Changes In Chromatin Structure Research Articles

Erythropoietin Regulates Transcription and YY1 Dynamics in a Pre-established Chromatin Architecture.

SummaryThe three-dimensional architecture of the genome plays an essential role in establishing and maintaining cell identity. However, the magnitude and temporal kinetics of changes in chromatin structure that arise during cell differentiation remain poorly understood. Here, we leverage a murine model of erythropoiesis to study the relationship between chromatin conformation, the epigenome, and transcription in erythroid cells. We discover that acute transcriptional responses induced by erythropoietin (EPO), the hormone necessary for erythroid differentiation, occur within an invariant chromatin topology. Within this pre-established landscape, Yin Yang 1 (YY1) occupancy dynamically redistributes to sites in proximity of EPO-regulated genes. Using HiChIP, we identify chromatin contacts mediated by H3K27ac and YY1 that are enriched for enhancer-promoter interactions of EPO-responsive genes. Taken together, these data are consistent with an emerging model that rapid, signal-dependent transcription occurs in the context of a pre-established chromatin architecture.

Changes in H3K27ac at Gene Regulatory Regions in Porcine Alveolar Macrophages Following LPS or PolyIC Exposure.

Changes in chromatin structure, especially in histone modifications (HMs), linked with chromatin accessibility for transcription machinery, are considered to play significant roles in transcriptional regulation. Alveolar macrophages (AM) are important immune cells for protection against pulmonary pathogens, and must readily respond to bacteria and viruses that enter the airways. Mechanism(s) controlling AM innate response to different pathogen-associated molecular patterns (PAMPs) are not well defined in pigs. By combining RNA sequencing (RNA-seq) with chromatin immunoprecipitation and sequencing (ChIP-seq) for four histone marks (H3K4me3, H3K4me1, H3K27ac and H3K27me3), we established a chromatin state map for AM stimulated with two different PAMPs, lipopolysaccharide (LPS) and Poly(I:C), and investigated the potential effect of identified histone modifications on transcription factor binding motif (TFBM) prediction and RNA abundance changes in these AM. The integrative analysis suggests that the differential gene expression between non-stimulated and stimulated AM is significantly associated with changes in the H3K27ac level at active regulatory regions. Although global changes in chromatin states were minor after stimulation, we detected chromatin state changes for differentially expressed genes involved in the TLR4, TLR3 and RIG-I signaling pathways. We found that regions marked by H3K27ac genome-wide were enriched for TFBMs of TF that are involved in the inflammatory response. We further documented that TF whose expression was induced by these stimuli had TFBMs enriched within H3K27ac-marked regions whose chromatin state changed by these same stimuli. Given that the dramatic transcriptomic changes and minor chromatin state changes occurred in response to both stimuli, we conclude that regulatory elements (i.e. active promoters) that contain transcription factor binding motifs were already active/poised in AM for immediate inflammatory response to PAMPs. In summary, our data provides the first chromatin state map of porcine AM in response to bacterial and viral PAMPs, contributing to the Functional Annotation of Animal Genomes (FAANG) project, and demonstrates the role of HMs, especially H3K27ac, in regulating transcription in AM in response to LPS and Poly(I:C).

Natural Products Impacting DNA Methyltransferases and Histone Deacetylases.

Epigenetics refers to heritable changes in gene expression and chromatin structure without change in a DNA sequence. Several epigenetic modifications and respective regulators have been reported. These include DNA methylation, chromatin remodeling, histone post-translational modifications, and non-coding RNAs. Emerging evidence has revealed that epigenetic dysregulations are involved in a wide range of diseases including cancers. Therefore, the reversible nature of epigenetic modifications concerning activation or inhibition of enzymes involved could be promising targets and useful tools for the elucidation of cellular and biological phenomena. In this review, emphasis is laid on natural products that inhibit DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) making them promising candidates for the development of lead structures for anticancer-drugs targeting epigenetic modifications. However, most of the natural products targeting HDAC and/or DNMT lack isoform selectivity, which is important for determining their potential use as therapeutic agents. Nevertheless, the structures presented in this review offer the well-founded basis that screening and chemical modifications of natural products will in future provide not only leads to the identification of more specific inhibitors with fewer side effects, but also important features for the elucidation of HDAC and DNMT function with respect to cancer treatment.

