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.     

Higher-order chromatin structure: bridging physics and biology

Advances in microscopy and genomic techniques have provided new insight into spatial chromatin organization inside of the nucleus. In particular, chromosome conformation capture data has highlighted the relevance of polymer physics for high-order chromatin organization. In this context, we review basic polymer states, discuss how an appropriate polymer model can be determined from experimental data, and examine the success and limitations of various polymer models of higher-order interphase chromatin organization. By taking into account topological constraints acting on the chromatin fiber, recently developed polymer models of interphase chromatin can reproduce the observed scaling of distances between genomic loci, chromosomal territories, and probabilities of contacts between loci measured by chromosome conformation capture methods. Polymer models provide a framework for the interpretation of experimental data as ensembles of conformations rather than collections of loops, and will be crucial for untangling functional implications of chromosomal organization.     

Forces driving the three‐dimensional folding of eukaryotic genomes

The last decade has radically renewed our understanding of higher order chromatin folding in the eukaryotic nucleus. As a result, most current models are in support of a mostly hierarchical and relatively stable folding of chromosomes dividing chromosomal territories into A‐ (active) and B‐ (inactive) compartments, which are then further partitioned into topologically associating domains (TADs), each of which is made up from multiple loops stabilized mainly by the CTCF and cohesin chromatin‐binding complexes. Nonetheless, the structure‐to‐function relationship of eukaryotic genomes is still not well understood. Here, we focus on recent work highlighting the biophysical and regulatory forces that contribute to the spatial organization of genomes, and we propose that the various conformations that chromatin assumes are not so much the result of a linear hierarchy, but rather of both converging and conflicting dynamic forces that act on it.     

The Role of Crowding Forces in Juxtaposing β-Globin Gene Domain Remote Regulatory Elements in Mouse Erythroid Cells

The extremely high concentration of macromolecules in a eukaryotic cell nucleus indicates that the nucleoplasm is a crowded macromolecular solution in which large objects tend to gather together due to crowding forces. It has been shown experimentally that crowding forces support the integrity of various nuclear compartments. However, little is known about their role in control of chromatin dynamics in vivo. Here, we experimentally addressed the possible role of crowding forces in spatial organization of the eukaryotic genome. Using the mouse β-globin domain as a model, we demonstrated that spatial juxtaposition of the remote regulatory elements of this domain in globin-expressing cells may be lost and restored by manipulation of the level of macromolecular crowding. In addition to proving the role of crowding forces in shaping interphase chromatin, our results suggest that the folding of the chromatin fiber is a major determinant in juxtaposing remote genomic elements.     

Structure and dynamics of interphase chromosomes

During interphase chromosomes decondense, but fluorescent in situ hybridization experiments reveal the existence of distinct territories occupied by individual chromosomes inside the nuclei of most eukaryotic cells. We use computer simulations to show that the existence and stability of territories is a kinetic effect that can be explained without invoking an underlying nuclear scaffold or protein-mediated interactions between DNA sequences. In particular, we show that the experimentally observed territory shapes and spatial distances between marked chromosome sites for human, Drosophila, and budding yeast chromosomes can be reproduced by a parameter-free minimal model of decondensing chromosomes. Our results suggest that the observed interphase structure and dynamics are due to generic polymer effects: confined Brownian motion conserving the local topological state of long chain molecules and segregation of mutually unentangled chains due to topological constraints.     

Specific staining of human chromosomes in Chinese hamster x man hybrid cell lines demonstrates interphase chromosome territories

