Structural Features Of Chromatin Research Articles

Epigenetic Modifications in Genome Help Remembering the Stress Tolerance Strategy Adopted by the Plant.

Genetic information in eukaryotic organisms is stored, replicated, transcribed, and inherited through the nucleus of a cell. Epigenetic modifications in the genetic material, including DNA methylation, histone modification, changes in non-coding RNA (ncRNA) biogenesis, and chromatin architecture play important roles in determining the genomic landscape and regulating gene expression. Genome architecture (structural features of chromatin, affected by epigenetic modifications) is a major driver of genomic functions/activities. Segregation of euchromatin (transcriptionally active) from heterochromatin (transcriptionally repressed chromosome) and positioning of genes in specific nuclear space in eukaryotic cells emphasise non-randomness in the organization of the genetic information. Not only does the base sequence of a gene carry the genetic information but the covalent modifications of bases, three-dimensional positioning of the genome, and chromatin loops are vital for switching on/off the gene and regulating its expression during growth/environmental stress. The epigenetic dynamics depend on the activities of writers and erasers under changing environmental conditions. The discovery of non-coding RNAs (one of the players in de novo methylation of DNA), increased DNA methylation protein (guide for the DNA demethylase), and methylation monitoring sequence (that helps keep a balance between DNA demethylation and methylation) have been some of the new developments in the era of epigenomics. To respond to environmental stimuli, plants depend on modulating gene expression through different mechanisms including biochemical, molecular, genetic, and epigenetic alterations. Studies on plants might provide better insights into epigenetic stress memory and molecular bases of adaptability to enable (epi)genome editing of crops for climate resilience and sustainable agriculture in the present era of multifaceted climate change.

Efficient Hi-C inversion facilitates chromatin folding mechanism discovery and structure prediction

Genome-wide chromosome conformation capture (Hi-C) experiments have revealed many structural features of chromatin across multiple length scales. Further understanding genome organization requires relating these discoveries to the mechanisms that establish chromatin structures and reconstructing these structures in three dimensions, but both objectives are difficult to achieve with existing algorithms that are often computationally expensive. To alleviate this challenge, we present an algorithm that efficiently converts Hi-C data into contact energies, which measure the interaction strength between genomic loci brought into proximity. Contact energies are local quantities unaffected by the topological constraints that correlate Hi-C contact probabilities. Thus, extracting contact energies from Hi-C contact probabilities distills the biologically unique information contained in the data. We show that contact energies reveal the location of chromatin loop anchors, support a phase separation mechanism for genome compartmentalization, and parameterize polymer simulations that predict three-dimensional chromatin structures. Therefore, we anticipate that contact energy extraction will unleash the full potential of Hi-C data and that our inversion algorithm will facilitate the widespread adoption of contact energy analysis.

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.

Four-Dimensional Chromosome Structure Prediction.

Chromatin conformation plays an important role in a variety of genomic processes, including genome replication, gene expression, and gene methylation. Hi-C data is frequently used to analyze structural features of chromatin, such as AB compartments, topologically associated domains, and 3D structural models. Recently, the genomics community has displayed growing interest in chromatin dynamics. Here, we present 4DMax, a novel method, which uses time-series Hi-C data to predict dynamic chromosome conformation. Using both synthetic data and real time-series Hi-C data from processes, such as induced pluripotent stem cell reprogramming and cardiomyocyte differentiation, we construct smooth four-dimensional models of individual chromosomes. These predicted 4D models effectively interpolate chromatin position across time, permitting prediction of unknown Hi-C contact maps at intermittent time points. Furthermore, 4DMax correctly recovers higher order features of chromatin, such as AB compartments and topologically associated domains, even at time points where Hi-C data is not made available to the algorithm. Contact map predictions made using 4DMax outperform naïve numerical interpolation in 87.7% of predictions on the induced pluripotent stem cell dataset. A/B compartment profiles derived from 4DMax interpolation showed higher similarity to ground truth than at least one profile generated from a neighboring time point in 100% of induced pluripotent stem cell experiments. Use of 4DMax may alleviate the cost of expensive Hi-C experiments by interpolating intermediary time points while also providing valuable visualization of dynamic chromatin changes.

