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: In this article, the authors used serial block-face scanning electron microscopy (SBFSEM) to understand chromosomal architectural organization within 3D nuclear space, and presented the first 3D karyotype built from the compact axial structure seen on the core of all prophase chromosomes.
Abstract: Three dimensional (3D) ultra-structural imaging is an important tool for unraveling the organizational structure of individual chromosomes at various stages of the cell cycle. Performing hitherto uninvestigated ultra-structural analysis of the human genome at prophase, we used serial block-face scanning electron microscopy (SBFSEM) to understand chromosomal architectural organization within 3D nuclear space. Acquired images allowed us to segment, reconstruct, and extract quantitative 3D structural information about the prophase nucleus and the preserved, intact individual chromosomes within it. Our data demonstrate that each chromosome can be identified with its homolog and classified into respective cytogenetic groups. Thereby, we present the first 3D karyotype built from the compact axial structure seen on the core of all prophase chromosomes. The chromosomes display parallel-aligned sister chromatids with familiar chromosome morphologies with no crossovers. Furthermore, the spatial positions of all 46 chromosomes revealed a pattern showing a gene density-based correlation and a neighborhood map of individual chromosomes based on their relative spatial positioning. A comprehensive picture of 3D chromosomal organization at the nanometer level in a single human lymphocyte cell is presented.
4 citations
TL;DR: In this article , the authors compare the spatial recruitment of HR factors upon double-stranded DNA breaks (DSBs) induced in human and mouse pericentromeric heterochromatin, which differ in their capacity to form clusters.
4 citations
Dissertation•
07 Feb 2014
TL;DR: Cette description nouvelle met sur the piste de mecanismes responsables de l'organisation particuliere des RCs, qui pourront etre etudies grâce a la methode d'analyse and aux observables que nous avons developpees.
Abstract: Le noyau des cellules est une structure tres organisee, dont l'organisation joue un role important dans la regulation de l'expression des genes. La comprehension des mecanismes a l'origine de cette organisation est donc essentielle a la comprehension du fonctionnement des genomes. De nombreuses experiences conduites chez la souris ont montre que les regions centromeriques (RC) des chromosomes jouent un role dans l'organisation du noyau. L'organisation spatiale des RCs humaines est beaucoup moins etudiee, principalement a cause de la complexite des sequences qui les composent, qui rend plus difficile leur detection. Nous avons developpe des outils de traitement et d'analyse quantitative d'image, qui, combines a des nouveaux marqueurs des RCs humaines, nous ont permis de mieux decrire deux aspects de leur organisation spatiale. D'une part nous avons montre qu'elles se positionnent preferentiellement en peripherie du noyau ou aux bords des nucleoles, avec des frequences qui dependent des chromosomes. D'autre part nous avons montre qu'elles s'agregent dans le noyau pour former un compartiment d'heterochromatine, qui presente des caracteristiques similaires a celui observe dans d'autres especes telles que la souris. Ces deux aspects sont tous deux inter-dependants et varient au cours du cycle cellulaire. Cette description nouvelle met sur la piste de mecanismes responsables de l'organisation particuliere des RCs, qui pourront etre etudies grâce a la methode d'analyse et aux observables que nous avons developpees. L'etude de ces mecanismes permettra de mieux comprendre la fonction des RCs humaines dans l'organisation du noyau.
3 citations
Dissertation•
30 Nov 2018
TL;DR: The chromatin structure of histones is studied as well as higher order structure methods and TADs, which investigate the role of chromatin remodelling complexes in the construction of histone structure.
Abstract: ..................................................................................................................................... VI TABLE OF CONTENTS .................................................................................................................. IX LIST OF FIGURES ........................................................................................................................ XIV LIST OF TABLES ......................................................................................................................... XVI ABBREVIATIONS ....................................................................................................................... XVII CHAPTER 1: INTRODUCTION .................................................................................................... 1 1.1 CHROMATIN CONSTITUENTS .............................................................................................................. 3 1.1.1 Core histones ................................................................................................................................. 3 1.1.2 Histone variants ............................................................................................................................ 5 1.1.3 Histone modifications ................................................................................................................ 8 1.1.4 Bromodomains ............................................................................................................................ 13 1.1.5 Chromatin remodelling complexes .................................................................................. 17 1.1.6 Topoisomerases ......................................................................................................................... 21 1.1.7 Cohesin and Condensin ........................................................................................................ 23 1.2 CHROMATIN STRUCTURE ...................................................................................................................... 28 1.2.1 Nucleosome arrays and the 30-nM fibre ...................................................................... 29 1.2.2 Large-scale chromatin structure ....................................................................................... 32 1.2.3 Higher order structure methods and TADs ................................................................. 34
3 citations
TL;DR: The structural and temporal organization of the genome within the nucleus of stem cells, together with specific features of epigenetic and transcriptional regulation are emerging as key players that influence pluripotency and differentiation.
Abstract: Fundamental features of genome regulation depend on the linear DNA sequence, cell type specific modification of DNA and chromatin-associated proteins, which locally control the expression of single genes. Architectural features of genome organization within the three-dimensional (3D) nuclear space establish preferential positioning of genes relative to nuclear subcompartments associated with specific biochemical activities, thereby influencing states of expression. The structural and temporal organization of the genome within the nucleus of stem cells, together with specific features of epigenetic and transcriptional regulation are emerging as key players that influence pluripotency and differentiation.1,2
3 citations
References
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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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