High Chromatin Density Research Articles

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.

A global high-density chromatin interaction network reveals functional long-range and trans-chromosomal relationships

BackgroundChromatin contacts are essential for gene-expression regulation; however, obtaining a high-resolution genome-wide chromatin contact map is still prohibitively expensive owing to large genome sizes and the quadratic scale of pairwise data. Chromosome conformation capture (3C)-based methods such as Hi-C have been extensively used to obtain chromatin contacts. However, since the sparsity of these maps increases with an increase in genomic distance between contacts, long-range or trans-chromatin contacts are especially challenging to sample.ResultsHere, we create a high-density reference genome-wide chromatin contact map using a meta-analytic approach. We integrate 3600 human, 6700 mouse, and 500 fly Hi-C experiments to create species-specific meta-Hi-C chromatin contact maps with 304 billion, 193 billion, and 19 billion contacts in respective species. We validate that meta-Hi-C contact maps are uniquely powered to capture functional chromatin contacts in both cis and trans. We find that while individual dataset Hi-C networks are largely unable to predict any long-range coexpression (median 0.54 AUC), meta-Hi-C networks perform comparably in both cis and trans (0.65 AUC vs 0.64 AUC). Similarly, for long-range expression quantitative trait loci (eQTL), meta-Hi-C contacts outperform all individual Hi-C experiments, providing an improvement over the conventionally used linear genomic distance-based association. Assessing between species, we find patterns of chromatin contact conservation in both cis and trans and strong associations with coexpression even in species for which Hi-C data is lacking.ConclusionsWe have generated an integrated chromatin interaction network which complements a large number of methodological and analytic approaches focused on improved specificity or interpretation. This high-depth “super-experiment” is surprisingly powerful in capturing long-range functional relationships of chromatin interactions, which are now able to predict coexpression, eQTLs, and cross-species relationships. The meta-Hi-C networks are available at https://labshare.cshl.edu/shares/gillislab/resource/HiC/.

Chromatin Compaction Leads to a Preference for Peripheral Heterochromatin

A layer of dense heterochromatin is found at the periphery of the nucleus. Because this peripheral heterochromatin functions as a repressive phase, mechanisms that relocate genes to the periphery play an important role in regulating transcription. Using Monte Carlo simulations, we show that an interaction that attracts euchromatin and heterochromatin equally to the nuclear envelope will still preferentially locate heterochromatin to the nuclear periphery. This observation considerably broadens the class of possible interactions that result in peripheral positioning to include boundary interactions that either weakly attract all chromatin or strongly bind to a randomly chosen 0.05% of nucleosomes. The key distinguishing feature of heterochromatin is its high chromatin density with respect to euchromatin. In our model, this densification is caused by heterochromatin protein 1’s preferential binding to histone H3 tails with a methylated lysine at the ninth residue, a hallmark of heterochromatin. We find that a global rearrangement of chromatin to place heterochromatin at the nuclear periphery can be accomplished by attaching a small subset of loci, even if these loci are uncorrelated with heterochromatin. Hence, factors that densify chromatin determine which genomic regions condense to form peripheral heterochromatin.

Nucleosome Crowding in Chromatin Slows the Diffusion but Can Promote Target Search of Proteins

Dynamics of nuclear proteins in crowded chromatin has only been poorly understood. Here, we address the diffusion, target search, and structural dynamics of three proteins in a model chromatin using coarse-grained molecular simulations run on the K computer. We prepared two structures of chromatin made of 20 nucleosomes with different nucleosome densities and investigated dynamics of two transcription factors, HMGB1 and p53, and one signaling protein, ERK, embedded in the chromatin. We found fast and normal diffusion of the nuclear proteins in the low-density chromatins and slow and subdiffusional movements in the high-density chromatin. The diffusion of the largest transcription factor, p53, is slowed by high-density chromatin most markedly. The on rates and off rates for DNA binding are increased and decreased, respectively, in the high-density chromatin. To our surprise, the DNA sequence search was faster in chromatin with high nucleosome density, though the diffusion is slower. We also found that the three nuclear proteins preferred to bind on the linker DNA and the entry and exit regions of nucleosomal DNA. In addition to these regions, HMGB1 and p53 also bound to the dyad.