Optimized Stochastic Optical Reconstruction Microscopy for Imaging Chromatin Structure in Pathological Tissue.

Direct visualization of higher-order chromatin structure at the molecular scale is of great importance for understanding the impact of chromatin organization on gene expression in many biological processes. Understanding the changes in chromatin structure during pathological processes requires the use of in vivo models and clinical samples, and formalin-fixed, paraffin-embedded (FFPE) tissue is the most widespread form of preservation. Here we describe the details of PathSTORM, an optimized stochastic optical reconstruction microscopy (STORM) protocol for high-quality super-resolution imaging of densely packed higher-order chromatin organization in pathological tissue. We discuss detailed methods for fluorescence staining of DNA and histone proteins, as well as the key technical factors for obtaining high-quality STORM images in pathological tissue samples. © 2020 Wiley Periodicals LLC Basic Protocol 1: Fluorescence staining of chromatin in pathological tissue Basic Protocol 2: STORM data processing Support Protocol 1: Drift correction Support Protocol 2: Image reconstruction Support Protocol 3: Hematoxylin & eosin (H&E) staining.

Lamin B receptor: role on chromatin structure, cellular senescence and possibly aging

The nuclear envelope is composed by an outer nuclear membrane and an inner nuclear membrane, which is underlain by the nuclear lamina that provides the nucleus with mechanical strength for maintaining structure and regulates chromatin organization for modulating gene expression and silencing. A layer of heterochromatin is beneath the nuclear lamina, attached by inner nuclear membrane integral proteins such as Lamin B receptor (LBR). LBR is a chimeric protein, having also a sterol reductase activity with which it contributes to cholesterol synthesis. Lukasova et al. showed that when DNA is damaged by ɣ-radiation in cancer cells, LBR is lost causing chromatin structure changes and promoting cellular senescence. Cellular senescence is characterized by terminal cell cycle arrest and the expression and secretion of various growth factors, cytokines, metalloproteinases, etc., collectively known as senescence-associated secretory phenotype (SASP) that cause chronic inflammation and tumor progression when they persist in the tissue. Therefore, it is fundamental to understand the molecular basis for senescence establishment, maintenance and the regulation of SASP. The work of Lukasova et al. contributed to our understanding of cellular senescence establishment and provided the basis that lead to the further discovery that chromatin changes caused by LBR reduction induce an up-regulated expression of SASP factors. LBR dysfunction has relevance in several diseases and possibly in physiological aging. The potential bifunctional role of LBR on cellular senescence establishment, namely its role in chromatin structure together with its enzymatic activity contributing to cholesterol synthesis, provide a new target to develop potential anti-aging therapies.

Analysis of accessible chromatin landscape in the inner cell mass and trophectoderm of human blastocysts

Early embryonic development is characterized by drastic changes in chromatin structure that affects the accessibility of the chromatin. In human, the chromosome reorganization and its involvement in the first linage segregation are poorly characterized due to the difficulties in obtaining human embryonic material and limitation on low input technologies. In this study, we aimed to explore the chromatin remodeling pattern in human preimplantation embryos and gain insight into the epigenetic regulation of inner cell mass (ICM) and trophectoderm (TE) differentiation. We optimized ATAC-seq (an assay for transposase-accessible chromatin using sequencing) to analyze the chromatin accessibility landscape for low DNA input. Sixteen preimplantation human blastocysts frozen on Day 6 were used. Our data showed that ATAC peak distributions of the promoter regions (<1 kb) and distal regions versus other regions were significantly different between ICM versus TE samples (P < 0.01). We detected that a higher percentage of accessible binding loci were located within 1 kb of the transcription start site in ICM compared to TE (P < 0.01). However, a higher percentage of accessible regions was detected in the distal region of TE compared to ICM (P < 0.01). In addition, eight differential peaks with a false discovery rate <0.05 between ICM and TE were detected. This is the first study to compare the landscape of the accessible chromatin between ICM and TE of human preimplantation embryos, which unveiled chromatin-level epigenetic regulation of cell lineage specification in early embryo development.

Targeted chromatin conformation analysis identifies novel distal neural enhancers of ZEB2 in pluripotent stem cell differentiation.

