reagents known to alter histone methylation or acetylation caused nuclei to grow less while still importing to a similar extent or to grow larger without significantly increasing import. Nuclear growth but not import was increased in live sea urchin embryos treated with the DNA methylator N-nitrosodimethylamine. These data suggest that nuclear import is not the primary driving force for nuclear growth. Instead, we observed that nuclear blebs expanded preferentially at sites of high chromatin density and lamin addition, whereas small Benzonase-treated nuclei lacking DNA exhibited reduced lamin incorporation into the nuclear envelope. In summary, we report experimental conditions where nuclear import is not sufficient to drive nuclear growth, hypothesizing that this uncoupling is a result of altered chromatin structure.

Automated Image Analysis of Transmission Electron Micrographs: Nanoscale Evaluation of Radiation-Induced DNA Damage in the Context of Chromatin.

Heavy ion irradiation (IR) with high-linear energy transfer (LET) is characterized by a unique depth dose distribution and increased biological effectiveness. Following high-LET IR, localized energy deposition along the particle trajectories induces clustered DNA lesions, leading to low electron density domains (LEDDs). To investigate the spatiotemporal dynamics of DNA repair and chromatin remodeling, we established the automated image analysis of transmission electron micrographs. Human fibroblasts were irradiated with high-LET carbon ions or low-LET photons. At 0.1 h, 0.5 h, 5 h, and 24 h post-IR, nanoparticle-labeled repair factors (53BP1, pKu70, pKu80, DNA-PKcs) were visualized using transmission electron microscopy in interphase nuclei to monitor the formation and repair of DNA damage in the chromatin ultrastructure. Using AI-based software tools, advanced image analysis techniques were established to assess the DNA damage pattern following low-LET versus high-LET IR. Low-LET IR induced single DNA lesions throughout the nucleus, and most DNA double-strand breaks (DSBs) were efficiently rejoined with no visible chromatin decondensation. High-LET IR induced clustered DNA damage concentrated along the particle trajectories, resulting in circumscribed LEDDs. Automated image analysis was used to determine the exact number of differently sized nanoparticles, their distance from one another, and their precise location within the micrographs (based on size, shape, and density). Chromatin densities were determined from grayscale features, and nanoparticles were automatically assigned to euchromatin or heterochromatin. High-LET IR-induced LEDDs were delineated using automated segmentation, and the spatial distribution of nanoparticles in relation to segmented LEDDs was determined. The results of our image analysis suggest that high-LET IR induces chromatin relaxation along particle trajectories, enabling the critical repair of successive DNA damage. Following exposure to different radiation qualities, automated image analysis of nanoparticle-labeled DNA repair proteins in the chromatin ultrastructure enables precise characterization of specific DNA damage patterns.

Comparison of FNA-based conventional cytology specimens and digital image analysis in assessment of pancreatic lesions.

Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) is one of the most important diagnostic tools for investigation of suspected pancreatic masses, although the interpretation of the results is controversial. In recent decades, digital image analysis (DIA) has been considered in pathology. The aim of this study was to assess the DIA in the evaluation of EUS-FNA based cytopathological specimens of pancreatic masses and comparing it with conventional cytology analysis by pathologist. This study was performed using cytological slides related to EUS-FNA samples of pancreatic lesions. The digital images were prepared and then analyzed by ImageJ software. Factors such as perimeter, circularity, area, minimum, maximum, mean, median of gray value, and integrated chromatin density of cell nucleus were extracted by software ImageJ and sensitivity, specificity, and cutoff point were evaluated in the diagnosis of malignant and benign lesions. In this retrospective study, 115 cytology samples were examined. Each specimen was reviewed by a pathologist and 150 images were prepared from the benign and malignant lesions and then analyzed by ImageJ software and a cut point was established by SPSS 26. The cutoff points for perimeter, integrated density, and the sum of three factors of perimeter, integrated density, and circularity to differentiate between malignant and benign lesions were reported to be 204.56, 131953, and 24643077, respectively. At this cutting point, the accuracy of estimation is based on the factors of perimeter, integrated density, and the sum of the three factors of perimeter, integrated density, and circularity were 92%, 92%, and 94%, respectively. The results of this study showed that digital analysis of images has a high accuracy in diagnosing malignant and benign lesions in the cytology of EUS-FNA in patients with suspected pancreatic malignancy and by obtaining cutoff points by software output factors; digital imaging can be used to differentiate between benign and malignant pancreatic tumors.

Identification of mammalian transcription factors that bind to inaccessible chromatin.

