Digitale Bildanalyse der radialen Verteilungen von spezifischen Subregionen im Zellkern

In der vorliegenden Arbeit wurde die Struktur von Zellkernen während der Interphase mit konfokaler Laser-Scanning-Mikroskopie und Software zur Kartierung des Kerns in konzentrischen Schalen untersucht. Besonderes Augenmerk wurde hierbei auf die Lage der Chromosomen Nr. 18 und Nr. 19 gerichtet, welche in etwa die gleiche genomische Größe, aber eine unterschiedliche Gendichte und einen unterschiedlichen Replikationszeitpunkt aufweisen. Es zeigte sich, dass das früher replizierende und gendichtere Chromosom Nr. 19 eher im Inneren des Kerns zu finden ist. Chromosom Nr. 18 hat in Fibroblasten eine innere Position und in Lymphozyten eine Randposition. (hierbei ist zu beachten, dass Lymphozyten kugelförmig sind und in 3D ausgewertet werden können, während bei den flachen Fibroblasten nur Raum für eine 2D Auswertung existiert). In Lymphozyten von 7 verschiedenen Primatenarten wurden diese Befunde bestätigt. Somit blieb die radiale Lage dieser Chromosomen über 30 Millionen Jahren Evolution erhalten. In Karzinomzellen traten meistens chromosomale Umbauten mit Abweichungen in der Verteilung von Chromosom Nr. 18 und Nr. 19 gegenüber gesunden Zellen korreliert auf. Zudem wurden Modelle von kugelförmigen menschlichen Zellkernen im PC erzeugt, und die Wirkung der eingesetzten Wahrscheinlichkeitsfunktionen für die Chromosomenpositionierung gemessen. Das Modell mit den realistischsten Verteilungen kann für weitergehende Berechnungen wie Translokationsraten nach Doppelstrangbrüchen verwendet werden. Es wurden auch die Verteilungen von Centromeren untersucht. Während in der G0 Phase alle Centromere zu den betrachteten 8 Chromosomen am Kernrand lagen, lagen sie in den anderen Interphasen zum Teil im Kerninneren, woraus auf eine Bewegung der Centromere während der Interphase des Teilungszyklus geschlossen wird. Schließlich wurde die räumliche Korrelation von RNA und DNA im Zellkern untersucht

Common themes and cell type specific variations of higher order chromatin arrangements in the mouse

BACKGROUND: Similarities as well as differences in higher order chromatin arrangements of human cell types were previously reported. For an evolutionary comparison, we now studied the arrangements of chromosome territories and centromere regions in six mouse cell types (lymphocytes, embryonic stem cells, macrophages, fibroblasts, myoblasts and myotubes) with fluorescence in situ hybridization and confocal laser scanning microscopy. Both species evolved pronounced differences in karyotypes after their last common ancestors lived about 87 million years ago and thus seem particularly suited to elucidate common and cell type specific themes of higher order chromatin arrangements in mammals. RESULTS: All mouse cell types showed non-random correlations of radial chromosome territory positions with gene density as well as with chromosome size. The distribution of chromosome territories and pericentromeric heterochromatin changed during differentiation, leading to distinct cell type specific distribution patterns. We exclude a strict dependence of these differences on nuclear shape. Positional differences in mouse cell nuclei were less pronounced compared to human cell nuclei in agreement with smaller differences in chromosome size and gene density. Notably, the position of chromosome territories relative to each other was very variable. CONCLUSION: Chromosome territory arrangements according to chromosome size and gene density provide common, evolutionary conserved themes in both, human and mouse cell types. Our findings are incompatible with a previously reported model of parental genome separation

The actin family member Arp6 and the histone variant H2A.Z are required for spatial positioning of chromatin in chicken cell nuclei

The spatial organization of chromatin in the nucleus contributes to genome function and is altered during the differentiation of normal and tumorigenic cells. Although nuclear actin-related proteins (Arps) have roles in the local alteration of chromatin structure, it is unclear whether they are involved in the spatial positioning of chromatin. In the interphase nucleus of vertebrate cells, gene-dense and gene-poor chromosome territories (CTs) are located in the center and periphery, respectively. We analyzed chicken DT40 cells in which Arp6 had been knocked out conditionally, and showed that the radial distribution of CTs was impaired in these knockout cells. Arp6 is an essential component of the SRCAP chromatin remodeling complex, which deposits the histone variant H2A.Z into chromatin. The redistribution of CTs was also observed in H2A.Z-deficient cells for gene-rich microchromosomes, but to lesser extent for gene-poor macrochromosomes. These results indicate that Arp6 and H2A.Z contribute to the radial distribution of CTs through different mechanisms. Microarray analysis suggested that the localization of chromatin to the nuclear periphery per se is insufficient for the repression of most genes