The transcription factor zinc finger E-box binding protein 2 (ZEB2) controls embryonic and adult cell fate decisions and cellular maturation in many stem/progenitor cell types. Defects in these processes in specific cell types underlie several aspects of Mowat–Wilson syndrome (MOWS), which is caused by ZEB2 haplo-insufficiency. Human ZEB2, like mouse Zeb2, is located on chromosome 2 downstream of a ±3.5 Mb-long gene-desert, lacking any protein-coding gene. Using temporal targeted chromatin capture (T2C), we show major chromatin structural changes based on mapping in-cis proximities between the ZEB2 promoter and this gene desert during neural differentiation of human-induced pluripotent stem cells, including at early neuroprogenitor cell (NPC)/rosette state, where ZEB2 mRNA levels increase significantly. Combining T2C with histone-3 acetylation mapping, we identified three novel candidate enhancers about 500 kb upstream of the ZEB2 transcription start site. Functional luciferase-based assays in heterologous cells and NPCs reveal co-operation between these three enhancers. This study is the first to document in-cis Regulatory Elements located in ZEB2’s gene desert. The results further show the usability of T2C for future studies of ZEB2 REs in differentiation and maturation of multiple cell types and the molecular characterization of newly identified MOWS patients that lack mutations in ZEB2 protein-coding exons.

Genome-wide analyses of chromatin interactions after the loss of Pol I, Pol II, and Pol III

BackgroundThe relationship between transcription and the 3D chromatin structure is debated. Multiple studies have shown that transcription affects global Cohesin binding and 3D genome structures. However, several other studies have indicated that inhibited transcription does not alter chromatin conformations.ResultsWe provide the most comprehensive evidence to date to demonstrate that transcription plays a relatively modest role in organizing the local, small-scale chromatin structures in mammalian cells. We show degraded Pol I, Pol II, and Pol III proteins in mESCs cause few or no changes in large-scale 3D chromatin structures, selected RNA polymerases with a high abundance of binding sites or active promoter-associated interactions appear to be relatively more affected after the degradation, transcription inhibition alters local, small loop domains, as indicated by high-resolution chromatin interaction maps, and loops with bound Pol II but without Cohesin or CTCF are identified and found to be largely unchanged after transcription inhibition. Interestingly, Pol II depletion for a longer time significantly affects the chromatin accessibility and Cohesin occupancy, suggesting that RNA polymerases are capable of affecting the 3D genome indirectly. These direct and indirect effects explain the previous inconsistent findings on the influence of transcription inhibition on the 3D genome.ConclusionsWe conclude that Pol I, Pol II, and Pol III loss alters local, small-scale chromatin interactions in mammalian cells, suggesting that the 3D chromatin structures are pre-established and relatively stable.

