Atomic force microscope imaging of chromatin assembled in <Emphasis Type="Italic">Xenopus laevis</Emphasis> egg extract

Gaps persist in our understanding of chromatin lower- and higher-order structures. Xenopus egg extracts provide a way to study essential chromatin components which are difficult to manipulate in living cells, but nanoscale imaging of chromatin assembled in extracts poses a challenge. We describe a method for preparing chromatin assembled in extracts for atomic force microscopy (AFM) utilizing restriction enzyme digestion followed by transferring to a mica surface. Using this method, we find that buffer dilution of the chromatin assembly extract or incubation of chromatin in solutions of low ionic strength results in loosely compacted chromatin fibers that are prone to unraveling into naked DNA. We also describe a method for direct AFM imaging of chromatin which does not utilize restriction enzymes and reveals higher-order fibers of varying widths. Due to the capability of controlling chromatin assembly conditions, we believe these methods have broad potential for studying physiologically relevant chromatin structures.     

Spatial confinement is a major determinant of the folding landscape of human chromosomes

The global architecture of the cell nucleus and the spatial organization of chromatin play important roles in gene expression and nuclear function. Single-cell imaging and chromosome conformation capture-based techniques provide a wealth of information on the spatial organization of chromosomes. However, a mechanistic model that can account for all observed scaling behaviors governing long-range chromatin interactions is missing. Here we describe a model called constrained self-avoiding chromatin (C-SAC) for studying spatial structures of chromosomes, as the available space is a key determinant of chromosome folding. We studied large ensembles of model chromatin chains with appropriate fiber diameter, persistence length and excluded volume under spatial confinement. We show that the equilibrium ensemble of randomly folded chromosomes in the confined nuclear volume gives rise to the experimentally observed higher-order architecture of human chromosomes, including average scaling properties of mean-square spatial distance, end-to-end distance, contact probability and their chromosome-to-chromosome variabilities. Our results indicate that the overall structure of a human chromosome is dictated by the spatial confinement of the nuclear space, which may undergo significant tissue- and developmental stage-specific size changes.     

Archaea: The Final Frontier of Chromatin

The three domains of life employ various strategies to organize their genomes. Archaea utilize features similar to those found in both eukaryotic and bacterial chromatin to organize their DNA. In this review, we discuss the current state of research regarding the structure–function relationships of several archaeal chromatin proteins (histones, Alba, Cren7, and Sul7d). We address individual structures as well as inferred models for higher-order chromatin formation. Each protein introduces a unique phenotype to chromatin organization, and these structures are put into the context of in vivo and in vitro data. We close by discussing the present gaps in knowledge that are preventing further studies of the organization of archaeal chromatin, on both the organismal and domain level.     

Induction of chromatin condensation by nuclear expression of a novel arginine-rich cationic protein genetically engineered from the enhanced green fluorescent protein

In the interphase nuclei of cultured cells, chromatin is compacted and organized in higher-order structures through the condensation and decondensation processes. Chromosomes in the interphase nucleus are known to occupy distinct territories. The chromosome territory-interchromatin compartment model premises that the interchromatin compartment is separated from compact higher-order chromatin domains and expands in between these chromatin-organized territories. Chromatin in cultured cells is compacted under some conditions, such as the stress of heat shock and high osmolarity, and Src-mediated nuclear tyrosine phosphorylation. We report here that a novel arginine-rich cationic protein is generated by frameshift mutation of enhanced green fluorescent protein (EGFP). The arginine-rich cationic protein is highly hydrophilic and contains potential arginine-based nuclear localization signals. Expression of the arginine-rich cationic protein shows its predominant localization to the nucleus and induces striking chromatin condensation in the interphase, which might be involved in interchromatin spacing or euchromatinization. Thus, the arginine-rich cationic protein as a new tool would be useful for dissecting chromatin architecture dynamics.     

