Nucleolus-associated Chromatin Research Articles

Sleeping Beauty Transposon Insertions into Nucleolar DNA by an Engineered Transposase Localized in the Nucleolus.

Transposons are nature's gene delivery vehicles that can be harnessed for experimental and therapeutic purposes. The Sleeping Beauty (SB) transposon shows efficient transposition and long-term transgene expression in human cells, and is currently under clinical development for gene therapy. SB transposition occurs into the human genome in a random manner, which carries a risk of potential genotoxic effects associated with transposon integration. Here, we evaluated an experimental strategy to manipulate SB's target site distribution by preferentially compartmentalizing the SB transposase to the nucleolus, which contains repetitive ribosomal RNA (rRNA) genes. We generated a fusion protein composed of the nucleolar protein nucleophosmin (B23) and the SB100X transposase, which was found to retain almost full transposition activity as compared to unfused transposase and to be predominantly localized to nucleoli in transfected human cells. Analysis of transposon integration sites generated by B23-SB100X revealed a significant enrichment into the p-arms of chromosomes containing nucleolus organizing regions (NORs), with preferential integration into the p13 and p11.2 cytobands directly neighboring the NORs. This bias in the integration pattern was accompanied by an enrichment of insertions into nucleolus-associated chromatin domains (NADs) at the periphery of nucleolar DNA and into lamina-associated domains (LADs). Finally, sub-nuclear targeting of the transposase resulted in preferential integration into chromosomal domains associated with the Upstream Binding Transcription Factor (UBTF) that plays a critical role in the transcription of 47S rDNA gene repeats of the NORs by RNA Pol I. Future modifications of this technology may allow the development of methods for specific gene insertion for precision genetic engineering.

Mapping nucleolus-associated chromatin interactions using nucleolus Hi-C reveals pattern of heterochromatin interactions

As the largest substructures in the nucleus, nucleoli are the sites of ribosome biogenesis. Increasing evidence indicates that nucleoli play a key role in the organization of 3D genome architecture, but systematic studies of nucleolus-associated chromatin interactions are lacking. Here, we developed a nucleolus Hi-C (nHi-C) experimental technique to enrich nucleolus-associated chromatin interactions. Using the nHi-C experiment, we identify 264 high-confidence nucleolus-associated domains (hNADs) that form strong heterochromatin interactions associated with the nucleolus and consist of 24% of the whole genome in HeLa cells. Based on the global hNAD inter-chromosomal interactions, we find five nucleolar organizer region (NOR)-bearing chromosomes formed into two clusters that show different interaction patterns, which is concordant with their epigenetic states and gene expression levels. hNADs can be divided into three groups that display distinct cis/trans interaction signals, interaction frequencies associated with nucleoli, distance from the centromeres, and overlap percentage with lamina-associated domains (LADs). Nucleolus disassembly caused by Actinomycin D (ActD) significantly decreases the strength of hNADs and affects compartment/TAD strength genome-wide. In summary, our results provide a global view of heterochromatin interactions organized around nucleoli and demonstrate that nucleoli act as an inactive inter-chromosomal hub to shape both compartments and TADs.

Heterochromatin Networks: Topology, Dynamics, and Function (a Working Hypothesis).

Open systems can only exist by self-organization as pulsing structures exchanging matter and energy with the outer world. This review is an attempt to reveal the organizational principles of the heterochromatin supra-intra-chromosomal network in terms of nonlinear thermodynamics. The accessibility of the linear information of the genetic code is regulated by constitutive heterochromatin (CHR) creating the positional information in a system of coordinates. These features include scale-free splitting-fusing of CHR with the boundary constraints of the nucleolus and nuclear envelope. The analysis of both the literature and our own data suggests a radial-concentric network as the main structural organization principle of CHR regulating transcriptional pulsing. The dynamic CHR network is likely created together with nucleolus-associated chromatin domains, while the alveoli of this network, including springy splicing speckles, are the pulsing transcription hubs. CHR contributes to this regulation due to the silencing position variegation effect, stickiness, and flexible rigidity determined by the positioning of nucleosomes. The whole system acts in concert with the elastic nuclear actomyosin network which also emerges by self-organization during the transcriptional pulsing process. We hypothesize that the the transcriptional pulsing, in turn, adjusts its frequency/amplitudes specified by topologically associating domains to the replication timing code that determines epigenetic differentiation memory.

