Distinctive nuclear organisation of centromeres and regions involved in pluripotency in human embryonic stem cells
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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TL;DR: A high-resolution map of the interaction sites of the entire genome with NL components in human fibroblasts is constructed and demonstrates that the human genome is divided into large, discrete domains that are units of chromosome organization within the nucleus.
Abstract: The architecture of human chromosomes in interphase nuclei is still largely unknown. Microscopy studies have indicated that specific regions of chromosomes are located in close proximity to the nuclear lamina (NL). This has led to the idea that certain genomic elements may be attached to the NL, which may contribute to the spatial organization of chromosomes inside the nucleus. However, sequences in the human genome that interact with the NL in vivo have not been identified. Here we construct a high-resolution map of the interaction sites of the entire genome with NL components in human fibroblasts. This map shows that genome-lamina interactions occur through more than 1,300 sharply defined large domains 0.1-10 megabases in size. These lamina-associated domains (LADs) are typified by low gene-expression levels, indicating that LADs represent a repressive chromatin environment. The borders of LADs are demarcated by the insulator protein CTCF, by promoters that are oriented away from LADs, or by CpG islands, suggesting possible mechanisms of LAD confinement. Taken together, these results demonstrate that the human genome is divided into large, discrete domains that are units of chromosome organization within the nucleus.
1,762 citations
TL;DR: It is suggested that hyperdynamic binding of structural chromatin proteins is a functionally important hallmark of pluripotent ES cells that contributes to the maintenance of plasticity in undifferentiated ES cells and to establishing higher-order chromatin structure.
1,003 citations
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...In another system, no significant differences in the extent of centromere clustering were observed between undifferentiated human ES cells and two diploid differentiated cell types, including a lymphoblastoid cell line (FATO LCL) and primary fibroblasts (Wiblin et al., 2005)....
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...However, centromeres in ES cells were mainly found within the nuclear interior, whereas, in differentiated cells, centromeres tend to localize at the nuclear periphery (Wiblin et al., 2005)....
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TL;DR: This work discusses how unique properties of chromatin in ES cells contribute to the maintenance of pluripotency and the determination of differentiation properties.
Abstract: What makes a stem cell is still poorly understood. Recent studies have uncovered that chromatin might hold some of the keys to how embryonic stem cells maintain their pluripotency, their ability to self-renew and induce lineage specification. Embryonic stem (ES) cells are unique in that they are pluripotent and have the ability to self-renew. The molecular mechanisms that underlie these two fundamental properties are largely unknown. We discuss how unique properties of chromatin in ES cells contribute to the maintenance of pluripotency and the determination of differentiation properties.
707 citations
Cites background from "Distinctive nuclear organisation of..."
...For example, comparable distributions of centromeres and of promyelocytic leukaemia (PML) bodies (which are implicated in transcription, apoptosis and cellular stress processes) are found in both human ES cells and differentiated cell...
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TL;DR: It is found that differentiated tissues show surprisingly large K9-modified regions (up to 4.9 Mb), which are large organized chromatin K9 modifications (LOCKs) and may provide a cell type–heritable mechanism for phenotypic plasticity in development and disease.
Abstract: Higher eukaryotes must adapt a totipotent genome to specialized cell types with stable but limited functions. One potential mechanism for lineage restriction is changes in chromatin, and differentiation-related chromatin changes have been observed for individual genes. We have taken a genome-wide view of histone H3 lysine 9 dimethylation (H3K9Me2) and find that differentiated tissues show surprisingly large K9-modified regions (up to 4.9 Mb). These regions are highly conserved between human and mouse and are differentiation specific, covering only approximately 4% of the genome in undifferentiated mouse embryonic stem (ES) cells, compared to 31% in differentiated ES cells, approximately 46% in liver and approximately 10% in brain. These modifications require histone methyltransferase G9a and are inversely related to expression of genes within the regions. We term these regions large organized chromatin K9 modifications (LOCKs). LOCKs are substantially lost in cancer cell lines, and they may provide a cell type-heritable mechanism for phenotypic plasticity in development and disease.
582 citations
TL;DR: It is demonstrated that oxygen concentrations affected many aspects of stem-cell physiology, including growth and in vitro development, and may be a critical parameter during expansion and differentiation.
509 citations
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TL;DR: It is concluded that nuclear functions in the studied cell types may not require reproducible side-by-side arrangements of specific homologous or non-homologous CTs, and that presently unknown factors may play a decisive role to enforce the different radial arrangements of large and small CTs observed in ellipsoid and spherical human cell nuclei.