Transcription factors (TFs) are proteins that affect gene expression by binding to regulatory regions of DNA in a sequence specific manner. The binding of TFs to DNA is controlled by many factors, including the DNA sequence, concentration of TF, chromatin accessibility and co-factors. Here, we systematically investigated the binding mechanism of hundreds of TFs by analysing ChIP-seq data with our explainable statistical model, ChIPanalyser. This tool uses as inputs the DNA sequence binding motif; the capacity to distinguish between strong and weak binding sites; the concentration of TF; and chromatin accessibility. We found that approximately one third of TFs are predicted to bind the genome in a DNA accessibility independent fashion, which includes TFs that can open the chromatin, their co-factors and TFs with similar motifs. Our model predicted this to be the case when the TF binds to its strongest binding regions in the genome, and only a small number of TFs have the capacity to bind dense chromatin at their weakest binding regions, such as CTCF, USF2 and CEBPB. Our study demonstrated that the binding of hundreds of human and mouse TFs is predicted by ChIPanalyser with high accuracy and showed that many TFs can bind dense chromatin.

Regulation of chromatin microphase separation by binding of protein complexes.

We show evidence of the association of RNA polymerase II (RNAP) with chromatin in a core-shell organization, reminiscent of microphase separation where the cores comprise dense chromatin and the shell, RNAP and chromatin with low density. These observations motivate our physical model for the regulation of core-shell chromatin organization. Here, we model chromatin as a multiblock copolymer, comprising active and inactive regions (blocks) that are both in poor solvent and tend to be condensed in the absence of binding proteins. However, we show that the solvent quality for the active regions of chromatin can be regulated by the binding of protein complexes (e.g., RNAP and transcription factors). Using the theory of polymer brushes, we find that such binding leads to swelling of the active chromatin regions which in turn modifies the spatial organization of the inactive regions. In addition, we use simulations to study spherical chromatin micelles, whose cores comprise inactive regions and shells comprise active regions and bound protein complexes. In spherical micelles the swelling increases the number of inactive cores and controls their size. Thus, genetic modifications affecting the binding strength of chromatin-binding protein complexes may modulate the solvent quality experienced by chromatin and regulate the physical organization of the genome.

Resveratrol ameliorates spermatogenesis by increasing protamine 1, 2 and HSPA2 expression in experimental varicocele rat model

ObjectivesVaricocele is a common cause of male infertility associated with an elevated testicular temperature that induces apoptosis, spermatogenesis dysfunction, and affects sperm parameters. In this study, we investigate the probable therapeutic effects of resveratrol (RES), a natural phytoalexin, against varicocele. Materials and methodsIn this study, 48 male Wistar rats randomly divided into 8 groups: normal, sham, normal+RES (20 and 50mg/kg), varicocele, varicocele+ethanol and varicocele+RES (20 and 50mg/kg). Incomplete closure of the left renal vein was used for varicocele induction and two months later, RES was administrated orally for 60 days. Then, sperm parameters, DNA fragmentations, chromatin density, and testis histopathology were analyzed. In addition, HSPA2, protamine 1, and 2 expression levels were evaluated using real-time PCR. ResultsAccording to our results, resveratrol treatment improved sperm parameters, testis histopathology, DNA fragmentation, and chromatin maturation which damaged follow varicocele (p≤0.05). Also, it increased HSPA2, protamine 1, and 2 expression levels significantly in both doses (p≤0.05). ConclusionResveratrol potentially attenuates varicocele-induced spermatogenic impairments by its antioxidant features and regulates spermatogenic gene expression undergoing DNA fragmentation, so leads histopathological properties of tissues to physiological parameters.

Histone H1 facilitates restoration of H3K27me3 during DNA replication by chromatin compaction

During cell renewal, epigenetic information needs to be precisely restored to maintain cell identity and genome integrity following DNA replication. The histone mark H3K27me3 is essential for the formation of facultative heterochromatin and the repression of developmental genes in embryonic stem cells. However, how the restoration of H3K27me3 is precisely achieved following DNA replication is still poorly understood. Here we employ ChOR-seq (Chromatin Occupancy after Replication) to monitor the dynamic re-establishment of H3K27me3 on nascent DNA during DNA replication. We find that the restoration rate of H3K27me3 is highly correlated with dense chromatin states. In addition, we reveal that the linker histone H1 facilitates the rapid post-replication restoration of H3K27me3 on repressed genes and the restoration rate of H3K27me3 on nascent DNA is greatly compromised after partial depletion of H1. Finally, our in vitro biochemical experiments demonstrate that H1 facilitates the propagation of H3K27me3 by PRC2 through compacting chromatin. Collectively, our results indicate that H1-mediated chromatin compaction facilitates the propagation and restoration of H3K27me3 after DNA replication.