Bioinformatics analysis of Alu components at the level of genome 3D structure

The interphase chromatin is folded in the nucleus in a hierarchical manner, including the nucleosome, the "beads on a string" structure composed of nucleosomes, the solenoid fiber structure, the chromatin/DNA loop structure (chromatin/DNA loop), and the topologically associated domain (TAD). Among them, TAD is considered to be the basic unit of the 3D structure of chromatin because it is relatively stable and conserved in different cell types. Alu elements occupy a large proportion in the mammalian genomes. There are a wide variety of Alu elements, but their functional characterizations are limited to date. This study investigates the role of Alu elements in the assembly of 3D chromatin conformation. The evolutionary process of the Alu subfamily was explored by the distance relationship of the 3D structure of chromatin. We found that the proportion of Alu elements in high-density chromatin interaction increased with higher similarity, indicating that Alu plays an important role in the construction of chromatin 3D structure. There is a certain positive correlation between the strength of the upper interaction and the evolutionary relationship. In sum, the Alu elements with relatively close distances in the 1D sequence will also be close to each other in the 3D structure of chromatin.

Visualization of chromosome condensation in plants with large chromosomes

BackgroundMost data concerning chromosome organization have been acquired from studies of a small number of model organisms, the majority of which are mammals. In plants with large genomes, the chromosomes are significantly larger than the animal chromosomes that have been studied to date, and it is possible that chromosome condensation in such plants was modified during evolution. Here, we analyzed chromosome condensation and decondensation processes in order to find structural mechanisms that allowed for an increase in chromosome size.ResultsWe found that anaphase and telophase chromosomes of plants with large chromosomes (average 2C DNA content exceeded 0.8 pg per chromosome) contained chromatin-free cavities in their axial regions in contrast to well-characterized animal chromosomes, which have high chromatin density in the axial regions. Similar to animal chromosomes, two intermediates of chromatin folding were visible inside condensing (during prophase) and decondensing (during telophase) chromosomes of Nigella damascena: approximately 150 nm chromonemata and approximately 300 nm fibers. The spatial folding of the latter fibers occurs in a fundamentally different way than in animal chromosomes, which leads to the formation of chromosomes with axial chromatin-free cavities.ConclusionDifferent compaction topology, but not the number of compaction levels, allowed for the evolution of increased chromosome size in plants.

The Influence of Spatial Variation in Chromatin Density Determined by X-Ray Tomograms on the Time to Find DNA Binding Sites

In this work, we examine how volume exclusion caused by regions of high chromatin density might influence the time required for proteins to find specific DNA binding sites. The spatial variation of chromatin density within mouse olfactory sensory neurons is determined from soft X-ray tomography reconstructions of five nuclei. We show that there is a division of the nuclear space into regions of low-density euchromatin and high-density heterochromatin. Volume exclusion experienced by a diffusing protein caused by this varying density of chromatin is modeled by a repulsive potential. The value of the potential at a given point in space is chosen to be proportional to the density of chromatin at that location. The constant of proportionality, called the volume exclusivity, provides a model parameter that determines the strength of volume exclusion. Numerical simulations demonstrate that the mean time for a protein to locate a binding site localized in euchromatin is minimized for a finite, nonzero volume exclusivity. For binding sites in heterochromatin, the mean time is minimized when the volume exclusivity is zero (the protein experiences no volume exclusion). An analytical theory is developed to explain these results. The theory suggests that for binding sites in euchromatin there is an optimal level of volume exclusivity that balances a reduction in the volume searched in finding the binding site, with the height of effective potential barriers the protein must cross during the search process.

