Changes In Chromatin Compaction Research Articles

Histone H3 mutations and their impact on genome stability maintenance.

Histones are essential for maintaining chromatin structure and function. Histone mutations lead to changes in chromatin compaction, gene expression, and the recruitment of DNA repair proteins to the DNA lesion. These disruptions can impair critical DNA repair pathways, such as homologous recombination and non-homologous end joining, resulting in increased genomic instability, which promotes an environment favorable to tumor development and progression. Understanding these mechanisms underscores the potential of targeting DNA repair pathways in cancers harboring mutated histones, offering novel therapeutic strategies to exploit their inherent genomic instability for better treatment outcomes. Here, we examine how mutations in histone H3 disrupt normal chromatin function and DNA damage repair processes and how these mechanisms can be exploited for therapeutic interventions.

3D Super-Resolution Nuclear Q-FISH Imaging Reveals Cell-Cycle-Related Telomere Changes.

We present novel workflows for Q-FISH nanoscopy with the potential for prognostic applications and resolving novel chromatin compaction changes. DNA-fluorescence in situ hybridization (DNA-FISH) is a routine application to visualize telomeres, repetitive terminal DNA sequences, in cells and tissues. Telomere attrition is associated with inherited and acquired diseases, including cancer and cardiomyopathies, and is frequently analyzed by quantitative (Q)-FISH microscopy. Recently, nanoscopic imaging techniques have resolved individual telomere dimensions and their compaction as a prognostic marker, in part leading to conflicting conclusions still unresolved to date. Here, we developed a comprehensive Q-FISH nanoscopy workflow to assess telomeres with PNA telomere probes and 3D-Stimulated Emission Depletion (STED) microscopy combined with Dynamic Intensity Minimum (DyMIN) scanning. We achieved single-telomere resolution at high, unprecedented telomere coverage. Importantly, our approach revealed a decrease in telomere signal density during mitotic cell division compared to interphase. Innovatively expanding FISH-STED applications, we conducted double FISH targeting of both telomere- and chromosome-specific sub-telomeric regions and accomplished FISH-STED in human cardiac biopsies. In summary, this work further advanced Q-FISH nanoscopy, detected a new aspect of telomere compaction related to the cell cycle, and laid the groundwork for future applications in complex cell types such as post-mitotic neurons and muscle cells.

Super-resolution microscopy reveals the nanoscale cluster architecture of the DEK protein cancer biomarker

DEK protein, a key chromatin regulator, is strongly overexpressed in various forms of cancer. While conventional microscopy revealed DEK as uniformly distributed within the cell nucleus, advanced super-resolution techniques uncovered cluster-like structures. However, a comprehensive understanding of DEK's cellular distribution and its implications in cancer and cell growth remained elusive. To bridge this gap, we employed single-molecule localization microscopy (SMLM) to dissect DEK's nanoscale organization in both normal-like and aggressive breast cancer cell lines. Our investigation included characteristics such as localizations per cluster, cluster areas, and intra-cluster localization densities (ICLDs). We elucidated how cluster features align with different breast cell types and how chromatin decompaction influences DEK clusters in these contexts. Our results indicate that DEK's intra-cluster localization density and nano-organization remain preserved and not significantly influenced by protein overexpression or chromatin compaction changes. This study advances the understanding of DEK's role in cancer and underscores its stable nanoscale behavior.

Live imaging reveals chromatin compaction transitions and dynamic transcriptional bursting during stem cell differentiation in vivo.