Nucleolus-associated chromatin domains are maintained under heat stress, despite nucleolar reorganization in Arabidopsis thaliana.

Several layers of mechanisms participate in plant adaptation to heat-stress. For example, the plant metabolism switches from cell growth mode to stress adaptation mode. Ribosome biogenesis is one of the most energy costly pathways. That biogenesis process occurs in the nucleolus, the largest nuclear compartment, whose structure is highly dependent on this pathway. We used a nucleolar marker to track the structure of the nucleolus, and revealed a change in its sub-nucleolar distribution under heat stress. In addition, the nucleolus is implicated in other cellular processes, such as genome organization within the nucleus. However, our analyses of nucleolus-associated chromatin domains under heat stress did not reveal significant differences compared to the control plants, suggesting a lack of connection between two of the main functions of the nucleolus: ribosome biogenesis and nuclear organization.

Recognition but no repair of abasic site in single-stranded DNA by human ribosomal uS3 protein residing within intact 40S subunit.

Isolated human ribosomal protein uS3 has extra-ribosomal functions including those related to base excision DNA repair, e.g. AP lyase activity that nicks double-stranded (ds) DNA 3΄ to the abasic (AP) site. However, the ability of uS3 residing within ribosome to recognize and cleave damaged DNA has never been addressed. Here, we compare interactions of single-stranded (ss) DNA and dsDNA bearing AP site with human ribosome-bound uS3 and with the isolated protein, whose interactions with ssDNA were not yet studied. The AP lyase activity of free uS3 was much higher with ssDNA than with dsDNA, whereas ribosome-bound uS3 was completely deprived of this activity. Nevertheless, an exposed peptide of ribosome-bound uS3 located far away from the putative catalytic center previously suggested for isolated uS3 cross-linked to full-length uncleaved ssDNA, but not to dsDNA. In contrast, free uS3 cross-linked mainly to the 5΄-part of the damaged DNA strand after its cleavage at the AP site. ChIP-seq analysis showed preferential uS3 binding to nucleolus-associated chromatin domains. We conclude that free and ribosome-bound uS3 proteins interact with AP sites differently, exhibiting their non-translational functions in DNA repair in and around the nucleolus and in regulation of DNA damage response in looped DNA structures, respectively.

Plant nucleolar DNA: Green light shed on the role of Nucleolin in genome organization

ABSTRACTThe nucleolus forms as a consequence of ribosome biogenesis, but it is also implicated in other cell functions. The identification of nucleolus-associated chromatin domains (NADs) in animal and plant cells revealed the presence of DNA sequences other than rRNA genes in and around the nucleolus. NADs display repressive chromatin signatures and harbour repetitive DNA, but also tRNA genes and RNA polymerase II-transcribed genes. Furthermore, the identification of NADs revealed a specific function of the nucleolus and the protein Nucleolin 1 (NUC1) in telomere biology. Here, we discuss the significance of these data with regard to nucleolar structure and to the role of the nucleolus and NUC1 in global genome organization and stability.

Identification of Nucleolus-Associated Chromatin Domains Reveals a Role for the Nucleolus in 3D Organization of the A. thaliana Genome

The nucleolus is the site of rRNA gene transcription, rRNA processing, and ribosome biogenesis. However, the nucleolus also plays additional roles in the cell. We isolated nucleoli using fluorescence-activated cell sorting (FACS) and identified nucleolus-associated chromatin domains (NADs) by deep sequencing, comparing wild-type plants and null mutants for the nucleolar protein NUCLEOLIN 1 (NUC1). NADs are primarily genomic regions with heterochromatic signatures and include transposable elements (TEs), sub-telomeric regions, and mostly inactive protein-coding genes. However, NADs also include active rRNA genes and the entire short arm of chromosome 4 adjacent to them. In nuc1 null mutants, which alter rRNA gene expression and overall nucleolar structure, NADs are altered, telomere association with the nucleolus is decreased, and telomeres become shorter. Collectively, our studies reveal roles for NUC1 and the nucleolus in the spatial organization of chromosomes as well as telomere maintenance.

Nucleolar chromatin organization at different activities of soybean root meristematic cell nucleoli.