The Impact of Mitotic versus Interphase Chromatin Architecture on the Molecular Flow of EGFP by Pair Correlation Analysis

Here we address the impact nuclear architecture has on molecular flow within the mitotic nucleus of live cells as compared to interphase by the pair correlation function method. The mitotic chromatin is found to allow delayed but continuous molecular flow of EGFP in and out of a high chromatin density region, which, by pair correlation function analysis, is shown as a characteristic arc shape that appears upon entry and exit. This is in contrast to interphase chromatin, which regulates flow between different density chromatin regions by means of a mechanism which turns on and off intermittently, generating discrete bursts of EGFP. We show that the interphase bursts are maintained by metabolic energy, whereas the mitotic mechanism of regulation responsible for the arc is not sensitive to ATP depletion. These two distinct routes of molecular flow were concomitantly measured in the Caenorhabditis elegans germ line, which indicates a conservation of mechanism on a scale more widespread than cell type or organism.

The influence of volume exclusion by chromatin on the time required to find specific DNA binding sites by diffusion

Within the nuclei of eukaryotic cells, the density of chromatin is nonuniform. We study the influence of this nonuniform density, which we derive from microscopic images [Schermelleh L, et al. (2008) Science 320:1332-1336], on the diffusion of proteins within the nucleus, under the hypothesis that chromatin density is proportional to an effective potential that tends to exclude the diffusing protein from regions of high chromatin density. The constant of proportionality, which we call the volume exclusivity of chromatin, is a model parameter that we can tune to study the influence of such volume exclusivity on the random time required for a diffusing particle to find its target. We consider randomly chosen binding sites located in regions of low (20th-30th percentile) chromatin density, and we compute the median time to find such a binding site by a protein that enters the nucleus at a randomly chosen nuclear pore. As the volume exclusivity of chromatin increases from zero, we find that the median time needed to reach the target binding site at first decreases to a minimum, and then increases again as the volume exclusivity of chromatin increases further. Random permutation of the voxel values of chromatin density abolishes the minimum, thus demonstrating that the speedup seen with increasing volume exclusivity at low to moderate volume exclusivity is dependent upon the spatial structure of chromatin within the nucleus.

Description of Meiosis in Female and Male Pacific White Shrimp Litopenaeus vannamei (Decapoda: Penaeidae)

Abstract The first meiotic prophase was analyzed in both genders of Pacific white shrimp Litopenaeus vannamei. Progression through meiosis, from the formation of the synaptonemal complex to diplotene is described during molting stages. Most of the meiotic cells in both genders were observed at pachytene, recognized by complete synapsis of bivalents. In both genders, the zygotene, pachytene, and diplotene stages occurred at inter-molt, pre-molt, and post-molt stages in all individuals, which suggests that there is no relationship between molting and the first meiotic prophase. Sperm counts from the vas deferens and spermatophores support a continuous production of male gametes. The nature of the synaptonemal complex in gonad cells shows that each pair of homologous chromosomes synapses end to end, revealing a high chromatin density and a complex tangled arrangement in the nucleus. Both genders have 44 bivalents, of which around 40 have an o-ring configuration, and the rest have a v-shape, meaning that most...

Signatures of polycomb repression and reduced H3K4 trimethylation are associated with p15INK4b DNA methylation in AML

AbstractDNA hypermethylation of the p15INK4b tumor suppressor gene is commonly observed in acute myeloid leukemia (AML). Repressive histone modifications and their associated binding proteins have been implicated in the regulation of DNA methylation and the transcriptional repression of genes with DNA methylation. We have used high-density chromatin immunoprecipitation-on-chip to determine the histone modifications that normally regulate p15INK4b expression in AML cells and how these marks are altered in cells that have p15INK4b DNA methylation. In AML patient blasts without p15INK4b DNA methylation, a bivalent pattern of active (H3K4me3) and repressive (H3K27me3) modifications exist at the p15INK4b promoter. AML patient blasts with p15INK4b DNA methylation lose H3K4me3 at p15INK4b and become exclusively marked by H3K27me3. H3K27me3, as well as EZH2, extends throughout p14ARF and p16INK4a, indicating that polycomb repression of p15INK4b is a common feature in all AML blasts irrespective of the DNA methylation status of the gene. Reactivation of p15INK4b expression in AML cell lines and patient blasts using 5-aza-2′-deoxycytidine (decitabine) and trichostatin A increased H3K4me3 and maintained H3K27me3 enrichment at p15INK4b. These data indicate that AML cells with p15INK4b DNA methylation have an altered histone methylation pattern compared with unmethylated samples and that these changes are reversible by epigenetic drugs.

