- Email: [email protected]
P74: Arrangements of chromosome territories in human cell clones
540
POSTER
D. Köhler et al. / European Journal of Medical Genetics 48 (2005) 537–542 PRESENTATIONS
P73: Impact of primate evolutionary chromosome rearrangements on the nuclear topology of genomic signatures Michaela Neusser, Thomas Cremer, Stefan Muller Department Biology II, Human Genetics, Ludwig-Maximilians University, Munich, Germany The impact of genomic signatures like gene density, base-pair composition and repetitive elements on the higher order nuclear architecture has been in the focus of numerous investigations. These included studies of their spatial orientation, DNA methylation status, histone modification and chromatin fibre compactness, with the aim to explain putative functional properties related to nuclear compartmentalization and epigenetic control mechanisms of gene regulation. Here we report on a comparative 3D FISH study of human and various higher primate species, to investigate the impact of evolutionary genomic changes on the nuclear organization of genomic signatures. Employing a panel of breakpoint flanking BAC clones we analyzed the side-by-side arrangement of evolutionary rearranged chromosomes. Objective of these experiments was to test whether preferential neighborhoods of ancestral chromosome forms may exist, which may have predisposed for evolutionary fusions or translocations. A second aspect of the present study was to investigate the topology of genes, which are differentially expressed in fibroblast nuclei of human and chimpanzee, but which reside in otherwise conserved genomic regions in both species. This study served to elucidate whether speciesspecific changes of the transcriptional status leads to a significant repositioning of the locus in the nucleus and in the respective CT. With this study we aim to illuminate the impact of genomic and expression level changes on the nuclear architecture and help clarify to which extent non-random spatial chromatin arrangements can affect the direction of genome evolution.
P74: Arrangements of chromosome territories in human cell clones Daniela Köhler a, Juntao Gao b, Julian Mattes b, Roland Eils b, Thomas Cremer a, Irina Solovei a a LMU, University of Munich, Munich, Germany b iBios, German Cancer Research Center (DKFZ), Heidelberg, Germany Arrangement of chromosome territories (CT) throughout mitosis is still discussed controversial (Gerlich et al., Cell 112 (2003) 751; Walter et al., J Cell Biol, 160 (2003) 685). Therefore we performed 3D-FISH experiments with sets of chromosome specific probes on human cell clones, including normal diploid fibroblasts (two-cell clones), normal epithelial cells (twoand four-cell clones) and the cancer cell lines DLD and HeLa (2-, 4-, 8-, 16-, and 32-cell clones). In addition we performed living cell experiments to trace clonally dividing cells over several cell cycles and identify their relationship by descent. To estimate the degree of similarity of CT arrangements between cells derived from the same clone we determined (i) the 3D distance differences between gravity centers of homologous CTs and (ii) the “bending energy” (BE), a virtual energy required to transform the CT arrangement of one nucleus into that of another. Both approaches consistently confirmed very similar CT arrangements in sister nuclei of two-cell clones. For clones with larger number of cells, higher values of both parameters were found, steadily increasing from 2- to 16-cell clones. In 16-cell clones dissimilarities
K. Teller et al. / European Journal of Medical Genetics 48 (2005) 537–542
541
reached the level observed for randomly chosen non-related cells from the entire cell population. As a control we performed replication-labeling experiments where we followed a large number of clearly defined replication foci over several hours, which provided an independent data set of bending energies. As expected, the measured BE was rather low. From these results we conclude that CT arrangement is inherited by daughter cells from the parent cell to a certain degree. On the other hand, CTs change their neighborhood in each successive mitosis. These changes accumulate and result in complete loss of similarity compared to the ancestor cell after only a few cell divisions.