Nucleolar chromatin, including nucleolus-associated chromatin as well as active and inactive condensed ribosomal DNA (rDNA) chromatin, derives mostly from secondary constrictions known as nucleolus organizer regions containing rDNA genes on nucleolus-forming chromosomes. This chromatin may occupy different nucleolar positions being in various condensation states which may imply different rDNA transcriptional competence. Sections of nucleoli originating from root meristematic cells of soybean seedlings grown at 25 °C (the control), then subjected to chilling stress (10 °C), and next transferred again to 25 °C (the recovery) were used to measure profile areas occupied by nucleolar condensed chromatin disclosed with sodium hydroxide methylation–acetylation plus uranyl acetate technique. The biggest total area of condensed chromatin was found in the nucleoli of chilled plants, while the smallest was found in those of recovered plants in relation to the amounts of chromatin in the control nucleoli. The condensed nucleolar chromatin, in the form of different-sized and different-shaped clumps, was mainly located in fibrillar centers. One can suppose that changes of condensed rDNA chromatin amounts might be a mechanism controlling the number of transcriptionally active rDNA genes as the nucleoli of plants grown under these experimental conditions show different transcriptional activity and morphology.

Initial Genomics of the Human Nucleolus

We report for the first time the genomics of a nuclear compartment of the eukaryotic cell. 454 sequencing and microarray analysis revealed the pattern of nucleolus-associated chromatin domains (NADs) in the linear human genome and identified different gene families and certain satellite repeats as the major building blocks of NADs, which constitute about 4% of the genome. Bioinformatic evaluation showed that NAD–localized genes take part in specific biological processes, like the response to other organisms, odor perception, and tissue development. 3D FISH and immunofluorescence experiments illustrated the spatial distribution of NAD–specific chromatin within interphase nuclei and its alteration upon transcriptional changes. Altogether, our findings describe the nature of DNA sequences associated with the human nucleolus and provide insights into the function of the nucleolus in genome organization and establishment of nuclear architecture.

Chromatin position in human HepG2 cells: although being non-random, significantly changed in daughter cells.

Mammalian chromosomes occupy chromosome territories within nuclear space the positions of which are generally accepted as non-random. However, it is still controversial whether position of chromosome territories/chromatin is maintained in daughter cells. We addressed this issue and investigated maintenance of various chromatin regions of unknown composition as well as nucleolus-associated chromatin, a significant part of which is composed of nucleolus organizer region-bearing chromosomes. The photoconvertible histone H4-Dendra2 was used to label such regions in transfected HepG2 cells, and its position was followed up to next interphase. The distribution of labeled chromatin in daughter cells exhibited a non-random character. However, its distribution in a vast majority of daughter cells extensively differed from the original ones and the labeled nucleolus-associated chromatin differently located into the vicinity of different nucleoli. Therefore, our results were not consistent with a concept of preservation chromatin position. This conclusion was supported by the finding that the numbers of nucleoli significantly differed between the two daughter cells. Our results support a view that while the transfected daughter HepG2 cells maintain some features of the parental cell chromosome organization, there is also a significant stochastic component associated with reassortment of chromosome territories/chromatin that results in their positional rearrangements.

In situ comparative studies on subnucleolar distribution and configuration of plant rDNA

The distribution and configurations of nucleolar DNA in Pisum sativum L., Allium sativum L., Triticum aestivum L. were analyzed by specific cytochemical staining using NAMA-Ur. It has been observed that in the nucleoli of different plant species, the DNA occupied different positions in different areas, which may imply a different status and strategy of rDNA transcription. Our results showed irregular clumps of rDNA surrounding FCs in semi-circular formations in P. sativum and T. aestivum, indicating a regular pattern of rDNA distribution and supporting the helix model of rDNA configuration. The rDNA was condensed in some regions and uncondensed in others. Nucleolus-associated chromatin extended from outside the nucleolus to the periphery of the FCs via nucleolar channels, which suggests a possible origin for nucleolar DNA.

Subnucleolar distribution and organization of Vicia faba L. rDNA in situ

The distribution and organization of nucleolar DNA in Vicia faba L. was analyzed by specific cytochemical staining using NAMA-Ur. The results showed that nucleolar DNA was distributed in the FCs and at the FC/DFC junctions. Statistical analysis showed that the rRNA genes occupied about one-third of the total dense fibrillar component region. The rDNA was condensed in some regions and uncondensed in others. Nucleolus-associated chromatin extended from outside the nucleolus to the periphery of the FCs via nucleolar channels, suggesting a possible origin for nucleolar DNA.

