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Journal ArticleDOI

Distinctive nuclear organisation of centromeres and regions involved in pluripotency in human embryonic stem cells

01 Sep 2005-Journal of Cell Science (The Company of Biologists Ltd)-Vol. 118, Iss: 17, pp 3861-3868
TL;DR: It is concluded that hES cell nuclei have a distinct nuclear architecture, especially at loci involved in maintaining pluripotency, which provides a framework within which other large-scale chromatin changes that may accompany differentiation can be considered.
Abstract: Nuclear organisation is thought to be important in regulating gene expression. Here we investigate whether human embryonic stem cells (hES) have a particular nuclear organisation, which could be important for maintaining their pluripotent state. We found that whereas the nuclei of hES cells have a general gene-density-related radial organisation of chromosomes, as is seen in differentiated cells, there are also distinctive localisations for chromosome regions and gene loci with a role in pluripotency. Chromosome 12p, a region of the human genome that contains clustered pluripotency genes including NANOG, has a more central nuclear localisation in ES cells than in differentiated cells. On chromosome 6p we find no overall change in nuclear chromosome position, but instead we detect a relocalisation of the OCT4 locus, to a position outside its chromosome territory. There is also a smaller proportion of centromeres located close to the nuclear periphery in hES cells compared to differentiated cells. We conclude that hES cell nuclei have a distinct nuclear architecture, especially at loci involved in maintaining pluripotency. Understanding this level of hES cell biology provides a framework within which other large-scale chromatin changes that may accompany differentiation can be considered.

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Citations
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Journal ArticleDOI
TL;DR: Current understanding of telomere dynamics in stem cell populations is discussed, and some data is provided to indicate that telomeres in human embryonic stem cells may be more stable and less prone to large-scale stochastic telomeric deletion.

38 citations


Cites background from "Distinctive nuclear organisation of..."

  • ...display a nuclear architecture, such that loci required for the maintenance of pluripotency, display specific nuclear locations; for example NANOG on 12p has a more central location in ES cells compared to differentiated cells, or 6p that maintains its chromosome position but the OCT4 locus on that chromosome is outside of its usual position [90]....

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Journal ArticleDOI
TL;DR: These analyses discriminate for the first time two contrasted types of nuclear organization in NT embryos, which may correspond to different functional states of the nuclei.
Abstract: Efficient reprograming of the donor cell genome in nuclear transfer (NT) embryos is linked to the ability of the embryos to sustain full-term development. As the nuclear architecture has recently emerged as a key factor in the regulation of gene expression, we questioned whether early bovine embryos obtained from transfer of cultured fibroblasts into enucleated oocytes would adopt an embryo-like nuclear organization. We studied the dynamics of constitutive heterochromatin in the stages prior to embryonic genome activation by distribution analysis of heterochromatin protein CBX1 (HP1), centromeric proteins CENPA and CENPB, and histone H3 three-methylated at lysine 9. Then we applied descriptive, quantitative, and co-localization analyses. A dramatic reorganization of heterochromatic blocks of somatic donor cells was first observed in the late one-cell stage NT embryos. Then at two- and four-cell stages, we found two types of NT embryos: one displaying noncondensed heterochromatin patches similar to IVF embryos, whereas the second type displayed condensed heterochromatin blocks, normally observed in IVF embryos only after the eight-cell stage. These analyses discriminate for the first time two contrasted types of nuclear organization in NT embryos, which may correspond to different functional states of the nuclei. The relationship with the somatic nucleus reprograming efficiency is discussed.

37 citations


Cites background from "Distinctive nuclear organisation of..."

  • ...However, the structure and distribution of chromocenters is cell type specific and may vary among mammalian species, as well as the associated epigenetic marks (Solovei et al. 2004, Brero et al. 2005, Mayer et al. 2005, Wiblin et al. 2005)....