P75: Analysis of expression related changes of a Tat inducible HIV construct Claudia Weierich a, Manja Ziegler b, Ruth Brack-Werner b, Thomas Cremer a a Department of Biology II, LMU Munich, Institute of Human Genetics, Munich, Germany b GSF-Institute of Molecular Virology, Germany Recent studies demonstrate that HIV-DNA can be expressed not only in a chromosomally integrated but also in an episomal state (Wu and Marsh, 2003). To study these phenomena in a model system, HeLa cells were transfected with a DNA construct connecting a Tat-inducible HIV promoter with a reporter gene. This gene encodes a red fluorescent protein (RFP) fused to a nuclear export signal. The 3D location of the construct in the nucleus is visualized by binding of mRFP-LacI to multiple LacI binding sites upstream of the reporter gene, while its transcriptional activity is indicated by RFP accumulation in the cytoplasm. A 3D positional analysis is presently carried out in nuclei of living HeLa cells under a variety of conditions, including the episomal or chromosomally integrated state of the construct. Furthermore the non-induced or Tat-induced status of its reporter gene is investigated in its 3D position relative to nuclear reference structures like centromeres and splicing factor-containing speckles. Live cell experiments are complemented by 3D FISH and immunocytochemistry.
P76: Kissing not required for maintenance of imprinting in cycling human cells Kathrin Teller a, Irina Solovei a, Stefan Stein b, Karin Buiting c, Bernhard Horsthemke c, Thomas Cremer, Thomas Cremer a, Thomas Cremer a a Department of Biology II, Ludwig Maximilians University, 82152 Martinsried, Germany b Kirchhoff Institute of Physics, University of Heidelberg, 69120 Heidelberg, Germany c Institute of Human Genetics, University of Essen, 45122 Essen, Germany In 1996 spatial association of oppositely imprinted regions in late S-phase but not at other stages of the cell cycle was reported for the Prader-Willi/Angelman syndrome regions on chromosome 15q11–13 in human cells (LaSalle and Lalande, Science 272 (1996) 725). This so called “kissing model” is postulated to be an important mechanism to maintain the right imprinting status and the correct expression of genes during the cell cycle. In 1999 this homologous association was also reported for an imprinted region on mouse chromosome 7 (Riesselmann and Haaf, Cytogenet Cell Genet 86 (1) (1999) 39). We re-investigated this “kissing model” using 3D-Fluorescence in Situ Hybridization (3D-FISH) with locus specific probes. A precise S-phase staging was achieved for every individual cell using replication labeling with BrdU. 3D image stacks were acquired with a confocal laser-scanning microscope and distance measurements were done on these stacks. Our results demonstrate that imprinted PWS/AS re-
POSTER
D. Köhler et al. / European Journal of Medical Genetics 48 (2005) 537–542 PRESENTATIONS
P73: Impact of primate evolutionary chromosome rearrangements on the nuclear topology of genomic signatures Michaela Neusser, Thomas Cremer, Stefan Muller Department Biology II, Human Genetics, Ludwig-Maximilians University, Munich, Germany The impact of genomic signatures like gene density, base-pair composition and repetitive elements on the higher order nuclear architecture has been in the focus of numerous investigations. These included studies of their spatial orientation, DNA methylation status, histone modification and chromatin fibre compactness, with the aim to explain putative functional properties related to nuclear compartmentalization and epigenetic control mechanisms of gene regulation. Here we report on a comparative 3D FISH study of human and various higher primate species, to investigate the impact of evolutionary genomic changes on the nuclear organization of genomic signatures. Employing a panel of breakpoint flanking BAC clones we analyzed the side-by-side arrangement of evolutionary rearranged chromosomes. Objective of these experiments was to test whether preferential neighborhoods of ancestral chromosome forms may exist, which may have predisposed for evolutionary fusions or translocations. A second aspect of the present study was to investigate the topology of genes, which are differentially expressed in fibroblast nuclei of human and chimpanzee, but which reside in otherwise conserved genomic regions in both species. This study served to elucidate whether speciesspecific changes of the transcriptional status leads to a significant repositioning of the locus in the nucleus and in the respective CT. With this study we aim to illuminate the impact of genomic and expression level changes on the nuclear architecture and help clarify to which extent non-random spatial chromatin arrangements can affect the direction of genome evolution.
