Changes In Nuclear Organization Research Articles

Reorganization of lamina-associated domains in early mouse embryos is regulated by RNA polymerase II activity.

Fertilization in mammals is accompanied by an intense period of chromatin remodeling and major changes in nuclear organization. How the earliest events in embryogenesis, including zygotic genome activation (ZGA) during maternal-to-zygotic transition, influence such remodeling remains unknown. Here, we have investigated the establishment of nuclear architecture, focusing on the remodeling of lamina-associated domains (LADs) during this transition. We report that LADs reorganize gradually in two-cell embryos and that blocking ZGA leads to major changes in nuclear organization, including altered chromatin and genomic features of LADs and redistribution of H3K4me3 toward the nuclear lamina. Our data indicate that the rearrangement of LADs is an integral component of the maternal-to-zygotic transition and that transcription contributes to shaping nuclear organization at the beginning of mammalian development.

Changes in genotoxic stress response, ribogenesis and PAP (3'-phosphoadenosine 5'-phosphate) levels are associated with loss of desiccation tolerance in overprimed Medicago truncatula seeds.

Re‐establishment of desiccation tolerance is essential for the survival of germinated seeds facing water deficit in the soil. The molecular and ultrastructural features of desiccation tolerance maintenance and loss within the nuclear compartment are not fully resolved. In the present study, the impact of desiccation‐induced genotoxic stress on nucleolar ultrastructure and ribogenesis was explored along the rehydration−dehydration cycle applied in standard seed vigorization protocols. Primed and overprimed Medicago truncatula seeds, obtained through hydropriming followed by desiccation (dry‐back), were analysed. In contrast to desiccation‐tolerant primed seeds, overprimed seeds enter irreversible germination and do not survive dry‐back. Reactive oxygen species, DNA damage and expression profiles of antioxidant/DNA Damage Response genes were measured, as main hallmarks of the seed response to desiccation stress. Nuclear ultrastructural features were also investigated. Overprimed seeds subjected to dry‐back revealed altered rRNA accumulation profiles and up‐regulation of genes involved in ribogenesis control. The signal molecule PAP (3′‐phosphoadenosine 5′‐phosphate) accumulated during dry‐back only in primed seeds, as a distinctive feature of desiccation tolerance. The presented results show the molecular and ultrastructural landscapes of the seed desiccation response, including substantial changes in nuclear organization.

Active RB causes visible changes in nuclear organization.

RB restricts G1/S progression by inhibiting E2F. Here, we show that sustained expression of active RB, and prolonged G1 arrest, causes visible changes in chromosome architecture that are not directly associated with E2F inhibition. Using FISH probes against two euchromatin RB-associated regions, two heterochromatin domains that lack RB-bound loci, and two whole-chromosome probes, we found that constitutively active RB (ΔCDK-RB) promoted a more diffuse, dispersed, and scattered chromatin organization. These changes were RB dependent, were driven by specific isoforms of monophosphorylated RB, and required known RB-associated activities. ΔCDK-RB altered physical interactions between RB-bound genomic loci, but the RB-induced changes in chromosome architecture were unaffected by dominant-negative DP1. The RB-induced changes appeared to be widespread and influenced chromosome localization within nuclei. Gene expression profiles revealed that the dispersion phenotype was associated with an increased autophagy response. We infer that, after cell cycle arrest, RB acts through noncanonical mechanisms to significantly change nuclear organization, and this reorganization correlates with transitions in cellular state.

DNA methylation changes during long-term in vitro cell culture are caused by epigenetic drift

Culture expansion of primary cells evokes highly reproducible DNA methylation (DNAm) changes. We have identified CG dinucleotides (CpGs) that become continuously hyper- or hypomethylated during long-term culture of mesenchymal stem cells (MSCs) and other cell types. Bisulfite barcoded amplicon sequencing (BBA-seq) demonstrated that DNAm patterns of neighboring CpGs become more complex without evidence of continuous pattern development and without association to oligoclonal subpopulations. Circularized chromatin conformation capture (4C) revealed reproducible changes in nuclear organization between early and late passages, while there was no enriched interaction with other genomic regions that also harbor culture-associated DNAm changes. Chromatin immunoprecipitation of CTCF did not show significant differences during long-term culture of MSCs, however culture-associated hypermethylation was enriched at CTCF binding sites and hypomethylated CpGs were devoid of CTCF. Taken together, our results support the notion that DNAm changes during culture-expansion are not directly regulated by a targeted mechanism but rather resemble epigenetic drift.