Strong interactions between highly dynamic lamina-associated domains and the nuclear envelope stabilize the 3D architecture of Drosophila interphase chromatin

BackgroundInteractions among topologically associating domains (TADs), and between the nuclear envelope (NE) and lamina-associated domains (LADs) are expected to shape various aspects of three-dimensional (3D) chromatin structure and dynamics; however, relevant genome-wide experiments that may provide statistically significant conclusions remain difficult.ResultsWe have developed a coarse-grained dynamical model of D. melanogaster nuclei at TAD resolution that explicitly accounts for four distinct epigenetic classes of TADs and LAD–NE interactions. The model is parameterized to reproduce the experimental Hi-C map of the wild type (WT) nuclei; it describes time evolution of the chromatin over the G1 phase of the interphase. The simulations include an ensemble of nuclei, corresponding to the experimentally observed set of several possible mutual arrangements of chromosomal arms. The model is validated against multiple structural features of chromatin from several different experiments not used in model development. Predicted positioning of all LADs at the NE is highly dynamic—the same LAD can attach, detach and move far away from the NE multiple times during interphase. The probabilities of LADs to be in contact with the NE vary by an order of magnitude, despite all having the same affinity to the NE in the model. These probabilities are mostly determined by a highly variable local linear density of LADs along the genome, which also has the same strong effect on the predicted positioning of individual TADs -- higher probability of a TAD to be near NE is largely determined by a higher linear density of LADs surrounding this TAD. The distribution of LADs along the chromosome chains plays a notable role in maintaining a non-random average global structure of chromatin. Relatively high affinity of LADs to the NE in the WT nuclei substantially reduces sensitivity of the global radial chromatin distribution to variations in the strength of TAD–TAD interactions compared to the lamin depleted nuclei, where a small (0.5 kT) increase of cross-type TAD–TAD interactions doubles the chromatin density in the central nucleus region.ConclusionsA dynamical model of the entire fruit fly genome makes multiple genome-wide predictions of biological interest. The distribution of LADs along the chromatin chains affects their probabilities to be in contact with the NE and radial positioning of highly mobile TADs, playing a notable role in creating a non-random average global structure of the chromatin. We conjecture that an important role of attractive LAD–NE interactions is to stabilize global chromatin structure against inevitable cell-to-cell variations in TAD–TAD interactions.

High-LET targeted microbeam irradiation induces local chromatin reorganization in living cells showing active basal mechanisms at highly complex DNA damage sites

DNA repair eukaryotic cells have additional protective mechanisms that avoid uncontrolled interaction of different parts of the chromatin and damaged regions. Key factors here are the regulation of chromatin density and mobility. The 4D (temporal and spatial) organization of chromatin is controlling this security barrier by regulating the accessibility of genes, flexibility of DNA, and its ability to move inside the nucleus. How this regulation mechanisms are involved in DNA repair upon radiation damage is until now rarely known but an important part to understand the enhanced effectiveness of high linear energy transfer (LET) particles. The damage recognition via PARP1 and the subsequent chromatin decondensation via PARylation is a crucial step in the DNA damage response (DDR). Upon We used the SNAKE microbeam with a beam spot size of <1 µm to induce highly localized DNA damage in living cells using 55 MeV Carbon ions to investigate the chromatin rearrangements in the early stage of DDR. The nuclei were irradiated with a cross pattern consisting of 1000 ions per spot and 25 spots per cell either with one (11 000 Gy), two (22 000 Gy), or three crosses (33 000 Gy). The chromatin rearrangement was imaged live for several minutes after irradiation at the beam using SiR chromatin stain. Upon 91% of the cells show a localized decondensation starting from a few seconds up to minutes after irradiation. The chromatin is decondensed by 6%-8% in the beam path with a local condensation at the edges of up to 8%. Our results suggest that chromatin decondensation is a fast process in the first few seconds after damage induction. Furthermore, decondensation status does not change over minutes, which gives evidence that this process and therefore DDR is paused or even stopped. In combination with the existing knowledge about early reactions to damage induction our data support the model of PARP induced chromatin decondensation. Furthermore, it is evident that also ultra-high doses of radiation are, in first place not able to inactivate initial basal mechanisms as response to damage induction.