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
Cites background from "Distinctive nuclear organisation of..."
...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
References
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TL;DR: This data indicates that heritable gene inactivation plays an important role in determining cell lineage fates during development and defining their temporal order, and recent advances in the area of chromatin and chromosome organisation may have an impact on the understanding of cellular differentiation.
112 citations
"Distinctive nuclear organisation of..." refers background in this paper
...In turn the localisation of centromeres within the nucleus may influence mechanisms of gene silencing (Fisher and Merkenschlager, 2002)....
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...important to determine how the genome is organised in the nucleus of pluripotent cells, and particularly in stem cells (Fisher and Merkenschlager, 2002)....
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...Jo ur na l o f C el l S ci en ce 3862 important to determine how the genome is organised in the nucleus of pluripotent cells, and particularly in stem cells (Fisher and Merkenschlager, 2002)....
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TL;DR: The results demonstrate that significant spatial genome reorganization occurs during differentiation and indicate that the relationship between dynamic genome topology and single gene regulation is highly complex.
Abstract: The spatial organization of genomes within the mammalian cell nucleus is non-random. The functional relevance of spatial genome organization might be in influencing gene expression programs as cells undergo changes during development and differentiation. To gain insight into the plasticity of genomes in space and time and to correlate the activity of specific genes with their nuclear position, we systematically analyzed the spatial genome organization in differentiating mouse T-cells. We find significant global reorganization of centromeres, chromosomes and gene loci during the differentiation process. Centromeres were repositioned from a preferentially internal distribution in undifferentiated cells to a preferentially peripheral position in differentiated CD4+ and CD8+ cells. Chromosome 6, containing the differentially expressed T-cell markers CD4 and CD8, underwent differential changes in position depending on whether cells differentiated into CD4+ or CD8+ thymocytes. Similarly, the two marker loci CD4 and CD8 showed distinct behavior in their position relative to the chromosome 6 centromere at various stages of differentiation. Our results demonstrate that significant spatial genome reorganization occurs during differentiation and indicate that the relationship between dynamic genome topology and single gene regulation is highly complex.
111 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 the distribution of centromeric alpha-satellite DNA in human lymphoid cells synchronized at G(0)/G(1) is unique for most individual chromosomes, and a model that predicts the intranuclear positioning of centromeres for each individual chromosome predicts that facultative heterochromatinization of distinct genomic regions may contribute to cell-type specific patterns of centRomere localization.
Abstract: Gene expression can be silenced by proximity to heterochromatin blocks containing centromeric α-satellite DNA. This has been shown experimentally through cis-acting chromosome rearrangements resulting in linear genomic proximity, or through trans-acting changes resulting in intranuclear spatial proximity. Although it has long been been established that centromeres are nonrandomly distributed during interphase, little is known of what determines the three-dimensional organization of these silencing domains in the nucleus. Here, we propose a model that predicts the intranuclear positioning of centromeric heterochromatin for each individual chromosome. With the use of fluorescence in situ hybridization and confocal microscopy, we show that the distribution of centromeric α-satellite DNA in human lymphoid cells synchronized at G0/G1 is unique for most individual chromosomes. Regression analysis reveals a tight correlation between nuclear distribution of centromeric α-satellite DNA and the presence of G-dark bands in the corresponding chromosome. Centromeres surrounded by G-dark bands are preferentially located at the nuclear periphery, whereas centromeres of chromosomes with a lower content of G-dark bands tend to be localized at the nucleolus. Consistent with the model, a t(11; 14) translocation that removes G-dark bands from chromosome 11 causes a repositioning of the centromere, which becomes less frequently localized at the nuclear periphery and more frequently associated with the nucleolus. The data suggest that “chromosomal environment” plays a key role in the intranuclear organization of centromeric heterochromatin. Our model further predicts that facultative heterochromatinization of distinct genomic regions may contribute to cell-type specific patterns of centromere localization.
78 citations
"Distinctive nuclear organisation of..." refers background in this paper
...Centromeres are also generally found at the nuclear periphery, or around nucleoli (Carvalho et al., 2001; Weierich et al., 2003; Gilchrist et al., 2004), whereas telomeres are mainly found in the nuclear interior (Weierich et al., 2003)....
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...Human centromeres are localised close to either the nuclear periphery or the nucleolus (Carvalho et al., 2001; Weierich et al., 2003)....
