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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: Results indicate that Arp6 and H2A.Z contribute to the radial distribution of CTs through different mechanisms, which indicates that the localization of chromatin to the nuclear periphery per se is insufficient for the repression of most genes.
Abstract: The spatial organization of chromatin in the nucleus contributes to genome function and is altered during the differentiation of normal and tumorigenic cells. Although nuclear actin-related proteins (Arps) have roles in the local alteration of chromatin structure, it is unclear whether they are involved in the spatial positioning of chromatin. In the interphase nucleus of vertebrate cells, gene-dense and gene-poor chromosome territories (CTs) are located in the center and periphery, respectively. We analyzed chicken DT40 cells in which Arp6 had been knocked out conditionally, and showed that the radial distribution of CTs was impaired in these knockout cells. Arp6 is an essential component of the SRCAP chromatin remodeling complex, which deposits the histone variant H2A.Z into chromatin. The redistribution of CTs was also observed in H2A.Z-deficient cells for gene-rich microchromosomes, but to lesser extent for gene-poor macrochromosomes. These results indicate that Arp6 and H2A.Z contribute to the radial distribution of CTs through different mechanisms. Microarray analysis suggested that the localization of chromatin to the nuclear periphery per se is insufficient for the repression of most genes.

14 citations


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

  • ...Z to development and tumorigenesis through nuclear organization Previous studies have shown that the intranuclear repositioning of chromosomal loci is linked to the differentiation of normal and tumorigenic cells (Cremer et al., 2003; Meaburn et al., 2009; Wiblin et al., 2005; Wiech et al., 2005)....

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  • ...linked to the differentiation of normal and tumorigenic cells (Cremer et al., 2003; Meaburn et al., 2009; Wiblin et al., 2005; Wiech et al., 2005)....

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Journal ArticleDOI
TL;DR: The results suggest the existence of mechanisms that drive the nuclear positioning of centromeres, and provide an accurate characterization of how their organization varies as a function of the proliferation state in human lymphoblastoid cells.
Abstract: The cell nucleus is a highly organized structure and plays an important role in gene regulation. Understanding the mechanisms that sustain this organization is therefore essential for understanding genome function. Centromeric regions (CRs) of chromosomes have been known for years to adopt specific nuclear positioning patterns, but the significance of this observation is not yet completely understood. Here, using a combination of fluorescence in situ hybridization and immunochemistry on fixed human cells and high-throughput imaging, we directly and quantitatively investigated the nuclear positioning of specific human CRs. We observe differential attraction of individual CRs toward both the nuclear border and the nucleoli, the former being enhanced in nonproliferating cells and the latter being enhanced in proliferating cells. Similar positioning patterns are observed in two different lymphoblastoid cell lines. Moreover, the positioning of CRs differs from that of noncentromeric regions, and CRs display specific orientations within chromosome territories. These results suggest the existence of not-yet-characterized mechanisms that drive the nuclear positioning of CRs and therefore pave the way toward a better understanding of how CRs affect nuclear organization.

14 citations


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

  • ...…the lack of reference points in the nucleus, many studies have focused on the radial distribution of nuclear structures, which are computed by measuring distance to either the nuclear border (Wiblin et al., 2005) or a computed nucleus center that has no biological meaning (Weierich et al., 2003)....

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  • ...…with the nuclear lamina, as well as with the nucleolus, in both mouse and human cells and that their distribution in the interphase nucleus is modified in relation to the cell cycle, as well as to physiological and differentiation states (reviewed in Pluta et al., 1995; Wiblin et al., 2005)....

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  • ...Owing to the lack of reference points in the nucleus, many studies have focused on the radial distribution of nuclear structures, which are computed by measuring distance to either the nuclear border (Wiblin et al., 2005) or a computed nucleus center that has no biological meaning (Weierich et al....

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  • ...It will be interesting to check whether the previously reported increased association of CRs with the nuclear border observed during cell differentiation reflects global reorganization of heterochromatin in these cells or is just the consequence of a higher proportion of nonproliferating cells (Wiblin et al., 2005)....