Stem cell differentiation requires dramatic changes in gene expression and global remodeling of chromatin architecture. How and when chromatin remodels relative to the transcriptional, behavioral, and morphological changes during differentiation remain unclear, particularly in an intact tissue context. Here, we develop a quantitative pipeline which leverages fluorescently-tagged histones and longitudinal imaging to track large-scale chromatin compaction changes within individual cells in a live mouse. Applying this pipeline to epidermal stem cells, we reveal that cell-to-cell chromatin compaction heterogeneity within the stem cell compartment emerges independent of cell cycle status, and instead is reflective of differentiation status. Chromatin compaction state gradually transitions over days as differentiating cells exit the stem cell compartment. Moreover, establishing live imaging of Keratin-10 (K10) nascent RNA, which marks the onset of stem cell differentiation, we find that Keratin-10 transcription is highly dynamic and largely precedes the global chromatin compaction changes associated with differentiation. Together, these analyses reveal that stem cell differentiation involves dynamic transcriptional states and gradual chromatin rearrangement.

Impact of Histone Modifications and Their Therapeutic Targeting in Hematological Malignancies.

Hematologic malignancies are a large and heterogeneous group of neoplasms characterized by complex pathogenetic mechanisms. The abnormal regulation of epigenetic mechanisms and specifically, histone modifications, has been demonstrated to play a central role in hematological cancer pathogenesis and progression. A variety of epigenetic enzymes that affect the state of histones have been detected as deregulated, being either over- or underexpressed, which induces changes in chromatin compaction and, subsequently, affects gene expression. Recent advances in the field of epigenetics have revealed novel therapeutic targets, with many epigenetic drugs being investigated in clinical trials. The present review focuses on the biological impact of histone modifications in the pathogenesis of hematologic malignancies, describing a wide range of therapeutic agents that have been discovered to target these alterations and are currently under investigation in clinical trials.

Lamin A/C-dependent chromatin architecture safeguards naïve pluripotency to prevent aberrant cardiovascular cell fate and function

Tight control of cell fate choices is crucial for normal development. Here we show that lamin A/C plays a key role in chromatin organization in embryonic stem cells (ESCs), which safeguards naïve pluripotency and ensures proper cell fate choices during cardiogenesis. We report changes in chromatin compaction and localization of cardiac genes in Lmna−/− ESCs resulting in precocious activation of a transcriptional program promoting cardiomyocyte versus endothelial cell fate. This is accompanied by premature cardiomyocyte differentiation, cell cycle withdrawal and abnormal contractility. Gata4 is activated by lamin A/C loss and Gata4 silencing or haploinsufficiency rescues the aberrant cardiovascular cell fate choices induced by lamin A/C deficiency. We uncover divergent functions of lamin A/C in naïve pluripotent stem cells and cardiomyocytes, which have distinct contributions to the transcriptional alterations of patients with LMNA-associated cardiomyopathy. We conclude that disruption of lamin A/C-dependent chromatin architecture in ESCs is a primary event in LMNA loss-of-function cardiomyopathy.

Phenotypic selection during laboratory evolution of yeast populations leads to a genome-wide sustainable chromatin compaction shift.

In a previous study, we have shown how microbial evolution has resulted in a persistent reduction in expression after repeatedly selecting for the lowest PGAL1-YFP-expressing cells. Applying the ATAC-seq assay on samples collected from this 28-day evolution experiment, here we show how genome-wide chromatin compaction changes during evolution under selection pressure. We found that the chromatin compaction was altered not only on GAL network genes directly impacted by the selection pressure, showing an example of selection-induced non-genetic memory, but also at the whole-genome level. The GAL network genes experienced chromatin compaction accompanying the reduction in PGAL1-YFP reporter expression. Strikingly, the fraction of global genes with differentially compacted chromatin states accounted for about a quarter of the total genome. Moreover, some of the ATAC-seq peaks followed well-defined temporal dynamics. Comparing peak intensity changes on consecutive days, we found most of the differential compaction to occur between days 0 and 3 when the selection pressure was first applied, and between days 7 and 10 when the pressure was lifted. Among the gene sets enriched for the differential compaction events, some had increased chromatin availability once selection pressure was applied and decreased availability after the pressure was lifted (or vice versa). These results intriguingly show that, despite the lack of targeted selection, transcriptional availability of a large fraction of the genome changes in a very diverse manner during evolution, and these changes can occur in a relatively short number of generations.