Role of Chromatin Architecture in Plant Stress Responses: An Update.

Sessile plants possess an assembly of signaling pathways that perceive and transmit environmental signals, ultimately resulting in transcriptional reprogramming. Histone is a key feature of chromatin structure. Numerous histone-modifying proteins act under different environmental stress conditions to help modulate gene expression. DNA methylation and histone modification are crucial for genome reprogramming for tissue-specific gene expression and global gene silencing. Different classes of chromatin remodelers including SWI/SNF, ISWI, INO80, and CHD are reported to act upon chromatin in different organisms, under diverse stresses, to convert chromatin from a transcriptionally inactive to a transcriptionally active state. The architecture of chromatin at a given promoter is crucial for determining the transcriptional readout. Further, the connection between somatic memory and chromatin modifications may suggest a mechanistic basis for a stress memory. Studies have suggested that there is a functional connection between changes in nuclear organization and stress conditions. In this review, we discuss the role of chromatin architecture in different stress responses and the current evidence on somatic, intergenerational, and transgenerational stress memory.

Genetic Landscape of Papillary Thyroid Carcinoma and Nuclear Architecture: An Overview Comparing Pediatric and Adult Populations.

Simple SummaryPapillary thyroid carcinoma (PTC) represents 80–90% of all differentiated thyroid carcinomas. PTC has a high rate of gene fusions and mutations, which can influence clinical and biological behavior in both children and adults. In this review, we focus on the comparison between pediatric and adult PTC, highlighting genetic alterations, telomere-related genomic instability and changes in nuclear organization as novel biomarkers for thyroid cancers.Thyroid cancer is a rare malignancy in the pediatric population that is highly associated with disease aggressiveness and advanced disease stages when compared to adult population. The biological and molecular features underlying pediatric and adult thyroid cancer pathogenesis could be responsible for differences in the clinical presentation and prognosis. Despite this, the clinical assessment and treatments used in pediatric thyroid cancer are the same as those implemented for adults and specific personalized target treatments are not used in clinical practice. In this review, we focus on papillary thyroid carcinoma (PTC), which represents 80–90% of all differentiated thyroid carcinomas. PTC has a high rate of gene fusions and mutations, which can influence the histologic subtypes in both children and adults. This review also highlights telomere-related genomic instability and changes in nuclear organization as novel biomarkers for thyroid cancers.

Analysis of Cellular EMT States Using Molecular Biology and High Resolution FISH Labeling.

Metastasis results from the ability of cancer cells to grow and to spread beyond the primary tumor to distant organs. Epithelial-to-Mesenchymal Transition (EMT), a fundamental developmental process, is reactivated in cancer cells, and causes epithelial properties to evolve into mesenchymal and invasive ones. EMT changes cellular characteristics between two distinct states, yet, the process is not binary but rather reflects a broad spectrum of partial EMT states in which cells exhibit various degrees of intermediate epithelial and mesenchymal phenotypes. EMT is a complex multistep process that involves cellular reprogramming through numerous signaling pathways, alterations in gene expression, and changes in chromatin morphology. Therefore, expression of key proteins, including cadherins, occludin, or vimentin must be precisely regulated. A comprehensive understanding of how changes in nuclear organization, at the level of single genes clusters, correlates with these processes during formation of metastatic cells is still missing and yet may help personalized prognosis and treatment in the clinic. Here, we describe methods to correlate physiological and molecular states of cells undergoing an EMT process with chromatin rearrangements observed via FISH labeling of specific domains.

