Joanna M. Bridger

Re-modelling of nuclear architecture in quiescent and senescent human fibroblasts

Spatial organisation of the genome within the nucleus can play a role in maintaining the expressed or silent state of some genes [1]. There are distinct addresses for specific chromosomes, which have different functional characteristics, within the nuclei of dividing populations of human cells [2]. Here, we demonstrate that this level of nuclear architecture is altered in cells that have become either quiescent or senescent. Upon cell cycle exit, a gene-poor human chromosome moves from a location at the nuclear periphery to a more internal site in the nucleus, and changes its associations with nuclear substructures. The chromosome moves back toward the edge of the nucleus at a distinctive time after re-entry into the cell cycle. There is a 2-4 hour period at the beginning of G1 when the spatial organisation of these human chromosomes is established. Lastly, these experiments provide evidence that temporal control of DNA replication can be independent of spatial chromosome organisation. We conclude that the sub-nuclear organisation of chromosomes in quiescent or senescent mammalian somatic cells is fundamentally different from that in proliferating cells and that the spatial organisation of the genome is plastic.

The genome and the nucleus: a marriage made by evolution

Genomes are housed within cell nuclei as individual chromosome territories. Nuclei contain several architectural structures that interact and influence the genome. In this review, we discuss how the genome may be organised within its nuclear environment with the position of chromosomes inside nuclei being either influenced by gene density or by chromosomes size. We compare interphase genome organisation in diverse species and reveal similarities and differences between evolutionary divergent organisms. Genome organisation is also discussed with relevance to regulation of gene expression, development and differentiation and asks whether large movements of whole chromosomes are really observed during differentiation. Literature and data describing alterations to genome organisation in disease are also discussed. Further, the nuclear structures that are involved in genome function are described, with reference to what happens to the genome when these structures contain protein from mutant genes as in the laminopathies.

Aging of Hutchinson -Gilford progeria syndrome fibroblasts is characterised by hyperproliferation and increased apoptosis

Hutchinson-Gilford progeria syndrome is a rare genetic disorder that mimics certain aspects of aging prematurely. Recent work has revealed that mutations in the lamin A gene are a cause of the disease. We show here that cellular aging of Hutchinson-Gilford progeria syndrome fibroblasts is characterised by a period of hyperproliferation and terminates with a large increase in the rate of apoptosis. The occurrence of cells with abnormal nuclear morphology reported by others is shown to be a result of cell division since the fraction of these abnormalities increases with cellular age. Similarly, the proportion of cells with an abnormal or absent A-type lamina increases with age. These data provide clues as to the cellular basis for premature aging in HGPS and support the view that cellular senescence and tissue homeostasis are important factors in the normal aging process.

Rapid chromosome territory relocation by nuclear motor activity in response to serum removal in primary human fibroblasts

BackgroundRadial chromosome positioning in interphase nuclei is nonrandom and can alter according to developmental, differentiation, proliferation, or disease status. However, it is not yet clear when and how chromosome repositioning is elicited.ResultsBy investigating the positioning of all human chromosomes in primary fibroblasts that have left the proliferative cell cycle, we have demonstrated that in cells made quiescent by reversible growth arrest, chromosome positioning is altered considerably. We found that with the removal of serum from the culture medium, chromosome repositioning took less than 15 minutes, required energy and was inhibited by drugs affecting the polymerization of myosin and actin. We also observed that when cells became quiescent, the nuclear distribution of nuclear myosin 1β was dramatically different from that in proliferating cells. If we suppressed the expression of nuclear myosin 1β by using RNA-interference procedures, the movement of chromosomes after 15 minutes in low serum was inhibited. When high serum was restored to the serum-starved cultures, chromosome repositioning was evident only after 24 to 36 hours, and this coincided with a return to a proliferating distribution of nuclear myosin 1β.ConclusionsThese findings demonstrate that genome organization in interphase nuclei is altered considerably when cells leave the proliferative cell cycle and that repositioning of chromosomes relies on efficient functioning of an active nuclear motor complex that contains nuclear myosin 1β.