DNA damage reduces heterogeneity and coherence of chromatin motions

Chromatin motions depend on and may regulate genome functions, in particular the DNA damage response. In yeast, DNA double-strand breaks (DSBs) globally increase chromatin diffusion, whereas in higher eukaryotes the impact of DSBs on chromatin dynamics is more nuanced. We mapped the motions of chromatin microdomains in mammalian cells using diffractive optics and photoactivatable chromatin probes and found a high level of spatial heterogeneity. DNA damage reduces heterogeneity and imposes spatially defined shifts in motions: Distal to DNA breaks, chromatin motions are globally reduced, whereas chromatin retains higher mobility at break sites. These effects are driven by context-dependent changes in chromatin compaction. Photoactivated lattices of chromatin microdomains are ideal to quantify microscale coupling of chromatin motion. We measured correlation distances up to 2 µm in the cell nucleus, spanning chromosome territories, and speculate that this correlation distance between chromatin microdomains corresponds to the physical separation of A and B compartments identified in chromosome conformation captureexperiments. After DNA damage, chromatin motions become less correlated, a phenomenon driven by phase separation at DSBs. Our data indicate tight spatial control of chromatin motions after genomic insults, which may facilitate repair at the break sites and prevent deleterious contacts of DSBs, thereby reducing the risk of genomic rearrangements.

Disruption of Chromatin Dynamics by Hypotonic Stress Suppresses HR and Shifts DSB Processing to Error-Prone SSA.

The processing of DNA double-strand breaks (DSBs) depends on the dynamic characteristics of chromatin. To investigate how abrupt changes in chromatin compaction alter these dynamics and affect DSB processing and repair, we exposed irradiated cells to hypotonic stress (HypoS). Densitometric and chromosome-length analyses show that HypoS transiently decompacts chromatin without inducing histone modifications known from regulated local chromatin decondensation, or changes in Micrococcal Nuclease (MNase) sensitivity. HypoS leaves undisturbed initial stages of DNA-damage-response (DDR), such as radiation-induced ATM activation and H2AX-phosphorylation. However, detection of ATM-pS1981, γ-H2AX and 53BP1 foci is reduced in a protein, cell cycle phase and cell line dependent manner; likely secondary to chromatin decompaction that disrupts the focal organization of DDR proteins. While HypoS only exerts small effects on classical nonhomologous end-joining (c-NHEJ) and alternative end-joining (alt-EJ), it markedly suppresses homologous recombination (HR) without affecting DNA end-resection at DSBs, and clearly enhances single-strand annealing (SSA). These shifts in pathway engagement are accompanied by decreases in HR-dependent chromatid-break repair in the G2-phase, and by increases in alt-EJ and SSA-dependent chromosomal translocations. Consequently, HypoS sensitizes cells to ionizing radiation (IR)-induced killing. We conclude that HypoS-induced global chromatin decompaction compromises regulated chromatin dynamics and genomic stability by suppressing DSB-processing by HR, and allowing error-prone processing by alt-EJ and SSA.

Transcriptional Activation of Heterochromatin by Recruitment of dCas9 Activators.

The transition from silenced heterochromatin to a biologically active state and vice versa is a fundamental part of the implementation of cell type-specific gene expression programs. To reveal structure-function relationships and dissect the underlying mechanisms, experiments that ectopically induce transcription are highly informative. In particular, the approach to perturb chromatin states by recruiting fusions of the catalytically inactive dCas9 protein in a sequence-specific manner to a locus of interest has been used in numerous applications. Here, we describe how this approach can be applied to activate pericentric heterochromatin (PCH) in mouse cells as a prototypic silenced state by providing protocols for the following workflow: (a) Recruitment of dCas9 fusion constructs with the strong transcriptional activator VPR to PCH. (b) Analysis of the resulting changes in chromatin compaction, epigenetic marks, and active transcription by fluorescence microscopy-based readouts. (c) Automated analysis of the resulting images with a set of scripts in the R programming language. Furthermore, we discuss how parameters for chromatin decondensation and active transcription are extracted from these experiments and can be combined with other readouts to gain insights into PCH activation.