Effect of hydrostatic pressure on ultrastructure of synovial fibroblasts from rat temporomandibular joint

To investigate the role of apoptosis in the remodeling of temporomandibular joint (TMJ) under pressure. Synovial fibroblasts obtained from rat temporomandibular joint were subjected to different hydrostatic pressure for 12 h. Changes of ultrastructure were observed by transmission electron microscope. At 30 kPa, the ultrastructure of synovial fibroblasts showed no obvious changes. At 60 kPa, the chromatin was condensated, the baryon took on crescent and the mitochondria seemed varicose. At 90 kPa, the apoptosis-like body was wrapped by membrane and embedded in the high density chromatin. Apoptosis-like change took place in ultrastructure of synovial fibroblasts under hydrostatic pressure, and the degree of the change was related to the hydrostatic pressure exerted.

Non-random distribution of high density chromatin detected at opposite ends of T-banded human metaphase chromosomes

Previous research using microdensitometric scanning and computer graphic image analysis showed that T-banded segments of human metaphase chromosomes usually exhibit an asymmetrical distribution of high density (HD) chromatin between sister chromatids. Here, we employed the same methods to analyze HD chromatin distribution at opposite ends of T-banded human lymphocyte chromosomes. This study revealed that in most chromosomes with an asymmetrical distribution of HD chromatin at both ends, the highest densities of each arm were located in opposite chromatids. The frequency of this configuration was 0.792 per chromosome, indicating that the highest chromatin densities of the terminal segments of T-banded human chromosomes were non-randomly distributed at opposite chromosome arms. The possible relationship of this observation to the mode of replication of the terminal chromosome region is briefly discussed.

Arachidonic acid hydroperoxide stimulates lipid peroxidation in rat liver nuclei and chromatin fractions

Arachidonic acid, the most abundant polyunsaturated fatty acid in rat liver nuclei phospholipids is a major target of free radical attack, which induces lipid peroxidation. The non-enzymatic lipid peroxidation process in intact rat liver nuclei and in several chromatin fractions indicated that the most sensitive fatty acid for peroxidation is arachidonic acid C20:4 n-6. In this study, the effect of different amounts of arachidonic acid hydroperoxide on the lipid peroxidation of rat liver nuclei and chromatin fractions was studied; rat liver nuclei and chromatin fractions deprived of exogenous added hydroperoxide were utilized as control. The addition of arachidonic acid hydroperoxide to liver nuclei produces a marked increase in light emission that was hydroperoxide concentration dependent. The maximal peak of chemiluminescence displayed by the different chromatin fractions analyzed was observed between 20 and 80 min of incubation. The highest value of light emission was displayed by the high-density chromatin fractions, the 27.5 K fraction showed intermediate values of light emission, whereas the lowest density fraction produced very low chemiluminescence. A high correlation between arachidonic acid hydroperoxide concentration and chemiluminescence in the different chromatin fractions was observed.

Ultrastructural morphology and localisation of cisplatin-induced platinum–DNA adducts in a cisplatin-sensitive and -resistant human small cell lung cancer cell line using electron microscopy

Ultrastructural morphology (transmission electron microscopy) and localisation of cisplatin-induced platinum (Pt)–DNA adducts (immunoelectron microscopy) were analysed in the human small cell lung cancer cell line GLC 4 and its 40-fold in vitro acquired cisplatin-resistant subline GLC 4-CDDP, which is characterised by, among other things, a decreased DNA platination. Immunolabelling of Pt–DNA adducts was performed with the polyclonal antibody GPt, known to detect the main Pt-containing intrastrand and interstrand DNA adducts. Morphological analysis of GLC 4 and GLC 4-CDDP at the ultrastructural level showed cells with a high nucleus/cytoplasm ratio with the majority of nuclei containing one or more nucleoli. GLC 4-CDDP showed, in contrast to GLC 4, an extensive Golgi apparatus and an increased number of mitochondria. DNA platination was detectable in both GLC 4 and GLC 4-CDDP. Immunoelectron microscopy showed Pt–DNA adducts primarily in the nucleus, preferentially at loci with high-density chromatin (e.g. heterochromatin, pars granulosa around nucleoli, condensed DNA in proliferating and apoptotic cells), and in mitochondria. The level of detectable Pt–DNA adducts was cell cycle status-dependent. In both cell lines, Pt–DNA adduct levels increased from non-dividing interphase cells to dividing cells and were highest in cells undergoing apoptosis. Overall localisation of Pt–DNA adducts was comparable in GLC 4 and GLC 4-CDDP cells.

