Microgravity and space radiation inhibit autophagy in human capillary endothelial cells, through either opposite or synergistic effects on specific molecular pathways.

Microgravity and space radiation (SR) are two highly influential factors affecting humans in space flight (SF). Many health problems reported by astronauts derive from endothelial dysfunction and impaired homeostasis. Here, we describe the adaptive response of human, capillary endothelial cells to SF. Reference samples on the ground and at 1g onboard permitted discrimination between the contribution of microgravity and SR within the combined responses to SF. Cell softening and reduced motility occurred in SF cells, with a loss of actin stress fibers and a broader distribution of microtubules and intermediate filaments within the cytoplasm than in control cells. Furthermore, in space the number of primary cilia per cell increased and DNA repair mechanisms were found to be activated. Transcriptomics revealed the opposing effects of microgravity from SR for specific molecular pathways: SR, unlike microgravity, stimulated pathways for endothelial activation, such as hypoxia and inflammation, DNA repair and apoptosis, inhibiting autophagic flux and promoting an aged-like phenotype. Conversely, microgravity, unlike SR, activated pathways for metabolism and a pro-proliferative phenotype. Therefore, we suggest microgravity and SR should be considered separately to tailor effective countermeasures to protect astronauts' health.

Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci.

The genome is associated with several structures inside cell nuclei, in order to regulate its activity and anchor it in specific locations. These structures are collectively known as the nucleoskeleton and include the nuclear lamina, the nucleoli, and nuclear bodies. Although many variants of fluorescence in situ hybridization (FISH) exist to study the genome and its organization, these are often limited by resolution and provide insufficient information on the genome's association with nuclear structures. The DNA halo method uses high salt concentrations and nonionic detergents to generate DNA loops that remain anchored to structures within nuclei through attachment regions within the genome. Here, soluble nuclear proteins, such as histones, lipids, and DNA not tightly bound to the nuclear matrix, are extracted. This leads to the formation of a halo of unattached DNA surrounding a residual nucleus which itself contains DNA closely associated with internal nuclear structures and extraction-resistant proteins. These extended DNA strands enable increased resolution and can facilitate physical mapping. In combination with FISH, this method has the added advantage of studying genomic interactions with all the structures that the genome is anchored by. This technique, termed HALO-FISH, is highly versatile whereby DNA halos can be coupled with nucleic acid probes to reveal gene loci, whole chromosomes, alpha satellite, telomeres and even RNA. This technique provides an insight into nuclear organization and function in normal cells and in disease progression such as with cancer.

Deletions of Chromosome 7q Affect Nuclear Organization and HLXB9Gene Expression in Hematological Disorders.

The radial spatial positioning of individual gene loci within interphase nuclei has been associated with up- and downregulation of their expression. In cancer, the genome organization may become disturbed due to chromosomal abnormalities, such as translocations or deletions, resulting in the repositioning of genes and alteration of gene expression with oncogenic consequences. In this study, we analyzed the nuclear repositioning of HLXB9 (also called MNX1), mapping at 7q36.3, in patients with hematological disorders carrying interstitial deletions of 7q of various extents, with a distal breakpoint in 7q36. We observed that HLXB9 remains at the nuclear periphery, or is repositioned towards the nuclear interior, depending upon the compositional properties of the chromosomal regions involved in the rearrangement. For instance, a proximal breakpoint leading the guanine-cytosine (GC)-poor band 7q21 near 7q36 would bring HLXB9 to the nuclear periphery, whereas breakpoints that join the GC-rich band 7q22 to 7q36 would bring HLXB9 to the nuclear interior. This nuclear repositioning is associated with transcriptional changes, with HLXB9 in the nuclear interior becoming upregulated. Here we report an in cis rearrangement, involving one single chromosome altering gene behavior. Furthermore, we propose a mechanistic model for chromatin reorganization that affects gene expression via the influences of new chromatin neighborhoods.

The non-random repositioning of whole chromosomes and individual gene loci in interphase nuclei and its relevance in disease, infection, aging, and cancer.

The genomes of a wide range of different organisms are non-randomly organized within interphase nuclei. Chromosomes and genes can be moved rapidly, with direction, to new non-random locations within nuclei upon a stimulus such as a signal to initiate differentiation, quiescence or senescence, or also the application of heat or an infection with a pathogen. It is now becoming increasingly obvious that chromosome and gene position can be altered in diseases such as cancer and other syndromes that are affected by changes to nuclear architecture such as the laminopathies. This repositioning seems to affect gene expression in these cells and may play a role in progression of the disease. We have some evidence in breast cancer cells and in the premature aging disease Hutchinson-Gilford Progeria that an aberrant nuclear envelope may lead to genome repositioning and correction of these nuclear envelope defects can restore proper gene positioning and expression in both disease situations.Although spatial positioning of the genome probably does not entirely control expression of genes, it appears that spatio-epigenetics may enhance the control over gene expression globally and/or is deeply involved in regulating specific sets of genes. A deviation from normal spatial positioning of the genome for a particular cell type could lead to changes that affect the future health of the cell or even an individual.