P0454MORPHODENSITOMETRIC FEATURES OF PERIPHERAL BLOOD LYMPHOCYTES IN PATIENTS WITH PRIMARY GLOMERULONEPHRITIS

Abstract Background and Aims Lymphocytes play an important role in the development of glomerulonephritis (GN). Their subpopulations are activated under the influence of certain cytokines that act on the activation factors of transcription of these cells, leading to the corresponding regulation of gene expression. However, in addition, in the regulatory processes of lymphocyte functions, epigenetic modifications of the degree of nuclear chromatin compaction are important. The morphological manifestation of the latter may be the state of the optical density of the nuclei and the range of its variability due to the ratio of chromatin condensation/decondensation. The study of the features of potentially reversible changes in chromatin structure in somatic cells becomes actual. One of the convenient objects for studying the epigenetic mechanisms of the genome functioning regulation are interphase nuclei of peripheral blood lymphocytes. Morphodensitometric study permits to establish compensatory changes at the cellular level and to evaluate the functional state of the hereditary apparatus. The aim of the research was to study the morphodensitometric features of genome status of peripheral blood lymphocytes in patients with primary GN. Method We examined 119 patients (76 men and 43 women) with primary GN. The average age of patients was (39.65±14.16) years. The patients underwent a renal biopsy to verify the morphological form of the disease. For comparison, a survey of 30 healthy individuals was conducted. Chromatin of nuclei and its changes were studied using an image analyzer based on the programm Image Tool for Windows (v. 3.0). The advantage of this method is the possibility of a combined assessment of morphometric (area, perimeter), spectrometric (optical and integral density) data with the analysis of the microanatomy of isolated chromatin blocks and the chromatin function at the same time. The study of the morphodensitometric characteristics of peripheral blood lymphocytes is an informative and accessible method for assessing the general state of the function and structure of the hereditary apparatus of the organism. Results The sexual characteristics of the morphometric and optical parameters of lymphocytes nuclei of patients were established. So, in all patients and especially women, a decrease in the perimeter of lymphocytes nuclei was registered in 1.25 and 1.31 times, respectively (p &amp;lt;0.05). The area of these cells nuclei also naturally decreased in 1.22 times in all patients (in 1.08 and 1.34 times in men and women, respectively) compared to the control (p&amp;lt;0.05). A significant decrease in the area of lymphocytes nuclei with an unchanged density of chromatin packing in these nuclei limits the space inside the nucleus for the interaction of individual chromosome loci in this space. Such limitations can be a serious obstacle to the normal functioning of the genome and its repairing processes. Meanwhile, differences in the optical density parameter of lymphocytes nuclei in both groups were defined. The minimum and maximum optical density were higher in the group of patients, and the range of optical density variability showed wider borders of functional heterogeneity of cells compared to the control. Conclusion Comparative analysis of morphodensitometric features of peripheral blood lymphocytes in patients with primary GN detected the changes in the ratio of chromatin condensation/decondensation and normal structure of nuclei, that may indicate functional disorders of the genotype due to exo- and endogenous factors.

Erythropoietin Regulates Transcription and YY1 Dynamics in a Pre-Established Chromatin Architecture

The three-dimensional architecture of the genome plays an essential role in establishing and maintaining cell identity. By contrast, the magnitude and temporal kinetics of changes in chromatin structure that arise during cell differentiation remain poorly understood. Here, we leverage a murine model of erythropoiesis to study the relationship between chromatin conformation, the epigenome, and transcription in erythroid cells. We discover that acute transcriptional responses induced by erythropoietin (EPO), the hormone necessary for erythroid differentiation, occur within an invariant chromatin topology. Within this pre-established landscape, Yin Yang 1 (YY1) occupancy dynamically redistributes to sites in proximity to EPO-regulated genes. Using HiChIP, we identify chromatin contacts mediated by H3K27ac and YY1 that are enriched for enhancer-promoter interactions of EPO-responsive genes. Taken together, these data are consistent with an emerging model that rapid, signal-dependent transcription occurs in the context of a pre-established chromatin architecture.

The vascular epigenome in patients with obesity and type 2 diabetes: opportunities for personalized therapies

Our genetic background provides limited information on individual risk of developing vascular complications overtime. New biological layers, namely epigenetic modifications, are now emerging as potent regulators of gene expression thus leading to altered transcriptional programs and vascular disease phenotypes. Such epigenetic modifications, defined as changes to the genome that do not involve changes in DNA sequence, are generally induced by environmental factors and poor lifestyle habits. Of note, adverse epigenetic signals acquired during life can be transmitted to the offspring thus leading to premature alterations of the epigenetic and transcriptional landscape eventually leading to early endothelial dysfunction and vascular senescence. Modifications of the epigenome play a pivotal role in the pathophysiology of cardiometabolic disturbances such as obesity and type 2 diabetes. In these patients, changes of DNA methylation and chromatin structure contribute to alter pathways regulating insulin sensitivity, glucose homeostasis, adipogenesis and vascular function. In this perspective, unveiling the ‘epigenetic landscape’ in cardiometabolic patients may help to identify new players implicated in obesity and diabetes-related vascular dysfunction and may pave the way for personalized therapies in this setting. In the present review, we discuss current knowledge of the epigenetic routes implicated in vascular damage and cardiovascular disease in patients with metabolic alterations.