Structures of nucleolus and transcription sites of rRNA genes in rat liver cells

We observed the ultrastructure of nucleolus in rat liver cells by conventional electron microscopy, and employed cytochemistry NAMA-Ur DNA specific stain method to analyze the distribution and position of nucleolar DNAin situ. The results showed that nucleolar DNA of rat liver cells comes from nucleolus-associated chromatin, and continuously extends in the dense fibrillar component (DFC) of nucleolus, localizes at the periphery of fibrillar center (FC) and in DFC. Furthermore, by employing anti-DNA/RNA hybrid antibodies, we directly and selectively labeled transcription sites of rRNA genes and testified that localization of transcription sites not only to DFC but also to the periphery of FC.

Three-dimensional analysis of the arrangement of compact chromatin in the nucleus of G0 rat lymphocytes.

The arrangement of compact chromatin of G0 lymphocytes was studied in three-dimensional reconstructions of the ensemble of the chromatin and of individual compact chromatin bodies. Rat spleen was serially cut and sections were contrasted with procedures preferential for DNA. Electron microscopy images were digitized, processed, and displayed using a commercial software package, complemented by a system for three-dimensional reconstruction and analysis developed by us on an IBM-compatible microcomputer provided with an image acquisition board. The reconstructions showed a continuous layer of compact chromatin in contact with the nuclear envelope that prevents the automatic recognition of individual chromatin clumps. The ensemble of the arrangement of compact chromatin was found to be very similar in different lymphocytes. After morphological filtering procedures, the initial mass was divided into individual bodies of compact chromatin, which were tagged. Most of these bodies contact the nuclear envelope. The number of bodies as well as the number of contacts with the envelope are similar and correspond to a haploid number of chromosomes. The largest body is always the one containing nucleolus-associated chromatin. When the cell has two nucleoli, the nucleolus-associated chromatin bodies contact the envelope in diametrically opposed areas. This feature was also described in rat liver cells. It is concluded that: (a) the individualized compact chromatin bodies do not correspond to an entire chromosome or to a pair of chromosomes; (b) the arrangement of compact chromatin is not identical in each G0 lymphocyte, but there are patterns that are repeated with limited changes; and (c) there are common features that appear in different cell types of individuals of the same species.

Physiologically Inactive Nucleoli Contain Nucleoplasmic Ribonucleoproteins: Immunoelectron Microscopy of Mouse Spermatids and Early Embryos

Physiologically inactivating or reactivating nucleoli represent a good model to investigate modifications of the nucleolar activity, which influence to a great extent the morphology of this organelle. We have studied the nucleoli of mouse spermatids during gradual inactivation, as well as those of early mouse preimplantation embryos, which undergo reactivation. In the 2-cell systems, inactive nucleoli are represented by homogeneously, finely fibrillar spherical bodies. With the aim of clarifying the composition of these nucleoli, we have analyzed them by means of immunoelectron microscopy using specific antibodies directed against nucleoplasmic snRNPs, hnRNPs, ribosomal proteins, or fibrillarin as well as by cytochemical methods for visualizing DNA and RNA. Our results indicate that RNA is present in zygote and 2-cell nucleolus precursor bodies (NPBs) as well as in 4- to 8-cell or more advanced embryo nucleoli, but not in inactive spermatid nucleoli. DNA is absent from inactive spermatid nucleoli and NPBs but is present within the nucleolus-associated chromatin and the nucleolonema of active nucleoli. The dense masses constituting the NPBs of the zygote and 2-cell embryos contain ribosomal proteins and fibrillarin but also hnRNPs and nucleoplasmic snRNPs. Of these, only fibrillarin is present in the spermatid residual nucleoli.

Transcriptional activity and nuclear ultrastructure of 8-cell bovine embryos developed by in vitro maturation and fertilization of oocytes from different growth categories of antral follicles.

The normalcy of nuclear differentiation in 8-cell bovine embryos derived by in vitro procedures from oocytes isolated from antral follicles of three different size categories was studied using autoradiographic localization of RNA synthesis and fine-structure nuclear morphology. In the few embryos derived from the oocytes from the small category of follicles (1-2 mm) the unlabeled nuclei prevailed. In contrast, the oocytes isolated from the more progressed follicles (medium and large size: 2-8 mm) yielded embryos with only slight, if at all detectable, differences to the picture expected in in vivo developed normal embryos. The diverging features probably concerned a slower onset of gene transcription, its consecutive pattern of localization as well as less pronounced progress of chromatin condensation and nucleolar differentiation. The assessment of these morphological changes and characteristics for individual embryos was obscured by marked differences in the status of the nuclei of particular blastomeres forming an embryo. Here the differences were seen in the fragmentation of nuclei, in the degree of chromatin condensation and in the absence of the nucleolus-associated chromatin in some of the blastomeres. It is suggested that most of the embryos from medium and large follicles have a comparable differentiation pattern of nuclear function development as embryos developing normally in vivo.