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Journal ArticleDOI
TL;DR: It is suggested that during activation of the caspase-8 pathway changes in the lamina structure precede changes in heterochromatin spatial organization, and the subsequent breakdown of lamina and PML-NB.
Abstract: Apoptosis is fundamental to the regulation of homeostasis of stem cells in vivo. Whereas the pathways underlying the molecular and biochemical details of nuclear breakdown that accompanies apoptosis have been elucidated, the precise nature of nuclear reorganization that precedes the demolition phase is not fully understood. Here, we expressed an inducible caspase-8 in human mesenchymal stem cells, and quantitatively followed the early changes in nuclear organization during apoptosis. We found that caspase-8 induces alteration of the nuclear lamina and a subsequent spatial reorganization of both centromeres, which are shifted towards a peripheral localization, and telomeres, which form aggregates. This nuclear reorganization correlates with caspase-3 sensitivity of lamina proteins, because the expression of lamin mutant constructs with caspase-3 hypersensitivity resulted in a caspase-8-independent appearance of lamina intranuclear structures and telomere aggregates, whereas application of a caspase inhibitor restrains these changes in nuclear reorganization. Notably, upon activation of apoptosis, we observed no initial changes in the spatial organization of the promyelocytic leukemia nuclear bodies (PML-NBs). We suggest that during activation of the caspase-8 pathway changes in the lamina structure precede changes in heterochromatin spatial organization, and the subsequent breakdown of lamina and PML-NB.

37 citations


Cites background or result from "Distinctive nuclear organisation of..."

  • ...Previous studies reported changes in the localization of telomeres and centromeres during the cell cycle (Chuang et al., 2004; Solovei et al., 2004) and cell differentiation (Gilchrist et al., 2004; Solovei et al., 2004; Wiblin et al., 2005)....

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  • ...Changes in the preferred distribution of centromeres and telomeres were found during the cell cycle and in differentiated cells (Solovei et al., 2004; Stadler et al., 2004; Wiblin et al., 2005; Weierich et al., 2003; Chuang et al., 2004)....

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Journal ArticleDOI
11 May 2015-PLOS ONE
TL;DR: The inter-chromosomal segment and gene contact networks in human and mouse embryonic stem cells do not appear to be conserved and a significant association between spatial proximity and the co-expression of genes in the human genome is found.
Abstract: The recent advent of conformation capture techniques has provided unprecedented insights into the spatial organization of chromatin. We present a large-scale investigation of the inter-chromosomal segment and gene contact networks in embryonic stem cells of two mammalian organisms: humans and mice. Both interaction networks are characterized by a high degree of clustering of genome regions and the existence of hubs. Both genomes exhibit similar structural characteristics such as increased flexibility of certain Y chromosome regions and co-localization of centromere-proximal regions. Spatial proximity is correlated with the functional similarity of genes in both species. We also found a significant association between spatial proximity and the co-expression of genes in the human genome. The structural properties of chromatin are also species specific, including the presence of two highly interactive regions in mouse chromatin and an increased contact density on short, gene-rich human chromosomes, thereby indicating their central nuclear position. Trans-interacting segments are enriched in active marks in human and had no distinct feature profile in mouse. Thus, in contrast to interactions within individual chromosomes, the inter-chromosomal interactions in human and mouse embryonic stem cells do not appear to be conserved.

34 citations


Cites background from "Distinctive nuclear organisation of..."

  • ...It is known that the centromeres in human ESCs (hESCs) are less likely to localize to the periphery and the nucleolus than those in differentiated human cells [26], implying that centromere co-localization is less pronounced in this undifferentiated cell type....

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  • ...In differentiated human cell types, the centromeres tend to be located at the nuclear periphery, or at the nucleolus, whereas they are located more centrally in hESCs [26]....

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  • ...[26], centromeres appear in 34 clusters in this cell type, thereby implying that some smaller spatial clusters of multiple chromosome centromeres do exist....