Age-related chromatin landscapes in mouse macrophages

Abstract Aging is associated with changes in gene expression. Age-related changes in chromatin structure may contribute to alterations in gene expression and to aging phenotypes in human. Macrophages play essential roles in tissue homeostasis and repair. Macrophage activation by bacterial LPS leads to induction of a complex inflammatory gene program dependent on numerous transcription factor (TF) families. We previously showed that chromatin accessibility of macrophages is massively increased after LPS treatment. Similar programs may also contribute to the chronically elevated inflammatory state in aging. Therefore, it is important to understand how macrophages are epigenetically and functionally altered during aging. To address the age-associated differences in the transcriptome and chromatin landscape of macrophages, we have performed RNA-seq and ATAC-seq of tissue-resident macrophages in the presence or absence of LPS. Our results revealed that age effect in macrophages seems cell type-specific with the strongest effect in microglia. The majority of age-dependent TF footprints in macrophages showed reduced TF occupancy, but with either reduced or increased chromatin accessibility. We are investigating the functional relevance of the candidate TFs identified in our analysis. Our data highlight the tissue-specificity of aging features that occur in resident macrophages in vivo.

Evaluation of fetal mesenchymal stromal/stem cells senescence during in vitro amplification for therapeutic purpose: choice of cell quality parameters

Background & Aim Mesenchymal stromal/stem cells (MSCs) are fibroblast-like multipotent cells capable of proliferation, self-renewal and differentiation into different mesodermal cell-lineages. They are known to be implicated in many key biological processes like tissue regeneration, secretion of bioactive factors which give them immunomodulatory and trophy properties. MSCs isolated from umbilical cord Wharton's Jelly (WJ-MSC) aroused much attention due to several advantages: multipotency capacity, painless, safety for allogenic therapeutic purposes and efficiency for treating certain diseases. In order to use MSCs for therapeutic purpose, these cells must be expanded in long-term, which inevitably triggers cell senescence, a process of cell proliferation arrest that occurs in vivo and in vitro as a response of cells to excessive stresses. This phenomenon is characterized by several markers and features and it can affect cell behavior. Because of senescent cells influence the outcome of a variety of physiological and pathological processes, and because of WJ-MSCs are used in cell therapy, the aim of the study was to characterize senescence WJ-MSCs after in vitro expansion, to control the quality of these cells in terms of function and phenotype. Methods, Results & Conclusion To induce senescence process, we used in vitro model called “replicative senescence”. Cells were cultured in normoxia (N) and hypoxia (H) conditions and analyzed in early and late passages. Proliferation analysis of MSCs demonstrated a high rate of proliferation in H condition. In addition, senescence analysis using a Beta-Galactosidase test indicated that MSCs in H condition senesce slower than those in N. On the other hand, cell-surface biomarker analysis revealed that all MSCs derived from WJ expressed the common surface markers among which CD44, CD105 without great changes during expansion. However other markers presented changes during expansion (like a decrease in CD146). Senescent cells presented variable immunosuppressive potential compared to MSCs in early passage. Known that senescent cells are characterized by changes in chromatin structure, we studied proteins implicated in chromatin remodeling like HMGB1. Immunofluorescence study showed a translocation of HMGB1 from MSC nucleus in senescent cells. All these results will improve the characterization of MSCs senescence and may provide new parameters for establishing the quality of MSCs prior to their therapeutic application.

Human monocyte chromatin accessibility variations identify inflammaging heterogeneity

Abstract Human aging is associated with low grade chronic inflammation termed, inflammaging, which is a significant risk factor for morbidity and mortality in elderly population. The innate immune system is implicated as master regulator of this process via expression of cytokines and other key mediators. We used ATAC-seq to identify chromatin signatures in monocytes obtained from healthy donors of the GESTALT cohort. State of differentially open chromatin (DOC) between young (&amp;lt;60y) and older (&amp;gt;60y) donors were identified. Older donors segregated in two clusters, young-like and distinct old-like cluster. Old-like DOC’s enriched for intergenic enhancer-like elements dominated by the DNA binding motif of the transcription factor NF-κB. The relationships of age-associated DOC to gene expression and DNA methylation reveled enrichment of cytokine mediated inflammatory pathways. Ongoing studies of cytokine expression using high-dimensional flowcytometry will be used to determine the functional consequences of chromatin structural changes associated with age.

Chromatin dynamics at the maternal to zygotic transition: recent advances from the zebrafish model.