The actin family member Arp6 and the histone variant H2A.Z are required for spatial positioning of chromatin in chicken cell nuclei

The spatial organization of chromatin in the nucleus contributes to genome function and is altered during the differentiation of normal and tumorigenic cells. Although nuclear actin-related proteins (Arps) have roles in the local alteration of chromatin structure, it is unclear whether they are involved in the spatial positioning of chromatin. In the interphase nucleus of vertebrate cells, gene-dense and gene-poor chromosome territories (CTs) are located in the center and periphery, respectively. We analyzed chicken DT40 cells in which Arp6 had been knocked out conditionally, and showed that the radial distribution of CTs was impaired in these knockout cells. Arp6 is an essential component of the SRCAP chromatin remodeling complex, which deposits the histone variant H2A.Z into chromatin. The redistribution of CTs was also observed in H2A.Z-deficient cells for gene-rich microchromosomes, but to lesser extent for gene-poor macrochromosomes. These results indicate that Arp6 and H2A.Z contribute to the radial distribution of CTs through different mechanisms. Microarray analysis suggested that the localization of chromatin to the nuclear periphery per se is insufficient for the repression of most genes

Folding and organization of a contiguous chromosome region according to the gene distribution pattern in primary genomic sequence

Specific mammalian genes functionally and dynamically associate together within the nucleus. Yet, how an array of many genes along the chromosome sequence can be spatially organized and folded together is unknown. We investigated the 3D structure of a well-annotated, highly conserved 4.3-Mb region on mouse chromosome 14 that contains four clusters of genes separated by gene “deserts.” In nuclei, this region forms multiple, nonrandom “higher order” structures. These structures are based on the gene distribution pattern in primary sequence and are marked by preferential associations among multiple gene clusters. Associating gene clusters represent expressed chromatin, but their aggregation is not simply dependent on ongoing transcription. In chromosomes with aggregated gene clusters, gene deserts preferentially align with the nuclear periphery, providing evidence for chromosomal region architecture by specific associations with functional nuclear domains. Together, these data suggest dynamic, probabilistic 3D folding states for a contiguous megabase-scale chromosomal region, supporting the diverse activities of multiple genes and their conserved primary sequence organization

Preservation of large-scale chromatin structure in FISH experiments

The nuclear organization of specific endogenous chromatin regions can be investigated only by fluorescence in situ hybridization (FISH). One of the two fixation procedures is typically applied: (1) buffered formaldehyde or (2) hypotonic shock with methanol acetic acid fixation followed by dropping of nuclei on glass slides and air drying. In this study, we compared the effects of these two procedures and some variations on nuclear morphology and on FISH signals. We analyzed mouse erythroleukemia and mouse embryonic stem cells because their clusters of subcentromeric heterochromatin provide an easy means to assess preservation of chromatin. Qualitative and quantitative analyses revealed that formaldehyde fixation provided good preservation of large-scale chromatin structures, while classical methanol acetic acid fixation after hypotonic treatment severely impaired nuclear shape and led to disruption of chromosome territories, heterochromatin structures, and large transgene arrays. Our data show that such preparations do not faithfully reflect in vivo nuclear architecture. ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00412-006-0084-2 and is accessible for authorized users

Ex Eliphasi Themanitae

EX ELIPHASI THEMANITAE Ex Eliphasi Themanitae ([1]) Titelseite ([1]) Widmung ([2]) Prooemium (3) Observationes Theologico-philologiacæ (6) Corollaria Respondentis (16

Preservation of large-scale chromatin structure

Abstract The nuclear organization of specific endogenous chromatin regions can be investigated only by fluorescence in situ hybridization (FISH). One of the two fixation procedures is typically applied: (1) buffered formaldehyde or (2) hypotonic shock with methanol acetic acid fixation followed by dropping of nuclei on glass slides and air drying. In this study, we compared the effects of these two procedures and some variations on nuclear morphology and on FISH signals. We analyzed mouse erythroleukemia and mouse embryonic stem cells because their clusters of subcentromeric heterochromatin provide an easy means to assess preservation of chromatin. Qualitative and quantitative analyses revealed that formaldehyde fixation provided good preservation of large-scale chromatin structures, while classical methanol acetic acid fixation after hypotonic treatment severely impaired nuclear shape and led to disruption of chromosome territories, heterochromatin structures, and large transgene arrays. Our data show that such preparations do not faithfully reflect in vivo nuclear architecture

Lebewohl der Ilm

LEBEWOHL DER ILM Lebewohl der Ilm (1) Titelseite (1) Noten (2