Appreciating What’s Unique about Every Stem Cell

Using single-cell techniques, the behaviors of individual stem cells are coming under ever-greater scrutiny. Such efforts are pushing the boundaries of technology development in cell biology and are getting to the heart of what defines cellular states, what drives cell-state changes, and what can we learn about biological systems from cell-to-cell variability. A prime example of this trend comes from recent work by Takahashi et al., 2019Takahashi S. Miura H. Shibata T. Nagao K. Okumura K. Ogata M. Obuse C. Takebayashi S.I. Hiratani I. Genome-wide stability of the DNA replication program in single mammalian cells.Nat Genet. 2019; 51: 529-540Crossref PubMed Scopus (40) Google Scholar, who have addressed whether replication timing across the genome is stereotyped between individual cells of the same cell type, and their results raise questions about the potential role of replication timing in the decision-making of differentiating stem cells. Previous studies with bulk sequencing of cell populations have revealed the basic principle that regions that replicate early tend to be euchromatic, actively transcribed, and in the nuclear interior, whereas late-replicating regions tend to be heterochromatic and at the nuclear periphery. Takahashi et al. have now designed and tested an approach to profile early- versus late-replicating regions at the single-cell level in mouse embryonic stem cells (mESCs), before and after differentiation and in an immortalized human retinal pigment epithelial cell line. Similar to another recent study by Dileep and Gilbert, 2018Dileep V. Gilbert D.M. Single-cell replication profiling to measure stochastic variation in mammalian replication timing.Nat. Commun. 2018; 9: 427Crossref PubMed Scopus (41) Google Scholar, the authors find that overall genome-wide replication timing is highly consistent between individual cells in a given state and that some regions exhibit more intrinsic variability than others. Going deeper, they reveal an interesting corollary: developmentally regulated genes exhibit higher-than-average variability in replication timing. Does this suggest a link between replication timing variability and developmental plasticity? The variability at these genes does in fact go away upon mESC differentiation. Future work may explore this facet of nuclear organization in stem cell “decision-making” or how this might be exploited to direct different differentiation endpoints. Getting to the molecular basis of the observations of studies such as Takahashi et al. will likely be aided by new approaches to study the behavior of proteins and protein complexes in single cells. In this vein, a recent study in our journal (Hainer et al., 2019Hainer S.J. Bošković A. McCannell K.N. Rando O.J. Fazzio T.G. Profiling of Pluripotency Factors in Single Cells and Early Embryos.Cell. 2019; 177: 1319-1329Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) adapts a nuclease-based method for mapping the binding of transcription factors, known as CUT&RUN (Skene and Henikoff, 2017Skene P.J. Henikoff S. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites.Elife. 2017; 6: e21856Crossref PubMed Scopus (584) Google Scholar), for use with very small cell numbers, even single cells. With this modified protocol, called ultra-low input CUT&RUN (uliCUT&RUN), Hainer and colleagues probe the distribution of transcription factors in mESCs and in vivo in pre-implantation embryos. In mESCs, they substantiate the long-standing conjecture that the fractional occupancy of transcription factors such as the pluripotency enabling NANOG and SOX2 in single cells is what underpins variable ChIP-seq (chromatin immunoprecipitation sequencing) peaks in bulk analyses. In early embryos, they further establish that NANOG binding in the inner cell mass requires the SWI/SNF chromatin remodeler, a feature not observed in similar analyses from cultured cells. This technological development will enable more accurate temporal reconstructions of transcription factor activity in individual cells and provides a new means of probing heterogeneity in vivo when tissue samples are limiting. One of the most powerful quantitative and high-throughput means of assessing levels of individual proteins in single stem cells comes from mass cytometry (cyTOF), in which antibodies to proteins of interest are conjugated to heavy metals to enable coupling of flow cytometry and time-of-flight mass spectrometry. A key advantage of cyTOF is the ability to monitor many proteins at once. Palii et al., 2019Palii C.G. Cheng Q. Gillespie M.A. Shannon P. Mazurczyk M. Napolitani G. Price N.D. Ranish J.A. Morrissey E. Higgs D.R. et al.Single-Cell Proteomics Reveal that Quantitative Changes in Co-expressed Lineage-SpecificTranscription Factors Determine Cell Fate.Cell Stem Cell. 2019; 24: 812-820Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar use this to impressive effect to assay the levels of 27 proteins across 13 time points (using temporal barcoding) during the differentiation of hematopoietic stem/progenitor cells (HSPCs). They specifically examine the trajectories of megakaryocyte-erythroid progenitors and find that both KLF1, which biases red blood cell fate, and FLI1, which biases megakaryocyte fate, are expressed in the same bipotential progenitors. They further show that the change in these lineage-specific transcription factors is gradual and not switch-like, as might be expected by a more rigid conceptualization of cell-fate decision-making. One anticipates that these gradual shifts in gene expression in developmental transitions (as observed by Palii et al.), the replication timing variability reported by Takahashi et al., and the conditional binding of transcription factors in early embryos (as seen by Hainer and colleagues) will all be reflected in changes in nuclear organization affecting the relevant loci. To confirm this and attain a more complete biophysical understanding of these transitions and their dynamics, will it one day be possible to track the compartmentalization and transcription factor occupancy of multiple developmentally important genetic loci in a single cell over time?