Early animal development is characterized by intense reorganization of the embryonic genome, including large-scale changes in chromatin structure and in the DNA and histone modifications that help shape this structure. Particularly profound shifts in the chromatin landscape are associated with the maternal-to-zygotic transition, when the zygotic genome is first transcribed and maternally loaded transcripts are degraded. The accessibility of the early zebrafish embryo facilitates the interrogation of chromatin during this critical window of development, making it an important model for early chromatin regulation. Here, we review our current understanding of chromatin dynamics during early zebrafish development, highlighting new advances as well as similarities and differences between early chromatin regulation in zebrafish and other species.

The impact of light and temperature on chromatin organization and plant adaptation.

Light and temperature shape the developmental trajectory and morphology of plants. Changes in chromatin organization and nuclear architecture can modulate gene expression and lead to short- and long-term plant adaptation to the environment. Here, we review recent reports investigating how changes in chromatin composition, structure, and topology modulate gene expression in response to fluctuating light and temperature conditions resulting in developmental and physiological responses. Furthermore, the potential application of novel revolutionary techniques, such Hi-C, RNA fluorescence in situ hybridization (FISH) and padlock-FISH, to study the impact of environmental stimuli such as light and temperature on nuclear compartmentalization in plants is discussed.

Chromatin Landscaping At Mitotic Exit Orchestrates Genome Function.

Chromatin architecture is highly dynamic during different phases of cell cycle to accommodate DNA-based processes. This is particularly obvious during mitotic exit, where highly condensed rod-like chromatids need to be rapidly decondensed. Such chromatin structural transitions are tightly controlled and organized as any perturbance in this dynamic process can lead to genome dysfunction which may culminate in loss of cellular fitness. However, the mechanisms underlying cell cycle-dependent chromatin structural changes are not fully understood. In this mini review, we highlight our current knowledge of chromatin structural organization, focusing on mitotic exit. In this regard, we examine how nuclear processes are orchestrated during chromatin unfolding and compartmentalization and discuss the critical importance of cell cycle-controlled chromatin landscaping in maintaining genome integrity.

Sycp2 is essential for synaptonemal complex assembly, early meiotic recombination and homologous pairing in zebrafish spermatocytes.

Meiotic recombination is essential for faithful segregation of homologous chromosomes during gametogenesis. The progression of recombination is associated with dynamic changes in meiotic chromatin structures. However, whether Sycp2, a key structural component of meiotic chromatin, is required for the initiation of meiotic recombination is still unclear in vertebrates. Here, we describe that Sycp2 is required for assembly of the synaptonemal complex and early meiotic events in zebrafish spermatocytes. Our genetic screening by N-ethyl-N-nitrosourea mutagenesis revealed that ietsugu (its), a mutant zebrafish line with an aberrant splice site in the sycp2 gene, showed a defect during meiotic prophase I. The its mutation appeared to be a hypomorphic mutation compared to sycp2 knockout mutations generated by TALEN mutagenesis. Taking advantage of these sycp2 hypomorphic and knockout mutant lines, we demonstrated that Sycp2 is required for the assembly of the synaptonemal complex that is initiated in the vicinity of telomeres in wild-type zebrafish spermatocytes. Accordingly, homologous pairing, the foci of the meiotic recombinases Dmc1/Rad51 and RPA, and γH2AX signals were largely diminished in sycp2 knockout spermatocytes. Taken together, our data indicate that Sycp2 plays a critical role in not only the assembly of the synaptonemal complex, but also early meiotic recombination and homologous pairing, in vertebrates.

Chromatin regulation of somatic abiotic stress memory.

In nature, plants are often subjected to periods of recurrent environmental stress that can strongly affect their development and productivity. To cope with these conditions, plants can remember a previous stress, which allows them to respond more efficiently to a subsequent stress, a phenomenon known as priming. This ability can be maintained at the somatic level for a few days or weeks after the stress is perceived, suggesting that plants can store information of a past stress during this recovery phase. While the immediate responses to a single stress event have been extensively studied, knowledge on priming effects and how stress memory is stored is still scarce. At the molecular level, memory of a past condition often involves changes in chromatin structure and organization, which may be maintained independently from transcription. In this review, we will summarize the most recent developments in the field and discuss how different levels of chromatin regulation contribute to priming and plant abiotic stress memory.

Epidermal structure and differentiation.

In this Primer, Moreci and Lechler follow the lifetime of an epidermal cell from its birth to its ultimate death, and detail how this journey is necessary for epidermal function.