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: The analysis reveals that rather than considering the morphologically distinct PH, PCH and PNH as individual subcompartments, they should be considered in aggregate as a functional compartment for late replicating chromatin, and suggests a functional equivalence or redundancy among the 3 subcomparts.
Abstract: The eukaryotic nucleus is structurally and functionally organized, as reflected in the distribution of its protein and DNA components. The genome itself is segregated into euchromatin and heterochromatin that replicate in a distinct spatio-temporal manner. We used a combination of fluorescence in situ hybridization (FISH) and DamID to investigate the localization of the early and late replicating components of the genome in a lymphoblastoid cell background. Our analyses revealed that the bulk of late replicating chromatin localizes to the nuclear peripheral heterochromatin (PH) in a chromosome size and gene density dependent manner. Late replicating DNA on small chromosomes exhibits a much lower tendency to localize to PH and tends to associate with alternate repressive subcompartments such as pericentromeric (PCH) and perinucleolar heterochromatin (PNH). Furthermore, multicolor FISH analysis revealed that late replicating loci, particularly on the smaller chromosomes, may associate with any of these 3 re...
34 citations
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...Moreover, due to the linear proximity of centromeres to NORs, PCH often colocalizes with PNH.(28-32) Figure 4....
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TL;DR: This review summarizes and discusses the recent progress that has highlighted the central role of histone variants in ESC pluripotency and ESC fate, focusing, in particular, on H1 variants, H2A variants H 2A.Z and macroH2A and H3 variant H3.3.
Abstract: Dynamic regulation of chromatin structure is an important mechanism for balancing the pluripotency and cell fate decision in embryonic stem cells (ESCs) Indeed ESCs are characterized by unusual chromatin packaging, and a wide variety of chromatin regulators have been implicated in control of pluripotency and differentiation Genome-wide maps of epigenetic factors have revealed a unique epigenetic signature in pluripotent ESCs and have contributed models to explain their plasticity In addition to the well known epigenetic regulation through DNA methylation, histone posttranslational modifications, chromatin remodeling, and non-coding RNA, histone variants are emerging as important regulators of ESC identity In this review, we summarize and discuss the recent progress that has highlighted the central role of histone variants in ESC pluripotency and ESC fate, focusing, in particular, on H1 variants, H2A variants H2AX, H2AZ and macroH2A and H3 variant H33
33 citations
TL;DR: In this article, the authors review the progress that has been made in modeling the human genome and conclude with an outlook on the opportunities and challenges in studying chromosome dynamics, and provide insight into the principles of whole-genome organization and enable de novo predictions of chromosome structures from epigenetic modifications.
Abstract: Three-dimensional (3D) organization of the human genome plays an essential role in all DNA-templated processes, including gene transcription, gene regulation, and DNA replication. Computational modeling can be an effective way of building high-resolution genome structures and improving our understanding of these molecular processes. However, it faces significant challenges as the human genome consists of over 6 × 109 base pairs, a system size that exceeds the capacity of traditional modeling approaches. In this perspective, we review the progress that has been made in modeling the human genome. Coarse-grained models parameterized to reproduce experimental data via the maximum entropy optimization algorithm serve as effective means to study genome organization at various length scales. They have provided insight into the principles of whole-genome organization and enabled de novo predictions of chromosome structures from epigenetic modifications. Applications of these models at a near-atomistic resolution further revealed physicochemical interactions that drive the phase separation of disordered proteins and dictate chromatin stability in situ. We conclude with an outlook on the opportunities and challenges in studying chromosome dynamics.
32 citations
TL;DR: A more comprehensive picture is drawn of how epigenetic modifiers orchestrate their cell‐intrinsic role with microenvironmental cues for proper skin development, homeostasis and wound repair.
Abstract: Cell-type- and cell-state-specific patterns of covalent modifications on DNA and histone tails form global epigenetic profiles that enable spatiotemporal regulation of gene expression. These epigenetic profiles arise from coordinated activities of transcription factors and epigenetic modifiers, which result in cell-type-specific outputs in response to dynamic environmental conditions and signalling pathways. Recent mouse genetic and functional studies have highlighted the physiological significance of global DNA and histone epigenetic modifications in skin. Importantly, specific epigenetic profiles are emerging for adult skin stem cells that are associated with their cell fate plasticity and proper activity in tissue regeneration. We can now begin to draw a more comprehensive picture of how epigenetic modifiers orchestrate their cell-intrinsic role with microenvironmental cues for proper skin development, homeostasis and wound repair. The field is ripe to begin to implement these findings from the laboratory into skin therapies.
32 citations
TL;DR: The findings showed that the nuclear arrangement of HP1 subtypes and TIF1β is differentiation specific, and seems to be more important than changes in the levels of these proteins, which were relatively stable during all the induced differentiation processes.
Abstract: Mammalian heterochromatin protein 1 (HP1α, HP1β, HP1γ subtypes) and transcriptional intermediary factor TIF1β play an important role in the regulation of chromatin structure and function. Here, we investigated the nuclear arrangement of these proteins during differentiation of embryonal carcinoma P19 cells into primitive endoderm and into the neural pathway. Additionally, the differentiation potential of trichostatin A (TSA) and 5-deoxyazacytidine (5-dAzaC) was studied. In 70% of the cells from the neural pathway and in 20% of TSA-stimulated cells, HP1α and HP1β co-localized and associated with chromocentres (clusters of centromeres), which correlated with clustering of TIF1β at these heterochromatic regions. The cell types that we studied were also characterized by a pronounced focal distribution of HP1γ. The above-mentioned nuclear patterns of HP1 and TIF1β proteins were completely different from the nuclear patterns observed in the remaining cell types investigated, in which HP1α was associated with chromocentres while HP1β and HP1γ were largely localized in distinct nuclear regions. Moreover, a dispersed nuclear distribution of TIF1β was observed. Our findings showed that the nuclear arrangement of HP1 subtypes and TIF1β is differentiation specific, and seems to be more important than changes in the levels of these proteins, which were relatively stable during all the induced differentiation processes.
32 citations
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...1h), which is considered an important marker of stem cell pluripotency (e.g. Hay et al. 2004; Wiblin et al. 2005)....
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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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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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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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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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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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