The human myometrium differentially expresses mTOR signalling components before and during pregnancy: evidence for regulation by progesterone.

Emerging studies implicate the signalling of the mammalian target of rapamycin (mTOR) in a number of reproductive functions. To this date, there are no data regarding the expression of mTOR signalling components in the human myometrium during pregnancy. We hypothesized that mTOR-related genes might be differentially expressed in term or preterm labour as well as in labour or non-labour myometria during pregnancy. Using quantitative RT-PCR we demonstrate for first time that there is a significant downregulation of mTOR, DEPTOR, and Raptor in preterm labouring myometria when compared to non-pregnant tissues taken from the same area (lower segment). We used an immortalized myometrial cell line (ULTR) as an in vitro model to dissect further mTOR signalling. In ULTR cells DEPTOR and Rictor had a cytoplasmic distribution, whereas mTOR and Raptor were detected in the cytoplasm and the nucleus, indicative of mTORC1 shuttling. Treatment with inflammatory cytokines caused only minor changes in gene expression of these components, whereas progesterone caused significant down-regulation. We performed a non-biased gene expression analysis of ULTR cells using Nimblegen human gene expression microarray (n=3), and selected genes were validated by quantitative RT-PCR in progesterone treated myometrial cells. Progesterone significantly down-regulated key components of the mTOR pathway. We conclude that the human myometrium differentially expresses mTOR signalling components and they can be regulated by progesterone. This article is part of a Special Issue entitled 'Pregnancy and Steroids'.

Relative proximity of chromosome territories influences chromosome exchange partners in radiation-induced chromosome rearrangements in primary human bronchial epithelial cells.

It is well established that chromosomes exist in discrete territories (CTs) in interphase and are positioned in a cell-type specific probabilistic manner. The relative localisation of individual CTs within cell nuclei remains poorly understood, yet many cancers are associated with specific chromosome rearrangements and there is good evidence that relative territorial position influences their frequency of exchange. To examine this further, we characterised the complexity of radiation-induced chromosome exchanges in normal human bronchial epithelial (NHBE) cells by M-FISH analysis of PCC spreads and correlated the exchanges induced with their preferred interphase position, as determined by 1/2-colour 2D-FISH analysis, at the time of irradiation. We found that the frequency and complexity of aberrations induced were reduced in ellipsoid NHBE cells in comparison to previous observations in spherical cells, consistent with aberration complexity being dependent upon the number and proximity of damaged CTs, i.e. lesion proximity. To ask if particular chromosome neighbourhoods could be identified we analysed all radiation-induced pair-wise exchanges using SCHIP (statistics for chromosome interphase positioning) and found that exchanges between chromosomes (1;13), (9;17), (9;18), (12;18) and (16;21) all occurred more often than expected assuming randomness. All of these pairs were also found to be either sharing similar preferred positions in interphase and/or sharing neighbouring territory boundaries. We also analysed a human small cell lung cancer cell line, DMS53, by M-FISH observing the genome to be highly rearranged, yet possessing rearrangements also involving chromosomes (1;13) and (9;17). Our findings show evidence for the occurrence of non-random exchanges that may reflect the territorial organisation of chromosomes in interphase at time of damage and highlight the importance of cellular geometry for the induction of aberrations of varying complexity after exposure to both low and high-LET radiation.

Interphase chromosome positioning in in vitro porcine cells and ex vivo porcine tissues.

BACKGROUND: In interphase nuclei of a wide range of species chromosomes are organised into their own specific locations termed territories. These chromosome territories are non-randomly positioned in nuclei which is believed to be related to a spatial aspect of regulatory control over gene expression. In this study we have adopted the pig as a model in which to study interphase chromosome positioning and follows on from other studies from our group of using pig cells and tissues to study interphase genome re-positioning during differentiation. The pig is an important model organism both economically and as a closely related species to study human disease models. This is why great efforts have been made to accomplish the full genome sequence in the last decade. RESULTS: This study has positioned most of the porcine chromosomes in in vitro cultured adult and embryonic fibroblasts, early passage stromal derived mesenchymal stem cells and lymphocytes. The study is further expanded to position four chromosomes in ex vivo tissue derived from pig kidney, lung and brain. CONCLUSIONS: It was concluded that porcine chromosomes are also non-randomly positioned within interphase nuclei with few major differences in chromosome position in interphase nuclei between different cell and tissue types. There were also no differences between preferred nuclear location of chromosomes in in vitro cultured cells as compared to cells in tissue sections. Using a number of analyses to ascertain by what criteria porcine chromosomes were positioned in interphase nuclei; we found a correlation with DNA content.

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.