Epstein-Barr Virus-Induced Nodules on Viral Replication Compartments Contain RNA Processing Proteins and a Viral Long Noncoding RNA.

Profound alterations in host cell nuclear architecture accompany the lytic phase of Epstein-Barr virus (EBV) infection. Viral replication compartments assemble, host chromatin marginalizes to the nuclear periphery, cytoplasmic poly(A)-binding protein translocates to the nucleus, and polyadenylated mRNAs are sequestered within the nucleus. Virus-induced changes to nuclear architecture that contribute to viral host shutoff (VHS) must accommodate selective processing and export of viral mRNAs. Here we describe additional previously unrecognized nuclear alterations during EBV lytic infection in which viral and cellular factors that function in pre-mRNA processing and mRNA export are redistributed. Early during lytic infection, before formation of viral replication compartments, two cellular pre-mRNA splicing factors, SC35 and SON, were dispersed from interchromatin granule clusters, and three mRNA export factors, Y14, ALY, and NXF1, were depleted from the nucleus. During late lytic infection, virus-induced nodular structures (VINORCs) formed at the periphery of viral replication compartments. VINORCs were composed of viral (BMLF1 and BGLF5) and cellular (SC35, SON, SRp20, and NXF1) proteins that mediate pre-mRNA processing and mRNA export. BHLF1 long noncoding RNA was invariably found in VINORCs. VINORCs did not contain other nodular nuclear cellular proteins (PML or coilin), nor did they contain viral proteins (BRLF1 or BMRF1) found exclusively within replication compartments. VINORCs are novel EBV-induced nuclear structures. We propose that EBV-induced dispersal and depletion of pre-mRNA processing and mRNA export factors during early lytic infection contribute to VHS; subsequent relocalization of these pre-mRNA processing and mRNA export proteins to VINORCs and viral replication compartments facilitates selective processing and export of viral mRNAs.IMPORTANCE In order to make protein, mRNA transcribed from DNA in the nucleus must enter the cytoplasm. Nuclear export of mRNA requires correct processing of mRNAs by enzymes that function in splicing and nuclear export. During the Epstein-Barr virus (EBV) lytic cycle, nuclear export of cellular mRNAs is blocked, yet export of viral mRNAs is facilitated. Here we report the dispersal and dramatic reorganization of cellular (SC35, SON, SRp20, Y14, ALY, and NXF1) and viral (BMLF1 and BGLF5) proteins that play key roles in pre-mRNA processing and export of mRNA. These virus-induced nuclear changes culminate in formation of VINORCs, novel nodular structures composed of viral and cellular RNA splicing and export factors. VINORCs localize to the periphery of viral replication compartments, where viral mRNAs reside. These EBV-induced changes in nuclear organization may contribute to blockade of nuclear export of host mRNA, while enabling selective processing and export of viral mRNA.