Abstract: A quantitative comparison of higher-order chromatin arrangements was performed in human cell types with three-dimensionally (3D) preserved, differently shaped nuclei. These cell types included flat-ellipsoid nuclei of diploid amniotic fluid cells and fibroblasts and spherical nuclei of B and T lymphocytes from peripheral human blood. Fluorescence in-situ hybridization (FISH) was performed with chromosome paint probes for large (#1–5) and small (#17–20) autosomes, and for the two sex chromosomes. Other probes delineated heterochromatin blocks of numerous larger and smaller human chromosomes. Shape differences correlated with distinct differences in higher order chromatin arrangements: in the spherically shaped lymphocyte nuclei we noted the preferential positioning of the small, gene dense #17, 19 and 20 chromosome territories (CTs) in the 3D nuclear interior – typically without any apparent connection to the nuclear envelope. In contrast, CTs of the gene-poor small chromosomes #18 and Y were apparently attached at the nuclear envelope. CTs of large chromosomes were also preferentially located towards the nuclear periphery. In the ellipsoid nuclei of amniotic fluid cells and fibroblasts, all tested CTs showed attachments to the upper and/or lower part of the nuclear envelope: CTs of small chromosomes, including #18 and Y, were located towards the centre of the nuclear projection (CNP), while the large chromosomes were positioned towards the 2D nuclear rim. In contrast to these highly reproducible radial arrangements, 2D distances measured between heterochromatin blocks of homologous and heterologous CTs were strikingly variable. These results as well as CT painting let us conclude that nuclear functions in the studied cell types may not require reproducible side-by-side arrangements of specific homologous or non-homologous CTs. 3D-modelling of statistical arrangements of 46 human CTs in spherical nuclei was performed under the assumption of a linear correlation between DNA content of each chromosome and its CT volume. In a set of modelled nuclei, we noted the preferential localization of smaller CTs towards the 3D periphery and of larger CTs towards the 3D centre. This distribution is in clear contrast to the experimentally observed distribution in lymphocyte nuclei. We conclude that presently unknown factors (other than topological constraints) may play a decisive role to enforce the different radial arrangements of large and small CTs observed in ellipsoid and spherical human cell nuclei.
391 citations
"Distinctive nuclear organisation of..." refers background in this paper
...This radial distribution is conserved amongst primates (Tanabe et al., 2002) and it is also applicable to other human chromosomes (Cremer et al., 2001; Boyle et al., 2001)....
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...However, a peripheral localisation of HSA18 was not seen in the very flat nuclei of amniotic fluid cells and quiescent fibroblasts (Bridger et al., 2000; Cremer et al., 2001)....
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..., 2002) and it is also applicable to other human chromosomes (Cremer et al., 2001; Boyle et al., 2001)....
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...(Cremer et al., 2001; Cremer et al., 2003)....
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...…and fibroblasts) with flat/ellipsoid-shaped nuclei, HSA18 can be found toward the nuclear centre rather than at the nuclear periphery, as is typical in cells with more spherical nuclei (lymphocytes, keratinocytes, colon and cervix epithelial cells) (Cremer et al., 2001; Cremer et al., 2003)....
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TL;DR: A systematic analysis of the spatial positioning of a subset of mouse chromosomes in several tissues shows that chromosomes exhibit tissue-specific organization, and points to a role for tissue- specific spatial genome organization in the formation of recurrent chromosome arrangements among tissues.
Abstract: Background: Genomes are organized in vivo in the form of chromosomes. Each chromosome occupies a distinct nuclear subvolume in the form of a chromosome territory. The spatial positioning of chromosomes within the interphase nucleus is often nonrandom. It is unclear whether the nonrandom spatial arrangement of chromosomes is conserved among tissues or whether spatial genome organization is tissue-specific. Results: Using two-dimensional and three-dimensional fluorescence in situ hybridization we have carried out a systematic analysis of the spatial positioning of a subset of mouse chromosomes in several tissues. We show that chromosomes exhibit tissue-specific organization. Chromosomes are distributed tissue-specifically with respect to their position relative to the center of the nucleus and also relative to each other. Subsets of chromosomes form distinct types of spatial clusters in different tissues and the relative distance between chromosome pairs varies among tissues. Consistent with the notion that nonrandom spatial proximity is functionally relevant in determining the outcome of chromosome translocation events, we find a correlation between tissue-specific spatial proximity and tissue-specific translocation prevalence. Conclusions: Our results demonstrate that the spatial organization of genomes is tissue-specific and point to a role for tissue-specific spatial genome organization in the formation of recurrent chromosome arrangements among tissues.
