Despite its size and rigidity, the cell nucleus can be moved or reorganized by cytoskeletal filaments under various conditions (for example, during viral infection). Moreover, whereas chromatin organizes into non-random domains, extensive heterogeneity at the single-cell level means that precisely how and why nuclei reorganize remains an area of intense investigation. Here we describe convolutional neural network-based automated cell classification and analysis pipelines, which revealed the extent to which human cytomegalovirus generates nuclear polarity through a virus-assembled microtubule-organizing centre.
View Article and Find Full Text PDFAn amendment to this paper has been published and can be accessed via a link at the top of the paper.
View Article and Find Full Text PDFThe three-dimensional organization of the genome in mammalian interphase nuclei is intrinsically linked to the regulation of gene expression. Whole chromosome territories and their encoded gene loci occupy preferential positions within the nucleus that changes according to the expression profile of a given cell lineage or stage. To further illuminate the relationship between chromosome organization, epigenetic environment, and gene expression, here we examine the functional organization of chromosome X and corresponding X-linked genes in a variety of healthy human and disease state X diploid (XX) cells.
View Article and Find Full Text PDFThe protein-DNA complexes that compose the end of mammalian chromosomes-telomeres-serve to stabilize linear genomic DNA and are involved in cellular and organismal aging. One mechanism that protects telomeres from premature degradation is the formation of structures called t-loops, in which the single-stranded 3' overhang present at the terminal end of telomeres loops back and invades medial double-stranded telomeric DNA. We identified looped structures formed between terminal chromosome ends and interstitial telomeric sequences (ITSs), which are found throughout the human genome, that we have termed interstitial telomeric loops (ITLs).
View Article and Find Full Text PDFSex chromosome gene dosage compensation is required to ensure equivalent levels of X-linked gene expression between males (46, XY) and females (46, XX). To achieve similar expression, X-chromosome inactivation (XCI) is initiated in female cells during early stages of embryogenesis. Within each cell, either the maternal or paternal X chromosome is selected for whole chromosome transcriptional silencing, which is initiated and maintained by epigenetic and chromatin conformation mechanisms.
View Article and Find Full Text PDFActin is abundant in the nucleus and it is clear that nuclear actin has important functions. However, mystery surrounds the absence of classical actin filaments in the nucleus. To address this question, we investigated how polymerizing nuclear actin into persistent nuclear actin filaments affected transcription by RNA polymerase II.
View Article and Find Full Text PDFHigher order chromatin structure establishes domains that organize the genome and coordinate gene expression. However, the molecular mechanisms controlling transcription of individual loci within a topological domain (TAD) are not fully understood. The cystic fibrosis transmembrane conductance regulator (CFTR) gene provides a paradigm for investigating these mechanisms.
View Article and Find Full Text PDFEver since the first demonstration of their repetitive sequence and unique replication pathway, telomeres have beguiled researchers with how they function in protecting chromosome ends. Of course much has been learned over the years, and we now appreciate that telomeres are comprised of the multimeric protein/DNA shelterin complex and that the formation of t-loops provides protection from DNA damage machinery. Deriving their name from D-loops, t-loops are generated by the insertion of the 3' overhang into telomeric repeats facilitated by the binding of TRF2.
View Article and Find Full Text PDFTelomeres protect the ends of linear genomes, and the gradual loss of telomeres is associated with cellular ageing. Telomere protection involves the insertion of the 3' overhang facilitated by telomere repeat-binding factor 2 (TRF2) into telomeric DNA, forming t-loops. We present evidence suggesting that t-loops can also form at interstitial telomeric sequences in a TRF2-dependent manner, forming an interstitial t-loop (ITL).
View Article and Find Full Text PDFHigher order chromatin structures across the genome are maintained in part by the architectural proteins CCCTC binding factor (CTCF) and the cohesin complex, which co-localize at many sites across the genome. Here, we examine the role of these proteins in mediating chromatin structure at the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR encompasses nearly 200 kb flanked by CTCF-binding enhancer-blocking insulator elements and is regulated by cell-type-specific intronic enhancers, which loop to the promoter in the active locus.
View Article and Find Full Text PDFα-Catenin (α-cat) is an actin-binding protein required for cell-cell cohesion. Although this adhesive function for α-cat is well appreciated, cells contain a substantial amount of nonjunctional α-cat that may be used for other functions. We show that α-cat is a nuclear protein that can interact with β-catenin (β-cat) and T-cell factor (TCF) and that the nuclear accumulation of α-cat depends on β-cat.