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Journal ArticleDOI
TL;DR: The results indicate that hPSCs exhibit a unique mechanism for maintaining genomic integrity by possessing the flexibility to reduce the amount of CENP-A required to maintain a functional centromere under self-renewing conditions, and maintaining a reserve of CenP- A mRNA to rebuild the centromer following differentiation or DNA damage.
Abstract: Human pluripotent stem cells (hPSCs) hold significant promise for use in regenerative medicine, or as a model to understand human embryo development. However, the basic mechanisms required for proliferation and self-renewal of hPSCs have not been fully uncovered. Proliferation in all eukaryotes is dependent upon highly regulated expression of the histone H3 variant Centromere protein A (CENP-A). In the current study, we demonstrate that hPSCs have a unique messenger ribonucleic acid (mRNA) reserve of CENP-A not found in somatic fibroblasts. Using short hairpin RNA technology to reduce but not ablate CENP-A, we show that CENP-A-depleted hPSCs are still capable of maintaining a functional centromeric mark, whereas fibroblasts are not. However, upon induction of differentiation or DNA damage, hPSCs with depleted CENP-A arrest in G2/M and undergo apoptosis. Analysis of CENP-A dynamics following DNA damage in hPSCs reveals that 60 min after irradiation, CENP-A is found in multiple small nuclear foci that are mutually exclusive to γH2AX as well as CENP-C. Furthermore, following irradiation, hPSCs with depleted CENP-A mount a normal apoptotic response at 6 h; however at 24 h, apoptosis is significantly increased in CENP-A-depleted hPSCs relative to control. Taken together, our results indicate that hPSCs exhibit a unique mechanism for maintaining genomic integrity by possessing the flexibility to reduce the amount of CENP-A required to maintain a functional centromere under self-renewing conditions, and maintaining a reserve of CENP-A mRNA to rebuild the centromere following differentiation or DNA damage.

34 citations

References
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Journal ArticleDOI
06 Nov 1998-Science
TL;DR: Human blastocyst-derived, pluripotent cell lines are described that have normal karyotypes, express high levels of telomerase activity, and express cell surface markers that characterize primate embryonic stem cells but do not characterize other early lineages.
Abstract: Human blastocyst-derived, pluripotent cell lines are described that have normal karyotypes, express high levels of telomerase activity, and express cell surface markers that characterize primate embryonic stem cells but do not characterize other early lineages. After undifferentiated proliferation in vitro for 4 to 5 months, these cells still maintained the developmental potential to form trophoblast and derivatives of all three embryonic germ layers, including gut epithelium (endoderm); cartilage, bone, smooth muscle, and striated muscle (mesoderm); and neural epithelium, embryonic ganglia, and stratified squamous epithelium (ectoderm). These cell lines should be useful in human developmental biology, drug discovery, and transplantation medicine.

15,555 citations


"Distinctive nuclear organisation of..." refers background or methods in this paper

  • ...Human ES cells have been derived from the inner cell mass of blastocysts, and as well as being able to self-renew, they have the ability to differentiate into all three embryonic germ layers when injected into severe combined immunodeficient mice (Thomson et al., 1998)....

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  • ...Human ES cell culture and analysis Human ES cell lines H1 (46XY), H7 and H9 (46XX) (Thomson et al., 1998) were grown as previously described, with minor modification (Xu et al., 2001)....

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  • ...Germ cells and stem cells in contrast have active telomerase, and robust telomerase activity is detected in hES cells (Thomson et al., 1998)....

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  • ...Human ES cell lines H1 (46XY), H7 and H9 (46XX) (Thomson et al., 1998) were grown as previously described, with minor modification (Xu et al....

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Journal ArticleDOI
TL;DR: A successful feeder-free hES culture system in which undifferentiated cells can be maintained for at least 130 population doublings and are suitable for scaleup production is demonstrated.
Abstract: Previous studies have shown that maintenance of undifferentiated human embryonic stem (hES) cells requires culture on mouse embryonic fibroblast (MEF) feeders. Here we demonstrate a successful feeder-free hES culture system in which undifferentiated cells can be maintained for at least 130 population doublings. In this system, hES cells are cultured on Matrigel or laminin in medium conditioned by MEF. The hES cells maintained on feeders or off feeders express integrin alpha6 and beta1, which may form a laminin-specific receptor. The hES cell populations in feeder-free conditions maintained a normal karyotype, stable proliferation rate, and high telomerase activity. Similar to cells cultured on feeders, hES cells maintained under feeder-free conditions expressed OCT-4, hTERT, alkaline phosphatase, and surface markers including SSEA-4, Tra 1-60, and Tra 1-81. In addition, hES cells maintained without direct feeder contact formed teratomas in SCID/beige mice and differentiated in vitro into cells from all three germ layers. Thus, the cells retain fundamental characteristics of hES cells in this culture system and are suitable for scaleup production.