370 citations
"Distinctive nuclear organisation of..." refers background in this paper
...In the mouse, differences in the spatial and radial distribution of chromosomes have been documented in different tissues of the animal (Parada et al., 2004) as well as during the differentiation of T cells (Kim et al., 2004)....
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...Chromosome 12p is located in the centre of the nucleus in ES cells Differences in the radial distribution of mouse chromosomes have been documented in different tissues and during T-cell differentiation (Parada et al., 2004; Kim et al., 2004)....
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...Differences in the radial distribution of mouse chromosomes have been documented in different tissues and during T-cell differentiation (Parada et al., 2004; Kim et al., 2004)....
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TL;DR: It is shown that pericentric heterochromatin in mammalian cells is specifically responsive to prolonged treatment with deacetylase inhibitors, and this data point to a crucial role of histone underacetylation within perICentric heterchromatin regions for their association with HP1 proteins, their nuclear compartmentalization and their contribution to centromere function.
Abstract: Histone modifications might act to mark and maintain functional chromatin domains during both interphase and mitosis. Here we show that pericentric heterochromatin in mammalian cells is specifically responsive to prolonged treatment with deacetylase inhibitors. These defined regions relocate at the nuclear periphery and lose their properties of retaining HP1 (heterochromatin protein 1) proteins. Subsequent defects in chromosome segregation arise in mitosis. All these changes can reverse rapidly after drug removal. Our data point to a crucial role of histone underacetylation within pericentric heterochromatin regions for their association with HP1 proteins, their nuclear compartmentalization and their contribution to centromere function.
368 citations
"Distinctive nuclear organisation of..." refers background in this paper
...Under some conditions, the levels of histone acetylation of centric heterochromatin can alter centromere position in human and mouse somatic cells (Taddei et al., 2001) and, in the mouse, histone hypoacetylation at satellite repeats only occurs upon the induction of differentiation of mES cells (Keohane et al....
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...Under some conditions, the levels of histone acetylation of centric heterochromatin can alter centromere position in human and mouse somatic cells (Taddei et al., 2001) and, in the mouse, histone hypoacetylation at satellite repeats only occurs upon the induction of differentiation of mES cells…...
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TL;DR: The current state of knowledge with regard to the organization of chromosomes within the nucleus and the positioning of active versus inactive genes is described and studies on the dynamics of chromosomes and specific genetic loci within living cells and its relationship to gene activity and the cell cycle are discussed.
Abstract: ▪ Abstract With the sequence of the human genome now complete, studies must focus on how the genome is functionally organized within the confines of the cell nucleus and the dynamic interplay between the genome and its regulatory factors to effectively control gene expression and silencing. In this review I describe our current state of knowledge with regard to the organization of chromosomes within the nucleus and the positioning of active versus inactive genes. In addition, I discuss studies on the dynamics of chromosomes and specific genetic loci within living cells and its relationship to gene activity and the cell cycle. Furthermore, our current understanding of the distribution and dynamics of RNA polymerase II transcription factors is discussed in relation to chromosomal loci and other nuclear domains.
343 citations
"Distinctive nuclear organisation of..." refers background in this paper
...In model organisms it is clear that nuclear organisation can regulate gene expression (Spector, 2003)....
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TL;DR: It is suggested that the large-scale geometry during the G0/G1 part of the cell cycle may consist of flexible chromatin loops, averaging approximately 3 million bp, with a random-walk backbone.
Abstract: Fluorescence in situ hybridization data on distances between defined genomic sequences are used to construct a quantitative model for the overall geometric structure of a human chromosome. We suggest that the large-scale geometry during the G0/G1 part of the cell cycle may consist of flexible chromatin loops, averaging approximately 3 million bp, with a random-walk backbone. A fully explicit, three-parametric polymer model of this random-walk/giant-loop structure can account well for the data. More general models consistent with the data are briefly discussed.
330 citations
"Distinctive nuclear organisation of..." refers methods in this paper
...In all cases, the distribution of d values conformed to that expected of a random-walk model of chromatin structure (s.d.=0.52-0.6; median/mean ~1.0) (Sachs et al., 1995; Chambeyron and Bickmore, 2004)....
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