Primary laminopathy fibroblasts display altered genome organization and apoptosis.

A number of diseases associated with specific tissue degeneration and premature aging have mutations in the nuclear envelope proteins A‐type lamins or emerin. Those diseases with A‐type lamin mutation are inclusively termed laminopathies. Due to various hypothetical roles of nuclear envelope proteins in genome function we investigated whether alterations to normal genomic behaviour are apparent in cells with mutations in A‐type lamins and emerin. Even though the distributions of these proteins in proliferating laminopathy fibroblasts appear normal, there is abnormal nuclear positioning of both chromosome 18 and 13 territories, from the nuclear periphery to the interior. This genomic organization mimics that found in normal nonproliferating quiescent or senescent cells. This finding is supported by distributions of modified pRb in the laminopathy cells. All laminopathy cell lines tested and an X‐linked Emery–Dreifuss muscular dystrophy cell line also demonstrate increased incidences of apoptosis. The most extreme cases of apoptosis occur in cells derived from diseases with mutations in the tail region of the LMNA gene, such as Dunningan‐type familial partial lipodystrophy and mandibuloacral dysplasia, and this correlates with a significant level of micronucleation in these cells.

Human acrocentric chromosomes with transcriptionally silent nucleolar organizer regions associate with nucleoli

Human ribosomal gene repeats are distributed among five nucleolar organizer regions (NORs) on the p arms of acrocentric chromosomes. On exit from mitosis, nucleoli form around individual active NORs. As cells progress through the cycle, these mini‐nucleoli fuse to form large nucleoli incorporating multiple NORs. It is generally assumed that nucleolar incorporation of individual NORs is dependent on ribosomal gene transcription. To test this assumption, we determined the nuclear location of individual human acrocentric chromosomes, and their associated NORs, in mouse> human cell hybrids. Human ribosomal genes are transcriptionally silent in this context. Combined immunofluorescence and in situ hybridization (immuno‐FISH) on three‐dimensional preserved nuclei showed that human acrocentric chromosomes associate with hybrid cell nucleoli. Analysis of purified nucleoli demonstrated that human and mouse NORs are equally likely to be within a hybrid cell nucleolus. This is supported further by the observation that murine upstream binding factor can associate with human NORs. Incorporation of silent NORs into mature nucleoli raises interesting issues concerning the maintenance of the activity status of individual NORs.

The nuclear lamina

The inner face of the nuclear envelope of metazoan cells is covered by a thin lamina consisting of a one‐layered network of intermediate filaments interconnecting with a complex set of transmembrane proteins and chromatin associating factors. The constituent proteins, the lamins, have recently gained tremendous recognition, because mutations in the lamin A gene, LMNA, are the cause of a complex group of at least 10 different diseases in human, including the Hutchinson–Gilford progeria syndrome. The analysis of these disease entities has made it clear that besides cytoskeletal functions, the lamina has an important role in the ‘behaviour’ of the genome and is, probably as a consequence of this function, intimately involved in cell fate decisions. Furthermore, these functions are related to the involvement of lamins in organizing the position and functional state of interphase chromosomes as well as to the occurrence of lamins and lamina‐associated proteins within the nucleoplasm. However, the structural features of these lamins and the nature of the factors that assist them in genome organization present an exciting challenge to modern biochemistry and cell biology.

The spatial repositioning of adipogenesis genes is correlated with their expression status in a porcine mesenchymal stem cell adipogenesis model system