Reduction in chromosome mobility accompanies nuclear organization during early embryogenesis in Caenorhabditis elegans

In differentiated cells, chromosomes are packed inside the cell nucleus in an organised fashion. In contrast, little is known about how chromosomes are packed in undifferentiated cells and how nuclear organization changes during development. To assess changes in nuclear organization during the earliest stages of development, we quantified the mobility of a pair of homologous chromosomal loci in the interphase nuclei of Caenorhabditis elegans embryos. The distribution of distances between homologous loci was consistent with a random distribution up to the 8-cell stage but not at later stages. The mobility of the loci was significantly reduced from the 2-cell to the 48-cell stage. Nuclear foci corresponding to epigenetic marks as well as heterochromatin and the nucleolus also appeared around the 8-cell stage. We propose that the earliest global transformation in nuclear organization occurs at the 8-cell stage during C. elegans embryogenesis.

Dynamic changes in the interchromosomal interaction of early histone gene loci during development of sea urchin.

The nuclear positioning and chromatin dynamics of eukaryotic genes are closely related to the regulation of gene expression, but they have not been well examined during early development, which is accompanied by rapid cell cycle progression and dynamic changes in nuclear organization, such as nuclear size and chromatin constitution. In this study, we focused on the early development of the sea urchin Hemicentrotus pulcherrimus and performed three-dimensional fluorescence in situ hybridization of gene loci encoding early histones (one of the types of histone in sea urchin). There are two non-allelic early histone gene loci per sea urchin genome. We found that during the morula stage, when the early histone gene expression levels are at their maximum, interchromosomal interactions were often formed between the early histone gene loci on separate chromosomes and that the gene loci were directed to locate to more interior positions. Furthermore, these interactions were associated with the active transcription of the early histone genes. Thus, such dynamic interchromosomal interactions may contribute to the efficient synthesis of early histone mRNA during the morula stage of sea urchin development.

Light behind the curtain: photoregulation of nuclear architecture and chromatin dynamics in plants.

SummaryLight is a powerful stimulus regulating many aspects of plant development and phenotypic plasticity. Plants sense light through the action of specialized photoreceptor protein families that absorb different wavelengths and intensities of light. Recent discoveries in the area of photobiology have uncovered photoreversible changes in nuclear organization correlated with transcriptional regulation patterns that lead to de‐etiolation and photoacclimation. Novel signalling components bridging photoreceptor activation with chromatin remodelling and regulation of gene expression have been discovered. Moreover, coregulated gene loci have been shown to relocate to the nuclear periphery in response to light. The study of photoinduced changes in nuclear architecture is a flourishing area leading to major discoveries that will allow us to better understand how highly conserved mechanisms underlying genomic reprogramming are triggered by environmental and endogenous stimuli. This review aims to discuss fundamental and innovative reports demonstrating how light triggers changes in chromatin and nuclear architecture during photomorphogenesis.

Building up the nucleus: nuclear organization in the establishment of totipotency and pluripotency during mammalian development.

In mammals, epigenetic reprogramming, the acquisition and loss of totipotency, and the first cell fate decision all occur within a 3-d window after fertilization from the one-cell zygote to the formation of the blastocyst. These processes are poorly understood in molecular detail, yet this is an essential prerequisite to uncover principles of stem cells, chromatin biology, and thus regenerative medicine. A unique feature of preimplantation development is the drastic genome-wide changes occurring to nuclear architecture. From studying somatic and in vitro cultured embryonic stem cells (ESCs) it is becoming increasingly established that the three-dimensional (3D) positions of genomic loci relative to each other and to specific compartments of the nucleus can act on the regulation of gene expression, potentially driving cell fate. However, the functionality, mechanisms, and molecular characteristics of the changes in nuclear organization during preimplantation development are only now beginning to be unraveled. Here, we discuss the peculiarities of nuclear compartments and chromatin organization during mammalian preimplantation development in the context of the transition from totipotency to pluripotency.