2,092 citations


"Distinctive nuclear organisation of..." refers methods in this paper

  • ...Human ES cell culture and analysis Human ES cell lines H1 (46XY), H7 and H9 (46XX) (Thomson et al., 1998) were grown as previously described, with minor modification (Xu et al., 2001)....

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  • ..., 1998) were grown as previously described, with minor modification (Xu et al., 2001)....

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Journal ArticleDOI
18 Oct 2002-Science
TL;DR: The transcriptional profiles of mouse embryonic, neural, and hematopoietic stem cells were compared to define a genetic program for stem cells and provide a foundation for a more detailed understanding of stem cell biology.
Abstract: The transcriptional profiles of mouse embryonic, neural, and hematopoietic stem cells were compared to define a genetic program for stem cells. A total of 216 genes are enriched in all three types of stem cells, and several of these genes are clustered in the genome. When compared to differentiated cell types, stem cells express a significantly higher number of genes (represented by expressed sequence tags) whose functions are unknown. Embryonic and neural stem cells have many similarities at the transcriptional level. These results provide a foundation for a more detailed understanding of stem cell biology.

1,776 citations


"Distinctive nuclear organisation of..." refers background in this paper

  • ..., 2002b) and hES cells (Ramalho-Santos et al., 2002), but which is located in a low gene-density region at 11p13 (32Mb), remains inside the CT (Table 1)....

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  • ...In contrast, RCN, which is expressed in both LCLs (Mahy et al., 2002b) and hES cells (Ramalho-Santos et al., 2002), but which is located in a low gene-density region at 11p13 (32Mb), remains inside the CT (Table 1)....

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Journal ArticleDOI
TL;DR: It is suggested that increased dosage of chromosome 17q and 12 gene(s) provides a selective advantage for the propagation of undifferentiated hES cells in transplantation therapies in which the use of aneuploid cells could be detrimental.
Abstract: We have observed karyotypic changes involving the gain of chromosome 17q in three independent human embryonic stem (hES) cell lines on five independent occasions. A gain of chromosome 12 was seen occasionally. This implies that increased dosage of chromosome 17q and 12 gene(s) provides a selective advantage for the propagation of undifferentiated hES cells. These observations are instructive for the future application of hES cells in transplantation therapies in which the use of aneuploid cells could be detrimental.

1,046 citations


"Distinctive nuclear organisation of..." refers background in this paper

  • ...It is interesting to note that recurrent gains of chromosome 12, including iso12p, have been found in human ES cells (Draper et al., 2004)....

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Journal ArticleDOI
TL;DR: It is demonstrated that the distribution of genomic sequences between chromosomes has implications for nuclear structure and the findings are discussed in relation to a model of the human nucleus that is functionally compartmentalized.
Abstract: Using fluorescence in situ hybridization we show striking differences in nuclear position, chromosome morphology, and interactions with nuclear substructure for human chromosomes 18 and 19. Human chromosome 19 is shown to adopt a more internal position in the nucleus than chromosome 18 and to be more extensively associated with the nuclear matrix. The more peripheral localization of chromosome 18 is established early in the cell cycle and is maintained thereafter. We show that the preferential localization of chromosomes 18 and 19 in the nucleus is reflected in the orientation of translocation chromosomes in the nucleus. Lastly, we show that the inhibition of transcription can have gross, but reversible, effects on chromosome architecture. Our data demonstrate that the distribution of genomic sequences between chromosomes has implications for nuclear structure and we discuss our findings in relation to a model of the human nucleus that is functionally compartmentalized.

914 citations


"Distinctive nuclear organisation of..." refers background or methods in this paper

  • ...HSA18 is found towards the nuclear periphery in a variety of differentiated cells and HSA19 is in the centre of the nucleus (Croft et al., 1999; Cremer et al., 2003)....

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  • ...Hybridisation was as described previously (Croft et al., 1999) but with the denaturing time reduced to 1....

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  • ...Chromosome paints were labelled with biotin-16-dUTP by nick translation or by PCR amplification (Croft et al., 1999) or obtained commercially (Cambio)....

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  • ...Slides were then subjected to freeze-thaw in 20% glycerol/PBS and FISH was carried out as described previously (Croft et al., 1999)....

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  • ...The radial distribution of CTs was determined in 2D specimens by an erosion script, as previously described (Croft et al., 1999)....

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