Alterations in the nuclear positioning of chromosomes and specific genes during differentiation and development have suggested strongly the existence of a relationship between non-random organization of the genome and its function. In this study, we have examined the genome organization in interphase nuclei during adipogenesis, using the pig as a model organism. We hypothesized that changes in the gene expression profile and chromatin remodeling which occur during cellular differentiation would elicit repositioning of whole chromosomes, moving specific genes on them to different regions of the nucleus. We established an in vitro adipogenesis differentiation system using mesenchymal stem cells, derived from porcine bone marrow. The nuclear position of seven adipogenesis genes (PPARG, SREBF1, FABP4, CEBPA, CEBPB, CREB, and GATA2), two control genes (SOX9 and MYL1), and six chromosomes carrying these gene loci (SSC4, SSC6, SSC12, SSC13, SSC15, and SSC17) was determined. We found that during adipogenesis, using the in vitro stem cell model system, in contrast to our original hypothesis, the nuclear position of genes involved in adipogenesis was altered radically with the up-regulation of gene expression correlating with these genes becoming more internally located within nuclei. Chromosome territories, containing these genes, were also found to alter their nuclear position during the in vitro adipogenesis model, with the most dramatic repositioning being SSC4 that moved from the nuclear periphery towards the nuclear interior. We found that during in vitro adipogenesis chromosome territories decondensed and the genes were found on loops and projections of chromatin, away from the main body of the chromosomes. From our data, it appears that the temporal repositioning of genes, emanating away from chromosomes, during adipogenesis is correlated with gene activity, supporting models of the involvement of spatial genome repositioning in regulating gene expression and the nuclear interior being an important region of the nucleus for transcription.

Association of pKi-67 with satellite DNA of the human genome in early G1 cells

AbstractpKi-67 is a nucleolar antigen that provides a specific marker for proliferating cells. It has been shown previously that pKi-67s distribution varies in a cell cycle-dependent manner: it coats all chromosomes during mitosis, accumulates in nuclear foci during G1 phase (type I distribution) and localizes within nucleoli in late G1 S and G2 phase (type II distribution). Although no function has as yet been ascribed to pKi-67, it has been found associated with centromeres in G1. In the present study the distribution pattern of pKi-67 during G1 in human dermal fibroblasts (HDFs) was analysed in more detail. Synchronization experiments show that in very early G1 cells pKi-67 coincides with virtually all satellite regions analysed, i.e. with centromeric (alpha-satellite), telomeric (minisatellite) and heterochromatic blocks (satellite III) on chromosomes 1 and Y (type Ia distribution). In contrast, later in the G1 phase, a smaller fraction of satellite DNA regions are found collocalized with pKi-67 foci (type Ib distribution). When all pKi-67 becomes localized within nucleoli, even fewer satellite regions remain associated with the pKi-67 staining. However, all centromeric and short arm regions of the acrocentric chromosomes, which are in very close proximity to or even contain the rRNA genes, are collocalized with anti-pKi-67 staining throughout the remaining interphase of the cell cycle. Thus, our data demonstrate that during post-mitotic reformation and nucleogenesis there is a progressive decline in the fraction of specific satellite regions of DNA that remain associated with pKi-67. This may be relevant to nucleolar reformation following mitosis.

Non-random chromosome positioning in mammalian sperm nuclei, with migration of the sex chromosomes during late spermatogenesis

Chromosomes are highly organized and compartmentalized in cell nuclei. The analysis of their position is a powerful way to monitor genome organization in different cell types and states. Evidence suggests that the organization of the genome could be functionally important for influencing different cellular and developmental processes, particularly at early stages of development (i.e. fertilization and the consequent entry of the sperm nucleus into the egg). The position of chromosomes in the sperm nucleus might be crucial, because their location could determine the time at which particular chromatin domains are decondensed and remodelled, allowing some epigenetic level of control or influence over subsequent paternal gene expression in the embryo. Here, we analyse genome organization by chromosome position in mammalian sperm nuclei from three breeds of pig, as a model species. We have mapped the preferential position of all chromosomes (bar one) in sperm nuclei in two dimensions and have established that the sex chromosomes are the most internally localized chromosomes in mature sperm. The distribution of two autosomes and chromosomes X and Y in sperm heads was compared in primary and secondary spermatocytes and spermatids in porcine testes. The sex chromosomes were found at the nuclear edge in primary spermatocytes, which correlates with the known position of the XY body and their position in somatic cells, whereas, in spermatids, the sex chromosomes were much more centrally located, mirroring the position of these chromosomes in ejaculated spermatozoa. This study reveals the temporal repositioning of chromosome territories in spermatogenesis.