Nuclear actin and myosins in adenovirus infection

Adenovirus serotypes have been shown to cause drastic changes in nuclear organization, including the transcription machinery, during infection. This ability of adenovirus to subvert transcription in the host cell facilitates viral replication. Because nuclear actin and nuclear myosin I, myosin V and myosin VI have been implicated as direct regulators of transcription and important factors in the replication of other viruses, we sought to determine how nuclear actin and myosins are involved in adenovirus infection. We first confirmed reorganization of the host's transcription machinery to viral replication centers. We found that nuclear actin also reorganizes to sites of transcription through the intermediate but not the advanced late phase of viral infection. Furthermore, nuclear myosin I localized with nuclear actin and sites of transcription in viral replication centers. Intriguingly, nuclear myosins V and VI, which also reorganized to viral replication centers, exhibited different localization patterns, suggesting specialized roles for these nuclear myosins. Finally, we assessed the role of actin in adenovirus infection and found both cytoplasmic and nuclear actin likely play roles in adenovirus infection and replication. Together our data suggest the involvement of actin and multiple myosins in the nuclear replication and late viral gene expression of adenovirus.

Replicative senescence is associated with nuclear reorganization and with DNA methylation at specific transcription factor binding sites.

BackgroundPrimary cells enter replicative senescence after a limited number of cell divisions. This process needs to be considered in cell culture experiments, and it is particularly important for regenerative medicine. Replicative senescence is associated with reproducible changes in DNA methylation (DNAm) at specific sites in the genome. The mechanism that drives senescence-associated DNAm changes remains unknown - it may involve stochastic DNAm drift due to imperfect maintenance of epigenetic marks or it is directly regulated at specific sites in the genome.ResultsIn this study, we analyzed the reorganization of nuclear architecture and DNAm changes during long-term culture of human fibroblasts and mesenchymal stromal cells (MSCs). We demonstrate that telomeres shorten and shift towards the nuclear center at later passages. In addition, DNAm profiles, either analyzed by MethylCap-seq or by 450k IlluminaBeadChip technology, revealed consistent senescence-associated hypermethylation in regions associated with H3K27me3, H3K4me3, and H3K4me1 histone marks, whereas hypomethylation was associated with chromatin containing H3K9me3 and lamina-associated domains (LADs). DNA hypermethylation was significantly enriched in the vicinity of genes that are either up- or downregulated at later passages. Furthermore, specific transcription factor binding motifs (e.g. EGR1, TFAP2A, and ETS1) were significantly enriched in differentially methylated regions and in the promoters of differentially expressed genes.ConclusionsSenescence-associated DNA hypermethylation occurs at specific sites in the genome and reflects functional changes in the course of replicative senescence. These results indicate that tightly regulated epigenetic modifications during long-term culture contribute to changes in nuclear organization and gene expression.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0057-5) contains supplementary material, which is available to authorized users.

Structural characterization of altered nucleoporin Nup153 expression in human cells by thin-section electron microscopy

Nuclear pore complexes (NPCs) span the 2 membranes of the nuclear envelope (NE) and facilitate nucleocytoplasmic exchange of macromolecules. NPCs have a roughly tripartite structural organization with the so-called nuclear basket emanating from the NPC scaffold into the nucleoplasm. The nuclear basket is composed of the 3 nucleoporins Nup153, Nup50, and Tpr, but their specific role for the structural organization of this NPC substructure is, however, not well established. In this study, we have used thin-section transmission electron microscopy to determine the structural consequences of altering the expression of Nup153 in human cells. We show that the assembly and integrity of the nuclear basket is not affected by Nup153 depletion, whereas its integrity is perturbed in cells expressing high concentrations of the zinc-finger domain of Nup153. Moreover, even mild over-expression of Nup153 is coinciding with massive changes in nuclear organization and it is the excess of the zinc-finger domain of Nup153 that is sufficient to induce these rearrangements. Our data indicate a central function of Nup153 in the organization of the nucleus, not only at the periphery, but throughout the entire nuclear interior.

Quantifying Changes in Nuclear Organization in Normal vs. Cancer Cells using X-ray Tomography

Quantifying Changes in Nuclear Organization in Normal vs. Cancer Cells using X-ray Tomography

Gold nanoparticles induce nuclear damage in breast cancer cells, which is further amplified by hyperthermia.