Summary In spite of Carl Rabl's (1885) and Theodor Boveri's (1909) early hypothesis that chromosomes occupy discrete territories or domains within the interphase nucleus, evidence in favor pf this hypothesis has been limited and indirect so far in higher plants and animals. The alternative possibility that the chromatin fiber of single chromosomes might be extended throughout the major part of even the whole interphase nucleus has been considered for many years. In the latter case, chromosomes would only exist as discrete chromatin bodies during mitosis but not during interphase. Both possibilities are compatible with Boveri's well established paradigm of chromosome individuality. Here we show that an active human X chromosome contained as the only human chromosome in a Chinese hamster x man hybrid cell line can be visualized both in metaphse plates and in interphase nuclei after in situ hybridization with either ³H- or biotin-labeled human genomic DNA. We demonstrate that this chromosome is organized as a distinct chromatin body throughout interphase. In addition, evidence for the territorial organization of human chromosomes is also presented for another hybrid cell line containing several autosomes and the human X chromosome. These findings are discussed in the context of our present knowledge of the organization and topography of interphase chromosomes. General applications of a strategy aimed at specific staining of individual chromosomes in experimental and clinical cytogenetics are briefly considered.     

Non-specific interactions are sufficient to explain the position of heterochromatic chromocenters and nucleoli in interphase nuclei

The organization of the eukaryote nucleus into functional compartments arises by self-organization both through specific protein–protein and protein–DNA interactions and non-specific interactions that lead to entropic effects, such as e.g. depletion attraction. While many specific interactions have so far been demonstrated, the contributions of non-specific interactions are still unclear. We used coarse-grained molecular dynamics simulations of previously published models for Arabidopsis thaliana chromatin organization to show that non-specific interactions can explain the in vivo localization of nucleoli and chromocenters. Also, we quantitatively demonstrate that chromatin looping contributes to the formation of chromosome territories. Our results are consistent with the previously published Rosette model for Arabidopsis chromatin organization and suggest that chromocenter-associated loops play a role in suppressing chromocenter clustering.     

Structural–Functional Domains of the Eukaryotic Genome

It is well known that DNA folding in the eukaryotic cell nucleus is tightly coupled with the operation of epigenetic mechanisms defining the repertoires of the genes expressed in different types of cells. To understand these mechanisms, it is important to know how DNA is packaged in chromatin. About 30 years ago a hypothesis was formulated, according to which epigenetic mechanisms operate not at the level of individual genes, but rather groups of genes localized in structurally and functionally isolated genomic segments that were called structural and functional domains. The question of what exactly these domains constitute has been re-examined multiple times as our knowledge of principles of chromatin folding has changed. In this review, we discuss structural and functional genomic domains in light of the current model of interphase chromosome organization based on the results of analysis of spatial proximity between remote genomic elements.     

Common pathway of chromosome condensation in Mammalian cells

Chromatin folding in the interphase nucleus is not known. We compared the pattern of chromatin condensation in Indian muntjac, Chinese hamster ovary, murine pre B, and K562 human erythroleukemia cells during the cell cycle. Fluorescent microscopy showed that chromosome condensation follows a general pathway. Synchronized cells were reversibly permeabilized and used to isolate interphase chromatin structures. Based on their structures two major categories of intermediates were distinguished: (1) decondensed chromatin and (2) condensed chromosomal forms. (1) Chromatin forms were found between the G1 and mid-S phase involving veil-like, supercoiled, fibrous, ribboned structures; (2) condensing chromosomal forms appeared in the late-S, G2, and M phase, including strings, chromatin bodies, elongated pre-chromosomes, pre-condensed chromosomes, and metaphase chromosomes. Results demonstrate that interphase chromosomes are clustered in domains; condensing interphase chromosomes are linearly arranged. Our results raise questions related to telomer sequences and to the chemical nature of chromosome connectivity.     

On the choreography of genome folding: A grand pas de deux of cohesin and CTCF

Cohesin and CTCF are key to the 3D folding of interphase chromosomes. Cohesin forms chromatin loops via loop extrusion, a process that involves the formation and enlargement of DNA loops. The architectural protein CTCF controls this process by acting as an anchor for chromatin looping. How CTCF controls cohesin has long been a mystery. Recent work shows that CTCF dictates chromatin looping via a direct interaction of its N-terminus with cohesin. CTCF's ability to regulate chromatin looping turns out to also be partially dependent on several RNA-binding domains. In this review, we discuss recent insights and consider how cohesin and CTCF together may orchestrate the folding of the genome into chromosomal loops.