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  • ...Since the pioneering work of Manuelidis and coworkers (Manuelidis, 1984, 1985), several groups have shown that centromeric regions are mobile structures that associate with the nuclear lamina, as well as with the nucleolus, in both mouse and human cells and that their distribution in the interphase nucleus is modified in relation to the cell cycle, as well as to physiological and differentiation states (reviewed in Pluta et al., 1995; Wiblin et al., 2005)....

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Journal ArticleDOI
TL;DR: During normal human hematopoietic differentiation, changes in gene subnuclear location relative to pericentromeric heterochromatin appear to be dictated by whether the gene will be permanently silenced or activated, rather than being predictive of commitment toward a given lineage.
Abstract: To further clarify the contribution of nuclear architecture in the regulation of gene expression patterns during differentiation of human multipotent cells, we analyzed expression status, histone modifications, and subnuclear positioning relative to repressive compartments, of hematopoietic loci in multipotent and lineage-committed primary human hematopoietic progenitors. We report here that positioning of lineage-affiliated loci relative to pericentromeric heterochromatin compartments (PCH) is identical in multipotent cells from various origins and is unchanged between multipotent and lineage-committed hematopoietic progenitors. However, during differentiation of multipotent hematopoietic progenitors, changes in gene expression and histone modifications at these loci occur in committed progenitors, prior to changes in gene positioning relative to pericentromeric heterochromatin compartments, detected at later stages in precursor and mature cells. Therefore, during normal human hematopoietic differentiation, changes in gene subnuclear location relative to pericentromeric heterochromatin appear to be dictated by whether the gene will be permanently silenced or activated, rather than being predictive of commitment toward a given lineage.

14 citations

Journal ArticleDOI
TL;DR: It is demonstrated that α5β1 integrin functional blockade induces cell migration of hepatic progenitor cells, and that this involves a dramatic remodeling of the nuclear landscape.
Abstract: Cell scattering is a physiological process executed by stem and progenitor cells during embryonic liver development and postnatal organ regeneration. Here, we investigated the genomic events occurring during this process induced by functional blockade of α5β1 integrin in liver progenitor cells. Cells treated with a specific antibody against α5β1 integrin exhibited cell spreading and scattering, over-expression of liver stem/progenitor cell markers and activation of the ERK1/2 and p38 MAPKs signaling cascades, in a similar manner to the process triggered by HGF/SF1 stimulation. Gene expression profiling revealed marked transcriptional changes of genes involved in cell adhesion and migration, as well as genes encoding chromatin remodeling factors. These responses were accompanied by conspicuous spatial reorganization of centromeres, while integrin genes conserved their spatial positioning in the interphase nucleus. Collectively, our results demonstrate that α5β1 integrin functional blockade induces cell migration of hepatic progenitor cells, and that this involves a dramatic remodeling of the nuclear landscape.

13 citations


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

  • ...For example, the stem cell specific genes Nanog and Oct4 acquire differential positioning in the nucleus as their expression levels change during differentiation of human embryonic stem cells [9]....

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Journal ArticleDOI
TL;DR: In this article, the authors developed a framework to dissect spatial interactions and organization principles by combining unbiased statistical tests, multiple spatial descriptors and new spatial models and used plant constitutive heterochromatin as a model system to demonstrate the potential of their framework.
Abstract: The spatial organization in the cell nucleus is tightly linked to genome functions such as gene regulation. Similarly, specific spatial arrangements of biological components such as macromolecular complexes, organelles and cells are involved in many biological functions. Spatial interactions among elementary components of biological systems define their relative positioning and are key determinants of spatial patterns. However, biological variability and the lack of appropriate spatial statistical methods and models limit our current ability to analyze these interactions. Here, we developed a framework to dissect spatial interactions and organization principles by combining unbiased statistical tests, multiple spatial descriptors and new spatial models. We used plant constitutive heterochromatin as a model system to demonstrate the potential of our framework. Our results challenge the common view of a peripheral organization of chromocenters, showing that chromocenters are arranged along both radial and lateral directions in the nuclear space and obey a multiscale organization with scale-dependent antagonistic effects. The proposed generic framework will be useful to identify determinants of spatial organizations and to question their interplay with biological functions.

12 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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