Cytochemical and immunocytochemical ultrastructural study of nucleoproteins during nucleologenesis in 8-cell bovine embryos.

The aim of this study was to characterize embryonic nucleologenesis by determining the appearance and localization of acid argyrophilic, basic lysine-rich and histone proteins in 8-cell bovine embryos. Two silver staining techniques, ethanolic phosphotungstic acid (PTA) and immunocytochemical methods using specific antibodies, were applied at the ultrastructural level. The silver-stained proteins were detected at the onset of nucleologenesis on the periphery of the dense nucleolus precursor bodies (NPBs). The amounts of these proteins increased during the transformation of the NPBs into the fibrillogranular nucleolus. At this stage the well-developed dense fibrillar components encircling fibrillar centres showed intense staining. PTA-positive (basic lysine-rich) proteins were present within most nucleolar structures during nucleologenesis as well as in the chromatin. Histones H2B, H3 and H4 were concentrated throughout the chromatin including the nucleolus-associated chromatin. At the onset of nucleologenesis, histones were absent in the NPBs. The first weak histone labelling was detected in the multivacuolated NPBs, both in the fibrous mass as well as inside the vacuoles. Nucleolar histones appeared with the massive penetration of DNA into the NPBs. We suggest that nucleologenesis may serve as a criterion of normal early embryonic development and that the proteins involved in the process of nucleologenesis and transcription could be used as chemical markers of nucleolar function.

Segregation of viral double-stranded and single-stranded DNA molecules in nucleo fo adenovirus infected cells as revealed by electron microscope in situ hybridization

Formation of progeny viruses in the nuclei of HeLa cells infected with adenovirus type 5 was studied at the ultrastructural level by in situ hybridization techniques allowing specific detection of either viral double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA). Prior to the initiation of replication of viral genomes, infective DNA molecules which entered the nucleus of the target cell were randomly distributed among host chromatin fibers including nucleolus-associated chromatin. They were double-stranded, that is, without single-strand breaks. Such association of viral DNA with host condensed chromatin also occurred in mitosis. The initiation of viral genome replication occufred simultaneously with the appearance in the nucleoplasm of small fibrillar regions containing intermingled viral dsDNA and ssDNA. Later, at the intermediate stage of nuclear transformation, viral dsDNA and ssDNA molecules were almost entirely separated into two contiguous substructures. At this stage, viruses were observed occasionally in the vicinity of viral ssDNA accumulation sites. Still later, an additional substructure developed in the centre of the nucleus which consisted of large quantities of viral dsDNA, traces of viral ssDNA and abundant viruses. Portions of viral ssDNA were attached to some viruses even at late stage of nuclear transformation, an association which strongly suggests the occurence of encapsidation of at least some of the viral genomes while they are still engaged in replication.

Structural differentiation of the nucleolus-organizing regions between mitotic and epidermal cells inVicia faba

SUMMARYThe nucleolar secondary constrictions of Vicia faba chromosomes were filled with a weakly stained substance when the sections were treated with conventional double staining and examined under an electron microscope. But both silver staining and ribonucleoprotein preferential staining contrasted these constrictions as positively stained areas. On the other hand, the nucleolus-associated chromatin blocks in the epidermal cells were clearly impregnated with silver although they were fully condensed, suggesting that ribosomal (r) RNA genes are organized into the condensed chromosomal segment when cells differentiate into specialized cells such as the epidermal cells.

Locating transcribed and non-transcribed rDNA spacer sequences within the nucleolus by in situ hybridization and immunoelectron microscopy

Immunoelectron microscopy and in situ hybridization have been used to investigate the precise location of transcribed and non-transcribed rDNA spacer sequences. Whereas a 5'-external transcribed spacer sequence is predominantly visualized in the fibrillar centers of nucleoli, a non-transcribed spacer sequence is preferentially detected in the interstices, in close contact with the fibrillar centers and which interrupt the surrounding dense fibrillar component. Occasionally these two spacers are also observed in clumps of dense nucleolus-associated chromatin. These observations provide insights into the organization of ribosomal repeats within the nucleolus.