Hho1p,the linker histone of Saccharomyces cerevisiae, is important for the proper chromatin organization in vivo

Despite the existence of certain differences between yeast and higher eukaryotic cells a considerable part of our knowledge on chromatin structure and function has been obtained by experimenting on Saccharomyces cerevisiae. One of the peculiarities of S. cerevisiae cells is the unusual and less abundant linker histone, Hho1p. Sparse is the information about Hho1p involvement in yeast higher-order chromatin organization. In an attempt to search for possible effects of Hho1p on the global organization of chromatin, we have applied Chromatin Comet Assay (ChCA) on HHO1 knock-out yeast cells. The results showed that the mutant cells exhibited highly distorted higher-order chromatin organization. Characteristically, linker histone depleted chromatin generally exhibited longer chromatin loops than the wild-type. According to the Atomic force microscopy data the wild-type chromatin appeared well organized in structures resembling quite a lot the “30-nm” fiber in contrast to HHO1 knock-out yeast.     

Balamuthia mandrillaris: The multiple nuclei of Balamuthia amebas; their location, activity, and site of development

Multiple nuclei were first noted in the pseudopodia of Balamuthia mandrillaris amebas feeding on mammalian cells. Phase microscope observations of live amebas in vitro reveal that while many amebas have a single nucleus, others have multiple nuclear-like structures, now confirmed as nuclei with hematoxylin and Feulgen stains. In the live cultures, two nuclei located near the tip of an extended pseudopodium were seen to fuse resulting in one larger morphologic unit. Such merging of nuclei has not been previously reported. Other nuclei were located at positions that subsequently became the site for the outgrowth of an additional pseudopod branch. A newly discovered large structure, a polyploid nucleus, was located in the mid-part of the ameba. Nucleoli of uniform size were seen to develop from the central mass of chromatin and each became surrounded by a vesicular component as they moved into the protoplasm as morphologically complete nuclei.     

Distinct polymer physics principles govern chromatin dynamics in mouse and Drosophila topological domains

Background

In higher eukaryotes, the genome is partitioned into large "Topologically Associating Domains" (TADs) in which the chromatin displays favoured long-range contacts. While a crumpled/fractal globule organization has received experimental supports at higher-order levels, the organization principles that govern chromatin dynamics within these TADs remain unclear. Using simple polymer models, we previously showed that, in mouse liver cells, gene-rich domains tend to adopt a statistical helix shape when no significant locus-specific interaction takes place.

Results

Here, we use data from diverse 3C-derived methods to explore chromatin dynamics within mouse and Drosophila TADs. In mouse Embryonic Stem Cells (mESC), that possess large TADs (median size of 840 kb), we show that the statistical helix model, but not globule models, is relevant not only in gene-rich TADs, but also in gene-poor and gene-desert TADs. Interestingly, this statistical helix organization is considerably relaxed in mESC compared to liver cells, indicating that the impact of the constraints responsible for this organization is weaker in pluripotent cells. Finally, depletion of histone H1 in mESC alters local chromatin flexibility but not the statistical helix organization. In Drosophila, which possesses TADs of smaller sizes (median size of 70 kb), we show that, while chromatin compaction and flexibility are finely tuned according to the epigenetic landscape, chromatin dynamics within TADs is generally compatible with an unconstrained polymer configuration.

Conclusions

Models issued from polymer physics can accurately describe the organization principles governing chromatin dynamics in both mouse and Drosophila TADs. However, constraints applied on this dynamics within mammalian TADs have a peculiar impact resulting in a statistical helix organization.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1786-8) contains supplementary material, which is available to authorized users.     