Monitoring global changes in chromatin compaction states upon localized DNA damage with tools of fluorescence anisotropy.

In the eukaryotic nucleus, DNA, packaged in the form of chromatin, is subject to continuous damage. Chromatin has to be remodeled in order to repair such damage efficiently. But compact chromatin may also be more refractory to damage. Chromatin responses during DNA double-strand break (DSB) repair have been studied with biochemistry or as indirect readouts for the physical state of the chromatin at the site of damage. Direct measures of global chromatin compaction upon damage are lacking. We used fluorescence anisotropy imaging of histone H2B-EGFP to interrogate global chromatin compaction changes in response to localized DSBs directly. Anisotropy maps were preserved in fixation and reported on underlying chromatin compaction states. Laser-induced clustered DSBs led to global compaction of even the undamaged chromatin. Live-cell dynamics could be coupled with fixed-cell assays. Repair factors, PARP1 and PCNA, were immediately recruited to the site of damage, though the local enrichment of PCNA persisted longer than that of PARP1. Subsequently, nodes of PCNA that incorporated deoxynucleotide analogs were observed in regions of low-anisotropy open chromatin, even away from the site of damage. Such fluorescence anisotropy–based readout of chromatin compaction may be used in the investigation of different forms of DNA damage.

High-Resolution Single-Cell Models of Ensemble Chromatin Structures During Drosophila Embryogenesis from Population HI-C

We describe a computational method for reconstruction of large 3D ensemble of single-cell chromatin conformations from population Hi-C. Our method identifies specific interactions constituting a small fraction (5-6% in Drosophila) of measured Hi-C frequencies. Yet, surprisingly, they are sufficient to drive chromatin folding, giving rise to much of observed Hi-C topological features. Our models reveal dramatic changes in chromatin compaction across three developmental stages of Drosophila, with %gt 50% of single cells likely maintaining TAD-like structures, possibly even in early embryos when no population TADs are discernable. Our models reveal details on how varying single-cell domain boundaries become fixated throughout development, with strong preferred-positioning at binding sites of insulator complexes during and after midblastula transition. Overall, our method can be used to re-interpret population Hi-C by generating ensembles of single-cell chromatin conformations at high resolution, facilitating better understanding of genome organization.

In Vivo Chromatin Compaction Changes as Detected by Water Dipolar Relaxations: the Molecular Crowding Role Revealed by the Acdan Fluorescence

In Vivo Chromatin Compaction Changes as Detected by Water Dipolar Relaxations: the Molecular Crowding Role Revealed by the Acdan Fluorescence

Application of fluorescence lifetime imaging microscopy of DNA binding dyes to assess radiation-induced chromatin compaction changes.

In recent years several approaches have been developed to address the chromatin status and its changes in eukaryotic cells under different conditions—but only few are applicable in living cells. Fluorescence lifetime imaging microscopy (FLIM) is a functional tool that can be used for the inspection of the molecular environment of fluorophores in living cells. Here, we present the use of single organic minor groove DNA binder dyes in FLIM for measuring chromatin changes following modulation of chromatin structure in living cells. Treatment with histone deacetylase inhibitors led to an increased fluorescence lifetime indicating global chromatin decompaction, whereas hyperosmolarity decreased the lifetime of the used dyes, thus reflecting the expected compaction. In addition, we demonstrate that time domain FLIM data based on single photon counting should be optimized using pile-up and counting loss correction, which affect the readout even at moderate average detector count rates in inhomogeneous samples. Using these corrections and utilizing Hoechst 34580 as chromatin compaction probe, we measured a pan nuclear increase in the lifetime following irradiation with X-rays in living NIH/3T3 cells thus providing a method to measure radiation-induced chromatin decompaction.