View Article and Find Full Text PDFBacterial artificial chromosomes (BACs) are widely used in transgenesis, particularly for the humanization of animal models. Moreover, due to their extensive capacity, BACs provide attractive tools to study distal regulatory elements associated with large gene loci. However, despite their widespread use, little is known about the integration dynamics of these large transgenes in mammalian cells.
View Article and Find Full Text PDFNuclear lamin B1 (LB1) is a major structural component of the nucleus that appears to be involved in the regulation of many nuclear functions. The results of this study demonstrate that LB1 expression in WI-38 cells decreases during cellular senescence. Premature senescence induced by oncogenic Ras also decreases LB1 expression through a retinoblastoma protein (pRb)-dependent mechanism.
View Article and Find Full Text PDFThe molecular events leading to human embryonic stem cell (hESC) differentiation are the subject of considerable scrutiny. Here, we characterize an in vitro model that permits analysis of the earliest steps in the transition of hESC colonies to squamous epithelium on basic fibroblast growth factor withdrawal. A set of markers (GSC, CK18, Gata4, Eomes, and Sox17) point to a mesendodermal nature of the epithelial cells with subsequent commitment to definitive endoderm (Sox17, Cdx2, nestin, and Islet1).
View Article and Find Full Text PDFTranscriptional noise has an important role in generating diversity in cellular populations that are seemingly identical. As this noise stems from the inherent stochasticity of gene expression, it has been unclear whether it is directly controlled. Dig1, a regulator of the budding yeast mating pathway, is now shown to prevent transcriptional noise by regulating the spatial organization of downstream gene targets.
View Article and Find Full Text PDFThe nuclei of differentiating cells exhibit several fundamental principles of self-organization. They are composed of many dynamical units connected physically and functionally to each other--a complex network--and the different parts of the system are mutually adapted and produce a characteristic end state. A unique cell-specific signature emerges over time from complex interactions among constituent elements that delineate coordinate gene expression and chromosome topology.
View Article and Find Full Text PDFCurr Opin Cell Biol
June 2010
The eukaryotic nucleus is functionally organized. Gene loci, for example, often reveal altered localization patterns according to their developmental regulation. Whole chromosomes also demonstrate non-random nuclear positions, correlated with inherent characteristics such as gene density or size.
View Article and Find Full Text PDFAlthough the importance of chromosome organization during mitosis is clear, it remains to be determined whether the nucleus assumes other functionally relevant chromosomal topologies. We have previously shown that homologous chromosomes have a tendency to associate during hematopoiesis according to their distribution of coregulated genes, suggesting cell-specific nuclear organization. Here, using the mathematical approaches of distance matrices and coupled oscillators, we model the dynamic relationship between gene expression and chromosomal associations during the differentiation of a multipotential hematopoietic progenitor.
View Article and Find Full Text PDFWhen transgenic mice are created by microinjection of DNA into the pronucleus, the sites of DNA integration into the mouse genome cannot be predicted. Most methods based on polymerase chain reaction (PCR) that have been used for determining the integration site of foreign DNA into a genome require specific reagents and/or complicated manipulations making routine use tedious. In this report we demonstrate the use of a PCR-based method-TAIL-PCR (Thermal Asymmetric Interlaced PCR) which relies on a series of PCR amplifications with gene specific and degenerate primers to reliably amplify the integration sites.
View Article and Find Full Text PDFGene loci are found in nuclear subcompartments that are related to their expression status. For instance, silent genes are often localized to heterochromatin and the nuclear periphery, whereas active genes tend to be found in the nuclear center. Evidence also suggests that chromosomes may be specifically positioned within the nucleus; however, the nature of this organization and how it is achieved are not yet fully understood.
View Article and Find Full Text PDFDuplication of the genome during the S phase of the cell cycle does not occur simultaneously; rather, different sequences are replicated at different times. The replication timing of specific sequences can change during development; however, the determinants of this dynamic process are poorly understood. To gain insights into the contribution of developmental state, genomic sequence, and transcriptional activity to replication timing, we investigated the timing of DNA replication at high resolution along an entire human chromosome (chromosome 22) in two different cell types.
View Article and Find Full Text PDFWhen considering the daunting complexity of eukaryotic genomes, some comfort can be found in the fact that the human genome may contain only 30,000 to 40,000 genes. Moreover, growing evidence suggests that genomes may be organized in such a way as to take advantage of space. A gene's location in the linear DNA sequence and its position in the three-dimensional nucleus can both be important in its regulation.
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