Zur histologischen Gradeinteilung der Chondrosarkome Eine qualitative und quantitative Analyse an 74 F�llen des Hamburger Knochentumorregisters

Chondrosarcomas are frequent malignant bone tumors. Aside from different subtypes, such as dedifferentiated, mesenchymal and clear-cell chondrosarcoma, chondrosarcomas (classical chondrosarcoma) show different grades of differentiation. The borderline between chondroma and classical chondrosarcoma is not clearly defined. The same chondrosarcoma can be graded differently at different institutes. Standardized therapy concepts are currently in preparation. As the Hamburg Bone Tumor Registry is often consulted for chondrogenic tumors, the histological criteria are based on a series of 74 chondrosarcomas recorded there. The emphasis has been laid on a classification which can be used in daily routine and which is reproducible and in agreement with the classifications of other international groups. Grade I chondrosarcomas (50%) can be distinguished only by growth criteria. The nuclei are small and show high chromatin density. Grade II chondrosarcomas (42%) have medium-sized, regular nuclei with loose chromatin structure. The chondrocytes of grade III cases (8%) show polymorphic nuclei. Binucleas forms, the number of mitoses and cellularity all show considerable overlap for all three grades. So far there are no immunohistological and molecular biological methods for reliable differentiation. The therapeutic consequences of the classification into grades are thorough curettage, in the case of grade I tumors, or complete resection, for grade II and III cases. The long-term results, however, need to be confirmed by a larger number of cases. From 1991 to 1995 the method was applied and proved to be easily practicable in daily diagnostic routine. Some 104 cases of classical chondrosarcomas (grade I 53%, grade II 39%, grade III 8%) were analyzed. Two pathologists both assigned the same grade in 90% of cases.

Age-related chromatin condensation in flight muscle nuclei of the adult male housefly, Musca domestica

Computer analysis of indirect flight muscle nuclei from the adult male housefly, M. domestica, has shown significant change with age in the chromatin condensation pattern. The pattern was analyzed by examining low, medium, and high density chromatin components (LDC, MDC, HDC). No significant change occurred in HDC with age, and the amount and distribution of LDC and MDC remain relatively stable between Day 1 and Day 4 post-eclosion. However, the analysis showed a significant increase in the amount of MDC with a corresponding decrease in the amount of LDC between Day 4 and Day 14. This exchange was accompanied by a significant redistribution of both components. These results are discussed with reference to the biochemical and ultrastructural profile of the flight muscle with age, and to age-related changes in the condensation pattern of specific brain and Malpighian tubule nuclei described earlier.

Texture analysis of cervical cell nuclei by segmentation of chromatin patterns.

Texture parameters of the nuclear chromatin pattern can contribute to the automated classification of specimens on the basis of single cell analysis in cervical cytology. Current texture parameters are abstract and therefore hamper understanding. In this paper texture parameters are described that can be derived from the chromatin pattern after segmentation of the nuclear image. These texture parameters are more directly related to the visual properties of the chromatin pattern. The image segmentation procedure is based on a region grow algorithm which specifically isolates high chromatin density. The texture analysis method has been tested on a data set of images of 112 cervical nuclei on photographic negatives digitized with a step size of 0.125 micron. The preliminary results of a classification trial indicate that these visually interpretable parameters have promising discriminatory power for the distinction between negative and positive specimens.