Chromatin features constrain structural variation across evolutionary timescales

The potential impact of structural variants includes not only the duplication or deletion of coding sequences, but also the perturbation of noncoding DNA regulatory elements and structural chromatin features, including topological domains (TADs). Structural variants disrupting TAD boundaries have been implicated both in cancer and developmental disease; this likely occurs via "enhancer hijacking," whereby removal of the TAD boundary exposes enhancers to new target transcription start sites (TSSs). With this functional role, we hypothesized that boundaries would display evidence for negative selection. Here we demonstrate that the chromatin landscape constrains structural variation both within healthy humans and across primate evolution. In contrast, in patients with developmental delay, variants occur remarkably uniformly across genomic features, suggesting a potentially broad role for enhancer hijacking in human disease.

Computational methods for analyzing and modeling genome structure and organization.

Recent advances in chromosome conformation capture technologies have led to the discovery of previously unappreciated structural features of chromatin. Computational analysis has been critical in detecting these features and thereby helping to uncover the building blocks of genome architecture. Algorithms are being developed to integrate these architectural features to construct better three-dimensional (3D) models of the genome. These computational methods have revealed the importance of 3D genome organization to essential biological processes. In this article, we review the state of the art in analytic and modeling techniques with a focus on their application to answering various biological questions related to chromatin structure. We summarize the limitations of these computational techniques and suggest future directions, including the importance of incorporating multiple sources of experimental data in building a more comprehensive model of the genome. This article is categorized under: Analytical and Computational Methods > Computational Methods Laboratory Methods and Technologies > Genetic/Genomic Methods Models of Systems Properties and Processes > Mechanistic Models.

The influence of sample preprocessing on in situ identification of 5-methylcytosine in metaphase chromosomes and interphase nuclei

Qualitative and quantitative analysis of DNA methylation in situ at the level of cells, chromosomes, and the chromosomal domain is extremely important in diagnosis and treatment of various pathologies, as well in studies of aging and the effects of environmental factors. Yet, the questions remain unresolved of whether the detectable in situ methylation patterns correspond to the actual DNA methylation per se and/or reflect the accessibility of DNA to antibodies, which depends on the structural features of chromatin and chromosome condensation. Thus, this phenomenon can result in an incorrect determination of the real DNA methylation pattern. In order to eliminate this disadvantage to the extent possible, we modified the commonly used methodology of in situ detection methylcytosine by means of monoclonal antibodies. In this study, we show that the efficiency of immunofluorescent labeling for 5-methylcytosin in centromeric heterochromatin, chromosome arms and sister chromatids is significantly affected by the conditions of pretreatment of chromosome preparations. We used undifferentiated murine embryonic F9 cells to show that variations in the conditions of storage of chromosome preparations can lead to a sharp reduction of labeling intensity and even disappearance of the fluorescence signal in centromeric heterochromatin. Using the developed method, we discovered asymmetric methylation of sister chromatids in F9 cells and in human peripheral blood lymphocytes. This phenomena can lead to asymmetric cell division and asymmetric transcriptional status in daughter cells. Thus, the modified methodology for detection of 5-methyl cytosine in situ can provide for a more precise assessment of methylation of chromosomes and chromosomal regions.