Linker histones and chromatin remodelling complexes maintain genome stability and control cellular ageing

Linker histones are major players in chromatin organization and per se are essential players in genome homeostasis. As the fifth class of histone proteins the linker histones not only interact with DNA and core histones but also with other chromatin proteins. These interactions prove to be essential for the higher levels of chromatin organization like chromatin loops, transcription factories and chromosome territories. Our recent results have proved that Saccharomyces cerevisiae linker histone – Hho1p, physically interacts with the actin-related protein 4 (Arp4) and that the abrogation of this interaction through the deletion of the gene for the linker histone in arp4 mutant cells leads to global changes in chromatin compaction. Here, we show that the healthy interaction between the yeast linker histone and Arp4p is critical for maintaining genome stability and for controlling cellular sensitivity to different types of stress. The abolished interaction between the linker histone and Arp4p leads the mutant yeast cells to premature ageing phenotypes. Cells die young and are more sensitive to stress. These results unambiguously prove the role of linker histones and chromatin remodelling in ageing by their cooperation in pertaining higher-order chromatin compaction and thus maintaining genome stability.

Nuclear parameters and chromatin remodeling in epithelial cells and lymphocytes from the palpebral conjunctiva of dogs with keratoconjunctivitis sicca.

To study parameters related to nuclear morphology and chromatin remodeling in epithelial cells and lymphocytes from the inferior palpebral conjunctiva of dogs with and without keratoconjunctivitis sicca (KCS). Thirty-two dogs (64 eyes) were included in the study. Based on the tear production measured by Schirmer tear test 1, the dogs were distributed into control and KCS groups. Epithelial cells and lymphocytes were collected by conjunctival brush cytology, fixed on glass slides, and subjected to the Feulgen reaction, a topochemical method specific for DNA/chromatin. Feulgen-stained cells were studied by microscopy and video image analysis to establish nuclear size (area and perimeter) and shape (relative nuclear roundness factor=RNRF), DNA content (ploidy), and compaction and texture of chromatin. Conjunctival samples in the KCS group showed infiltration of inflammatory and immune cells. Micronuclei, snake-like chromatin, aberrant chromosomes, and goblet cells were not detected. Compared with the controls, cells on the conjunctival surface of dogs with KCS showed altered nuclei. Conjunctival epithelial cells were more affected by KCS (changes in nuclear size, shape, DNA content, and chromatin compaction) than lymphocytes (changes in chromatin compaction, only). Significant chromatin decompaction was observed in both conjunctival epithelial cells and lymphocytes. Our results show that KCS promotes chromatin remodeling in epithelial cells and lymphocytes on the conjunctival surface of dogs. The changes described in this study are different from those reported for conjunctival cell nuclei of human KCS patients.

Transcriptome dynamics along axolotl regenerative development are consistent with an extensive reduction in gene expression heterogeneity in dedifferentiated cells.

Although in recent years the study of gene expression variation in the absence of genetic or environmental cues or gene expression heterogeneity has intensified considerably, many basic and applied biological fields still remain unaware of how useful the study of gene expression heterogeneity patterns might be for the characterization of biological systems and/or processes. Largely based on the modulator effect chromatin compaction has for gene expression heterogeneity and the extensive changes in chromatin compaction known to occur for specialized cells that are naturally or artificially induced to revert to less specialized states or dedifferentiate, I recently hypothesized that processes that concur with cell dedifferentiation would show an extensive reduction in gene expression heterogeneity. The confirmation of the existence of such trend could be of wide interest because of the biomedical and biotechnological relevance of cell dedifferentiation-based processes, i.e., regenerative development, cancer, human induced pluripotent stem cells, or plant somatic embryogenesis. Here, I report the first empirical evidence consistent with the existence of an extensive reduction in gene expression heterogeneity for processes that concur with cell dedifferentiation by analyzing transcriptome dynamics along forearm regenerative development in Ambystoma mexicanum or axolotl. Also, I briefly discuss on the utility of the study of gene expression heterogeneity dynamics might have for the characterization of cell dedifferentiation-based processes, and the engineering of tools that afforded better monitoring and modulating such processes. Finally, I reflect on how a transitional reduction in gene expression heterogeneity for dedifferentiated cells can promote a long-term increase in phenotypic heterogeneity following cell dedifferentiation with potential adverse effects for biomedical and biotechnological applications.