Changes of nuclear structure induced by increasing temperatures

Despite the recent improvement in understanding the higher-order structure of chromatin fibers, the organization of interphase chromosomes in specific nuclear domains emerged only recently and it is still controversial. This study took advantage of an integrated approach using complementary techniques in order to investigate the structure and organization of chromatin in interphase nucleus. Native CHO-K1 cells were progressively heated from 310 K to 410 K and the effects of increasing temperatures on nuclear chromatin were analyzed in situ by means of cytometric and calorimetric techniques. Distribution and organization of chromatin domains were analyzed by Fluorescence microscopy, while the mean condensation of nuclear chromatin was measured by Differential scanning calorimetry. The results show as changes of nuclear structures (envelope and matrix, namely) affect significantly organization and condensation of in situ chromatin. Moreover when volume is modified by an external force (the temperature gradient in our case) we observe significant alterations of chromatin structure. These data are in accordance with the hypothesis of an inverse relationship between nuclear volume and chromatin condensation.     

Fractal Characterization of Chromatin Decompaction in Live Cells

Chromatin organization has a fundamental impact on the whole spectrum of genomic functions. Quantitative characterization of the chromatin structure, particularly at submicron length scales where chromatin fractal globules are formed, is critical to understanding this structure-function relationship. Such analysis is currently challenging due to the diffraction-limited resolution of conventional light microscopy. We herein present an optical approach termed inverse spectroscopic optical coherence tomography to characterize the mass density fractality of chromatin, and we apply the technique to observe chromatin decompaction in live cells. The technique makes it possible for the first time, to our knowledge, to sense intracellular morphology with length-scale sensitivity from ∼30 to 450 nm, thus primarily probing the higher-order chromatin structure, without resolving the actual structures. We used chromatin decompaction due to inhibition of histone deacytelases and measured the subsequent changes in the fractal dimension of the intracellular structure. The results were confirmed by transmission electron microscopy and confocal fluorescence microscopy.     

Competition between histone H1 and HMGN proteins for chromatin binding sites

The ability of regulatory factors to access their nucleosomal targets is modulated by nuclear proteins such as histone H1 and HMGN (previously named HMG-14/-17 family) that bind to nucleosomes and either stabilize or destabilize the higher-order chromatin structure. We tested whether HMGN proteins affect the interaction of histone H1 with chromatin. Using microinjection into living cells expressing H1–GFP and photobleaching techniques, we found that wild-type HMGN, but not HMGN point mutants that do not bind to nucleosomes, inhibits the binding of H1 to nucleosomes. HMGN proteins compete with H1 for nucleosome sites but do not displace statically bound H1 from chromatin. Our results provide evidence for in vivo competition among chromosomal proteins for binding sites on chromatin and suggest that the local structure of the chromatin fiber is modulated by a dynamic interplay between nucleosomal binding proteins.     

Intra- and inter-nucleosome interactions of the core histone tail domains in higher-order chromatin structure

Eukaryotic chromatin is a hierarchical collection of nucleoprotein structures that package DNA to form chromosomes. The initial levels of packaging include folding of long strings of nucleosomes into secondary structures and array–array association into higher-order tertiary chromatin structures. The core histone tail domains are required for the assembly of higher-order structures and mediate short- and long-range intra- and inter-nucleosome interactions with both DNA and protein targets to direct their assembly. However, important details of these interactions remain unclear and are a subject of much interest and recent investigations. Here, we review work defining the interactions of the histone N-terminal tails with DNA and protein targets relevant to chromatin higher-order structures, with a specific emphasis on the contributions of H3 and H4 tails to oligonucleosome folding and stabilization. We evaluate both classic and recent experiments determining tail structures, effect of tail cleavage/loss, and posttranslational modifications of the tails on nucleosomes and nucleosome arrays, as well as inter-nucleosomal and inter-array interactions of the H3 and H4 N-terminal tails.     