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TL;DR: The pattern of expression of the Ig-CAMs in ES cells may have a role in defining the phenotype of differentiated and undifferentiated cells, as suggested by the observed patterns of expression seen on ES cells maintained on fibroblast feeder cells.
Abstract: The expression of cell adhesion molecules of the Ig superfamily (Ig-CAM) were examined on embryonic stem (ES) cells during culture in vitro. ES cells maintained an undifferentiated phenotype when cultured in the presence of leukemia inhibitory factor (LIF) or with fibroblast feeder cells; > 90% of cells reacted positively to an antibody (ECMA-7) that marks undifferentiated ES cells. Using flow cytometry, high concentrations of ICAM-1, VCAM-1, and NCAM antigens were detected on undifferentiated ES cells, but their specific receptors, Mac-1, LFA-1, and VLA-4, were not detected. There was also no class I or II major histocompatibility complex (MHC) antigen expression. The ICAM-1 expressed was functional, since anti-ICAM-1 significantly (p < 0.0001) blocked ES cell-lymphocyte binding. Ig-CAM and MHC-1 expression on undifferentiated ES cells was not up-regulated by treatment of cells with interferon-gamma (IFN-gamma), tumor necrosis factor alpha, or flavivirus infection, agents that up-regulate these molecules in other embryonic cell types. Twelve hours after LIF withdrawal, ICAM-1 and NCAM expression decreased significantly, while VCAM-1 was undetectable. However, morphology and ECMA-7 expression remained unchanged. Similar patterns of expression were seen on ES cells maintained on fibroblast feeder cells. This suggests that LIF or other cytokines may maintain the expression of Ig-CAMs on undifferentiated cells. Differentiation was induced by dimethyl sulfoxide treatment for 14 days. Cells changed from a colony-forming to a monolayer morphology, and approximately 60% of the cell population no longer expressed ECMA-7. In these cells, VCAM-1 was undetectable and ICAM-1 and NCAM had declined to low levels. In these differentiated cells, ICAM-1 and MHC-1 were inducible by IFN-gamma. This study suggests that the pattern of expression of the Ig-CAMs in ES cells may have a role in defining the phenotype of differentiated and undifferentiated cells.
77 citations
"Distinctive nuclear organisation of..." refers background in this paper
...Unlike mES cells, hES cells also express class I genes (Tian et al., 1997; Drukker et al., 2002; Draper et al., 2002; Carpenter et al., 2004)....
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TL;DR: The nuclear organization of histone modifications and the localization of centromeric domains in human cells before and after TSA treatment is quantified and it is concluded that the distinctive nuclear localization of 100% heterochromatin domains is independent of hist one acetylation.
Abstract: It is well established that modification of lysines in histone molecules correlates with gene expression and chromatin structure. It is not known whether this operates entirely at a local level, e.g. through the recruitment of specific proteins, or whether histone modifications might impact on more long-range aspects of chromatin organization. There is a distinctive organization of chromatin within the nucleus and the chromatin at the nuclear periphery of mammalian cells appears to be hypoacetylated. Previously it had been suggested that inhibition of histone deacetylases by TSA causes a gross remodeling of nuclear structure, specifically the recruitment of centromeric heterochromatin to the nuclear periphery. Here, we have quantified the nuclear organization of histone modifications and the localization of centromeric domains in human cells before and after TSA treatment. TSA alters the nuclear distribution of histone acetylation, but not that of histone methylation. TSA elevates levels of histone acetylation at the nuclear periphery but we see no alteration in the position of centromeric domains in the nuclei of treated cells. We conclude that the distinctive nuclear localization of centromeric domains is independent of histone acetylation.
65 citations
"Distinctive nuclear organisation of..." refers background in this paper
...Centromeres are also generally found at the nuclear periphery, or around nucleoli (Carvalho et al., 2001; Weierich et al., 2003; Gilchrist et al., 2004), whereas telomeres are mainly found in the nuclear interior (Weierich et al., 2003)....
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...Internal nuclear distribution of centromeres in hES cells In most human cell types, the predominant reported distribution of centromeres is toward the nuclear periphery (reviewed by Gilchrist et al., 2004)....
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...Centromeres are also generally found at the nuclear periphery, or around nucleoli (Carvalho et al., 2001; Weierich et al., 2003; Gilchrist et al., 2004), whereas telomeres are mainly found in the nuclear interior (Weierich et al....
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...However, changes of centromere distribution in relation to cell cycle, physiological or differentiation state have been reported (reviewed by Gilchrist et al., 2004)....
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