P74: Arrangements of chromosome territories in human cell clones Daniela Köhler a, Juntao Gao b, Julian Mattes b, Roland Eils b, Thomas Cremer a, Irina Solovei a a LMU, University of Munich, Munich, Germany b iBios, German Cancer Research Center (DKFZ), Heidelberg, Germany Arrangement of chromosome territories (CT) throughout mitosis is still discussed controversial (Gerlich et al., Cell 112 (2003) 751; Walter et al., J Cell Biol, 160 (2003) 685). Therefore we performed 3D-FISH experiments with sets of chromosome specific probes on human cell clones, including normal diploid fibroblasts (two-cell clones), normal epithelial cells (twoand four-cell clones) and the cancer cell lines DLD and HeLa (2-, 4-, 8-, 16-, and 32-cell clones). In addition we performed living cell experiments to trace clonally dividing cells over several cell cycles and identify their relationship by descent. To estimate the degree of similarity of CT arrangements between cells derived from the same clone we determined (i) the 3D distance differences between gravity centers of homologous CTs and (ii) the “bending energy” (BE), a virtual energy required to transform the CT arrangement of one nucleus into that of another. Both approaches consistently confirmed very similar CT arrangements in sister nuclei of two-cell clones. For clones with larger number of cells, higher values of both parameters were found, steadily increasing from 2- to 16-cell clones. In 16-cell clones dissimilarities
K. Teller et al. / European Journal of Medical Genetics 48 (2005) 537–542
541
reached the level observed for randomly chosen non-related cells from the entire cell population. As a control we performed replication-labeling experiments where we followed a large number of clearly defined replication foci over several hours, which provided an independent data set of bending energies. As expected, the measured BE was rather low. From these results we conclude that CT arrangement is inherited by daughter cells from the parent cell to a certain degree. On the other hand, CTs change their neighborhood in each successive mitosis. These changes accumulate and result in complete loss of similarity compared to the ancestor cell after only a few cell divisions.
P75: Analysis of expression related changes of a Tat inducible HIV construct Claudia Weierich a, Manja Ziegler b, Ruth Brack-Werner b, Thomas Cremer a a Department of Biology II, LMU Munich, Institute of Human Genetics, Munich, Germany b GSF-Institute of Molecular Virology, Germany Recent studies demonstrate that HIV-DNA can be expressed not only in a chromosomally integrated but also in an episomal state (Wu and Marsh, 2003). To study these phenomena in a model system, HeLa cells were transfected with a DNA construct connecting a Tat-inducible HIV promoter with a reporter gene. This gene encodes a red fluorescent protein (RFP) fused to a nuclear export signal. The 3D location of the construct in the nucleus is visualized by binding of mRFP-LacI to multiple LacI binding sites upstream of the reporter gene, while its transcriptional activity is indicated by RFP accumulation in the cytoplasm. A 3D positional analysis is presently carried out in nuclei of living HeLa cells under a variety of conditions, including the episomal or chromosomally integrated state of the construct. Furthermore the non-induced or Tat-induced status of its reporter gene is investigated in its 3D position relative to nuclear reference structures like centromeres and splicing factor-containing speckles. Live cell experiments are complemented by 3D FISH and immunocytochemistry.
P76: Kissing not required for maintenance of imprinting in cycling human cells Kathrin Teller a, Irina Solovei a, Stefan Stein b, Karin Buiting c, Bernhard Horsthemke c, Thomas Cremer, Thomas Cremer a, Thomas Cremer a a Department of Biology II, Ludwig Maximilians University, 82152 Martinsried, Germany b Kirchhoff Institute of Physics, University of Heidelberg, 69120 Heidelberg, Germany c Institute of Human Genetics, University of Essen, 45122 Essen, Germany In 1996 spatial association of oppositely imprinted regions in late S-phase but not at other stages of the cell cycle was reported for the Prader-Willi/Angelman syndrome regions on chromosome 15q11–13 in human cells (LaSalle and Lalande, Science 272 (1996) 725). This so called “kissing model” is postulated to be an important mechanism to maintain the right imprinting status and the correct expression of genes during the cell cycle. In 1999 this homologous association was also reported for an imprinted region on mouse chromosome 7 (Riesselmann and Haaf, Cytogenet Cell Genet 86 (1) (1999) 39). We re-investigated this “kissing model” using 3D-Fluorescence in Situ Hybridization (3D-FISH) with locus specific probes. A precise S-phase staging was achieved for every individual cell using replication labeling with BrdU. 3D image stacks were acquired with a confocal laser-scanning microscope and distance measurements were done on these stacks. Our results demonstrate that imprinted PWS/AS re-