Progressive polycomb assembly on H3K27me3 compartments generates polycomb bodies with developmentally regulated motion

Polycomb group (PcG) proteins are conserved chromatin factors that maintain silencing of key developmental genes outside of their expression domains. Recent genome-wide analyses showed a Polycomb (PC) distribution with binding to discrete PcG response elements (PREs). Within the cell nucleus, PcG proteins localize in structures called PC bodies that contain PcG-silenced genes, and it has been recently shown that PREs form local and long-range spatial networks. Here, we studied the nuclear distribution of two PcG proteins, PC and Polyhomeotic (PH). Thanks to a combination of immunostaining, immuno-FISH, and live imaging of GFP fusion proteins, we could analyze the formation and the mobility of PC bodies during fly embryogenesis as well as compare their behavior to that of the condensed fraction of euchromatin. Immuno-FISH experiments show that PC bodies mainly correspond to 3D structural counterparts of the linear genomic domains identified in genome-wide studies. During early embryogenesis, PC and PH progressively accumulate within PC bodies, which form nuclear structures localized on distinct euchromatin domains containing histone H3 tri-methylated on K27. Time-lapse analysis indicates that two types of motion influence the displacement of PC bodies and chromatin domains containing H2Av-GFP. First, chromatin domains and PC bodies coordinately undergo long-range motions that may correspond to the movement of whole chromosome territories. Second, each PC body and chromatin domain has its own fast and highly constrained motion. In this motion regime, PC bodies move within volumes slightly larger than those of condensed chromatin domains. Moreover, both types of domains move within volumes much smaller than chromosome territories, strongly restricting their possibility of interaction with other nuclear structures. The fast motion of PC bodies and chromatin domains observed during early embryogenesis strongly decreases in late developmental stages, indicating a possible contribution of chromatin dynamics in the maintenance of stable gene silencing.     

Suppression of liquid–liquid phase separation by 1,6-hexanediol partially compromises the 3D genome organization in living cells

Liquid–liquid phase separation (LLPS) contributes to the spatial and functional segregation of molecular processes within the cell nucleus. However, the role played by LLPS in chromatin folding in living cells remains unclear. Here, using stochastic optical reconstruction microscopy (STORM) and Hi-C techniques, we studied the effects of 1,6-hexanediol (1,6-HD)-mediated LLPS disruption/modulation on higher-order chromatin organization in living cells. We found that 1,6-HD treatment caused the enlargement of nucleosome clutches and their more uniform distribution in the nuclear space. At a megabase-scale, chromatin underwent moderate but irreversible perturbations that resulted in the partial mixing of A and B compartments. The removal of 1,6-HD from the culture medium did not allow chromatin to acquire initial configurations, and resulted in more compact repressed chromatin than in untreated cells. 1,6-HD treatment also weakened enhancer-promoter interactions and TAD insulation but did not considerably affect CTCF-dependent loops. Our results suggest that 1,6-HD-sensitive LLPS plays a limited role in chromatin spatial organization by constraining its folding patterns and facilitating compartmentalization at different levels.     

Spermatogenesis and distinctive mature sperm in the giant freshwater prawn, Macrobrachium rosenbergii (De Man, 1879)

The structures of differentiating male germ cells in the testis of the giant freshwater prawn, Macrobrachium rosenbergii, were studied by light and electron microscopy. Based on ultrastructural characteristics, the developing male germ cells are classified into 12 stages, including spermatogonia, six phases of primary spermatocytes (leptotene, zygotene, pachytene, diplotene, diakinesis and metaphase), secondary spermatocyte, three stages of spermatids and mature sperm. During spermatogenesis, the differentiating germ cells have characteristics similar to those of other invertebrates, but they exhibit some unique characteristics during spermiogenesis. In particular, an early spermatid has a round nucleus with highly condensed heterochromatin, appearing as thick interconnecting cords throughout the nucleus. In contrast to most invertebrates and vertebrates, the chromatin begins to decondense in one-half of the nucleus at the mid spermatid stage. In the late spermatid, the chromatin becomes almost entirely decondensed with only a small crescent-shaped heterochromatin patch remaining at the anterior pole of the nucleus. Mature sperm possess an everted umbrella-shaped plate with a spike covering the anterior pole of the nucleus, whose chromatin is totally decondensed as only small traces of histones H3 and H2B remain. The acrosome appears at the ruffled border of the spike plate as small sac-like structures. Few mitochondria remain in the cytoplasm at the posterior pole.