A new and improved algorithm for the quantification of chromatin condensation from microscopic data shows decreased chromatin condensation in regenerating axolotl limb cells.

The nuclear landscape plays an important role in the regulation of tissue and positional specific genes in embryonic and developing cells. Changes in this landscape can be dynamic, and are associated with the differentiation of cells during embryogenesis, and the de-differentiation of cells during induced pluripotent stem cell (iPSC) formation and in many cancers. However, tools to quantitatively characterize these changes are limited, especially in the in vivo context, where numerous tissue types are present and cells are arranged in multiple layers. Previous tools have been optimized for the monolayer nature of cultured cells. Therefore, we present a new algorithm to quantify the condensation of chromatin in two in vivo systems. We first developed this algorithm to quantify changes in chromatin compaction and validated it in differentiating spermatids in zebrafish testes. Our algorithm successfully detected the typical increase in chromatin compaction as these cells differentiate. We then employed the algorithm to quantify the changes that occur in amphibian limb cells as they participate in a regenerative response. We observed that the chromatin in the limb cells de-compacts as they contribute to the regenerating organ. We present this new tool as an open sourced software that can be readily accessed and optimized to quantify chromatin compaction in complex multi-layered samples.

Hormone-induced repression of genes requires BRG1-mediated H1.2 deposition at target promoters.

Eukaryotic gene regulation is associated with changes in chromatin compaction that modulate access to DNA regulatory sequences relevant for transcriptional activation or repression. Although much is known about the mechanism of chromatin remodeling in hormonal gene activation, how repression is accomplished is much less understood. Here we report that in breast cancer cells, ligand-activated progesterone receptor (PR) is directly recruited to transcriptionally repressed genes involved in cell proliferation along with the kinases ERK1/2 and MSK1. PR recruits BRG1 associated with the HP1γ-LSD1 complex repressor complex, which is further anchored via binding of HP1γ to the H3K9me3 signal deposited by SUV39H2. In contrast to what is observed during gene activation, only BRG1 and not the BAF complex is recruited to repressed promoters, likely due to local enrichment of the pioneer factor FOXA1. BRG1 participates in gene repression by interacting with H1.2, facilitating its deposition and stabilizing nucleosome positioning around the transcription start site. Our results uncover a mechanism of hormone-dependent transcriptional repression and a novel role for BRG1 in progestin regulation of breast cancer cell growth.

Quantitative analysis of chromatin interaction changes upon a 4.3 Mb deletion at mouse 4E2.

BackgroundCircular chromosome conformation capture (4C) has provided important insights into three dimensional (3D) genome organization and its critical impact on the regulation of gene expression. We developed a new quantitative framework based on polymer physics for the analysis of paired-end sequencing 4C (PE-4Cseq) data. We applied this strategy to the study of chromatin interaction changes upon a 4.3 Mb DNA deletion in mouse region 4E2.ResultsA significant number of differentially interacting regions (DIRs) and chromatin compaction changes were detected in the deletion chromosome compared to a wild-type (WT) control. Selected DIRs were validated by 3D DNA FISH experiments, demonstrating the robustness of our pipeline. Interestingly, significant overlaps of DIRs with CTCF/Smc1 binding sites and differentially expressed genes were observed.ConclusionsAltogether, our PE-4Cseq analysis pipeline provides a comprehensive characterization of DNA deletion effects on chromatin structure and function.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2137-5) contains supplementary material, which is available to authorized users.