Granulocyte maturation determines ability to release chromatin NETs and loss of DNA damage response; these properties are absent in immature AML granulocytes

Terminally-differentiated cells cease to proliferate and acquire specific sets of expressed genes and functions distinguishing them from less differentiated and cancer cells. Mature granulocytes show lobular structure of cell nuclei with highly condensed chromatin in which HP1 proteins are replaced by MNEI. These structural features of chromatin correspond to low level of gene expression and the loss of some important functions as DNA damage repair, shown in this work and, on the other hand, acquisition of a new specific function consisting in the release of chromatin extracellular traps in response to infection by pathogenic microbes. Granulocytic differentiation is incomplete in myeloid leukemia and is manifested by persistence of lower levels of HP1γ and HP1β isoforms. This immaturity is accompanied by acquisition of DDR capacity allowing to these incompletely differentiated multi-lobed neutrophils of AML patients to respond to induction of DSB by γ-irradiation. Immature granulocytes persist frequently in blood of treated AML patients in remission. These granulocytes contrary to mature ones do not release chromatin for NETs after activation with phorbol myristate-12 acetate-13 and do not exert the neutrophil function in immune defence. We suggest therefore the detection of HP1 expression in granulocytes of AML patients as a very sensitive indicator of their maturation and functionality after the treatment. Our results show that the changes in chromatin structure underlie a major transition in functioning of the genome in immature granulocytes. They show further that leukemia stem cells can differentiate ex vivo to mature granulocytes despite carrying the translocation BCR/ABL.

The ATAC acetyl transferase complex controls mitotic progression by targeting non-histone substrates

All DNA-related processes rely on the degree of chromatin compaction. The highest level of chromatin condensation accompanies transition to mitosis, central for cell cycle progression. Covalent modifications of histones, mainly deacetylation, have been implicated in this transition, which also involves transcriptional repression. Here, we show that the Gcn5-containing histone acetyl transferase complex, Ada Two A containing (ATAC), controls mitotic progression through the regulation of the activity of non-histone targets. RNAi for the ATAC subunits Ada2a/Ada3 results in delayed M/G1 transition and pronounced cell division defects such as centrosome multiplication, defective spindle and midbody formation, generation of binucleated cells and hyperacetylation of histone H4K16 and alpha-tubulin. We show that ATAC localizes to the mitotic spindle and controls cell cycle progression through direct acetylation of Cyclin A/Cdk2. Our data describes a new pathway in which the ATAC complex controls Cyclin A/Cdk2 mitotic function: ATAC/Gcn5-mediated acetylation targets Cyclin A for degradation, which in turn regulates the SIRT2 deacetylase activity. Thus, we have uncovered an essential function for ATAC in regulating Cyclin A activity and consequent mitotic progression.

DNA breakage detection-FISH (DBD-FISH) in human spermatozoa: technical variants evidence different structural features

Non-irradiated and X-irradiated (80 Gy) human spermatozoa were processed for in situ DNA breakage detection-FISH (DBD-FISH) of the whole genome, following two alternative variations of the basic technique. In the first, cells were initially incubated in the alkaline unwinding solution for transformation of DNA breaks into single-stranded DNA (ssDNA) to be hybridized, followed by the lysing solutions for protein removal. In the second, incubation in the lysing solutions was carried out before the denaturation step. The first approach yielded two subpopulations. While most sperm nuclei were faintly labeled and had chromocenters, a small subpopulation was strongly and homogeneously labeled, due to extensive DNA breakage. X-ray exposure increased the surface and mean fluorescence intensity. Otherwise, when the denaturation step was performed after protein extraction, all sperm nuclei yielded strong and dispersed FISH signals. Protein removal allows access of the unwinding solution to the DNA, which has abundant alkali-labile sites, and thus gives rise to large areas of ssDNA that are labeled by FISH. X-ray exposure increased the dispersion of FISH signals but decreased their mean fluorescence intensity. A linear dose-response was generated using the second experimental variant, being 30 Gy the lowest dose for detecting induction of damage by X-rays in mature sperm chromatin. These results indicate that DBD-FISH is not only useful for in situ detection of DNA breakage but also for revealing structural features of chromatin.

Multiple Sp1 sites efficiently drive transcription of the TATA-less promoter of the human glypican 3 (GPC3) gene.