Gold nanoparticles have emerged as promising tools for cancer research and therapy, where they can promote thermal killing. The molecular mechanisms underlying these events are not fully understood. The geometry and size of gold nanoparticles can determine the severity of cellular damage. Therefore, small and big gold nanospheres as well as gold nanoflowers were evaluated side-by-side. To obtain quantitative data at the subcellular and molecular level, we assessed how gold nanoparticles, either alone or in combination with mild hyperthermia, altered the physiology of cultured human breast cancer cells. Our analyses focused on the nucleus, because this organelle is essential for cell survival. We showed that all the examined gold nanoparticles associated with nuclei. However, their biological effects were quantitatively different. Thus, depending on the shape and size, gold nanoparticles changed multiple nuclear parameters. They redistributed stress-sensitive regulators of nuclear biology, altered the nuclear morphology, reorganized nuclear laminae and envelopes, and inhibited nucleolar functions. In particular, gold nanoparticles reduced the de novo biosynthesis of RNA in nucleoli, the subnuclear compartments that produce ribosomes. While small gold nanospheres and nanoflowers, but not big gold nanospheres, damaged the nucleus at normal growth temperature, several of these defects were further exacerbated by mild hyperthermia. Taken together, the toxicity of gold nanoparticles correlated with changes in nuclear organization and function. These results emphasize that the cell nucleus is a prominent target for gold nanoparticles of different morphologies. Moreover, we demonstrated that RNA synthesis in nucleoli provides quantitative information on nuclear damage and cancer cell survival.

Nanoscale changes in chromatin organization represent the initial steps of tumorigenesis: a transmission electron microscopy study

BackgroundNuclear alterations are a well-known manifestation of cancer. However, little is known about the early, microscopically-undetectable stages of malignant transformation. Based on the phenomenon of field cancerization, the tissue in the field of a tumor can be used to identify and study the initiating events of carcinogenesis. Morphological changes in nuclear organization have been implicated in the field of colorectal cancer (CRC), and we hypothesize that characterization of chromatin alterations in the early stages of CRC will provide insight into cancer progression, as well as serve as a biomarker for early detection, risk stratification and prevention.MethodsFor this study we used transmission electron microscopy (TEM) images of nuclei harboring pre-neoplastic CRC alterations in two models: a carcinogen-treated animal model of early CRC, and microscopically normal-appearing tissue in the field of human CRC. We quantify the chromatin arrangement using approaches with two levels of complexity: 1) binary, where chromatin is separated into areas of dense heterochromatin and loose euchromatin, and 2) grey-scale, where the statistics of continuous mass-density distribution within the nucleus is quantified by its spatial correlation function.ResultsWe established an increase in heterochromatin content and clump size, as well as a loss of its characteristic peripheral positioning in microscopically normal pre-neoplastic cell nuclei. Additionally, the analysis of chromatin density showed that its spatial distribution is altered from a fractal to a stretched exponential.ConclusionsWe characterize quantitatively and qualitatively the nanoscale structural alterations preceding cancer development, which may allow for the establishment of promising new biomarkers for cancer risk stratification and diagnosis. The findings of this study confirm that ultrastructural changes of chromatin in field carcinogenesis represent early neoplastic events leading to the development of well-documented, microscopically detectable hallmarks of cancer.

Physical clustering of FLC alleles during Polycomb-mediated epigenetic silencing in vernalization

Vernalization, the promotion of flowering by cold, involves Polycomb-mediated epigenetic silencing of FLOWERING LOCUS C (FLC). Cold progressively promotes cell-autonomous switching to a silenced state. Here, we used live-cell imaging of FLC-lacO to monitor changes in nuclear organization during vernalization. FLC-lacO alleles physically cluster during the cold and generally remain so after plants are returned to warm. Clustering is dependent on the Polycomb trans-factors necessary for establishment of the FLC silenced state but not on LIKE HETEROCHROMATIN PROTEIN 1, which functions to maintain silencing. These data support the view that physical clustering may be a common feature of Polycomb-mediated epigenetic switching mechanisms.