Simpson-Golabi-Behmel Syndrome (SGBS) is an X-linked disease characterized by pre- and postnatal overgrowth. Recently, we have shown that mutations in the glypican family gene, GPC3, cause SGBS. This gene is predominantly expressed in the same mesoderm-derived tissues that overgrow in its absence. To investigate the basis for promoter function, 3.3kb of GC-rich DNA 5' of the transcribed region were fused to a luciferase cDNA, transfected into Caco-2 and NT2 cells, and assayed for activity. Deletion analysis identified a 218-bp fragment upstream of the transcription start site that conferred more than 80% of maximal reporter gene activation. This fragment contains five putative Sp1 binding sites, three of which (centered at nt -14, -34, and -92) were active when assessed by DNaseI footprinting and gel shift/supershift assays. Additionally, Sp1 specifically transactivated transcription in Sp1-deficient Drosophila SL2 cells, demonstrating the functionality of Sp1 on the GPC3 promoter. A full-length promoter construct was also highly active in HeLa cells, which do not express endogenous GPC3. These results indicate that the GPC3 promoter is dependent on Sp1 for proper activation, but tissue-specific repression in non-expressing cells must involve either DNA that lies outside the region tested or auxiliary structural features of chromatin.

Theoretical mechanism for synthesis of carcinogen induced embryonic proteins: VIII transcriptional theory

In this paper an extension of prior writings on the mechanism by which changes in genic expression is presented. The previous development of these ideas led to a unifying concept of alterations in heterochromatin due to a variety of carcinogenic agents as a pivital process leading to potential re-expressions of genes (1). The following deals just with structural features of chromatin before continuing to finer control levels of gene expression, especially in reference to cancer cells. The DNA methylation status is presented as being an important step in an intermediate stage of chromatin expression along with protein acetylation, phosphorylation and methylation as other means of modifying chromatin status.

Deoxyribonuclease II as a probe for chromatin structure: II. Mode of cleavage

In the analysis of DNAase II digestion of chromatin, as described in the preceding paper, interactions between adjacent nucleosomes play an important part. In order to understand the mechanism of DNAase II cleavage we next investigated the role of histone H1 in these interactions and characterized the nucleoprotein particles arising in the course of DNAase II action. H1-free chromatin prepared by three different procedures, using either 0.6 m-NaCl, transfer RNA or an ion-exchange resin, can be cleaved by DNAase II only at the internucleosomal cleavage site leading to 200-bp † † Abbreviations used: bp, base-pairs; PMSF, phenylmethanesulfonyl fluoride; EGTA, ethyleneglycol bis(β-aminoethyl ether)- N,N′-tetraacetic acid. digestion patterns regardless of the ionic conditions. When H1 was added back to the three chromatin preparations the 100-bp cleavage pattern could be restored only with material prepared by the resin method at low concentrations of salt. Addition of polylysine instead of H1 has the same effect, but only with material prepared by that method. A direct correlation between extended and condensed states of chromatin as monitored by electron microscopy and DNAase II cleavage in the 200 and 100-bp modes, respectively, could be established. The continuity of the nucleosome chains in DNAase II-digested chromatin is maintained in spite of intranucleosomal cleavage in the terminal section of the core DNA, even in the absence of H1. Addition of 3 m-urea, however, disrupts the nucleosome chains at the intranucleosomal cleavage sites and leads to the formation of novel nucleoprotein particles as seen in sucrose gradient centrifugations. Those sedimenting between mononucleosomes and dinucleosomes contain, almost exclusively, DNA of 300 bp (mouse) or 315 bp (chicken erythrocyte). They can be formed from particles sedimenting in the absence of urea in the dinucleosome region by either a dissociation process or a massive conformational change. On the basis of the results presented here and in the preceding paper a mechanism for DNAase II cleavage of chromatin in the 200-bp and 100-bp modes is proposed and discussed in the context of structural features of chromatin recognized by DNAase II.