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Nuclear positioning and functional regulation of endogenous genes and transgenes in the fruit fly Drosophila melanogaster and in mammalian cells
Nuclear positioning and functional regulation of endogenous genes and transgenes in the fruit fly Drosophila melanogaster and in mammalian cells
The goal of the work was to address the role of higher order nuclear architecture in the functional regulation of endogenous genes and transgenes in different species. In the first part of the work, 3D distance measurements were performed to analyze in WT flies and in different transgenic lines of Drosophila melanogaster (Cavalli and Paro, 1998; Zink and Paro, 1995) the nuclear localization of endogenous genes and transgenic constructs containing the Polycomb Response Element (PRE) Fab-7 relative to the nuclear periphery and heterochromatin. Transgenic constructs containing the Fab-7 element and three endogenous genes, Abd-B, sd, and Ubx, were first analyzed at their inactive state. The results showed that they were preferentially associated with the nuclear periphery and did not display specific associations with heterochromatin. The localization of the transgenic Fab-7 element was further analyzed at different states of activity. Activation of the transgenic Fab-7 element resulted in frequent (up to ~50%) association with the boundary of the heterochromatic domain. The percentages of such associations were tissue- and fl y line-dependent. Further investigations of the boundary of heterochromatin showed that this region has a complex organization, where euchromatic sites enriched in the active form of RNA pol II and trimH3K4, sites enriched in dimH3K4 and Pc-binding sites, as well as pericentromeric satellite DNA are exposed and juxtaposed towards each other. The concentration and specific architecture of such sites at the boundary of heterochromatin might help to maintain the equilibrium between activation and repression at this boundary. This specific environment might be favourable for maintaining PREs in the active state. I also investigated in three transgenic lines whether endogenous and transgenic copies of the Fab-7 element interact physically, using 3D distance measurements. In five tissues analyzed, no pairing between endogenous and transgenic copies of the Fab-7 element was observed. Also enhancement of Pc-mediated silencing did not induce pairing. Additionally, the general organization of Pc-binding sites was addressed in six larval tissues. We used different methods of microscopy and image analyses to count the numbers of Pc foci in nuclei from these tissues. Our data did not indicate clustering of Pc-binding sites and formation of so called „Pc bodies“. However, corresponding analyses are not without problems at the current stage of methodology and results must be interpreted carefully. Together with the results of previously published studies, which investigated pairing between PREs (Bantignies et al., 2003; Vazquez et al., 2006), my data demonstrated that such pairing is a highly tissue-specific phenomenon and is likely not involved in the regulation of PREs in various tissues. Activity-related positioning of transgenes was also addressed in transgenic porcine cell lines (Hofmann et al., 2003). Results of 2D erosion analyses showed that the LV-PGK transgene was associated with the nuclear periphery in its inactive state, while it occupied more interior positions in its active state. This corresponds to my results obtained with transgenic Drosophila lines. My data also suggested that the active LV-PGK construct might be associated with heterochromatin in one case. However, further experiments would be necessary to confirm such associations. The results obtained with Drosophila and porcine cells suggested conserved mechanisms for tethering inactive loci to the nuclear periphery. These were further addressed using the human CFTR locus as a model, which is closely associated with the nuclear periphery in its inactive state (Zink et al., 2004). The question was addressed whether Tpr, a protein associated with the nuclear basket, plays a role in the perinuclear localization of the inactive CFTR locus. CFTR showed a high degree of association with the nuclear periphery in control cells in accordance with previous data (Zink et al., 2004). After knock-down of Tpr via RNAi CFTR displayed a more interior positioning. This suggests that Tpr is involved in the organization of inactive gene loci at the nuclear periphery. Moreover, since Drosophila Tpr has a high level of homology to the mammalian Tpr (Zimowska et al., 1997) and as it has been shown that the yeast Tpr homologs Mlp1 and Mlp2 are involved in tethering of inactive loci to the nuclear periphery in yeast cells (Galy et al., 2000), it is possible that Tpr is a part of a conserved mechanism anchoring inactive loci to the nuclear periphery in eukaryotic cells.
Nuclear architecture, Drosophila, Polycomb Response Elements, chromatin, the nuclear periphery
Rybakina, Elena
2006
Englisch
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Rybakina, Elena (2006): Nuclear positioning and functional regulation of endogenous genes and transgenes in the fruit fly Drosophila melanogaster and in mammalian cells. Dissertation, LMU München: Fakultät für Biologie
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Abstract

The goal of the work was to address the role of higher order nuclear architecture in the functional regulation of endogenous genes and transgenes in different species. In the first part of the work, 3D distance measurements were performed to analyze in WT flies and in different transgenic lines of Drosophila melanogaster (Cavalli and Paro, 1998; Zink and Paro, 1995) the nuclear localization of endogenous genes and transgenic constructs containing the Polycomb Response Element (PRE) Fab-7 relative to the nuclear periphery and heterochromatin. Transgenic constructs containing the Fab-7 element and three endogenous genes, Abd-B, sd, and Ubx, were first analyzed at their inactive state. The results showed that they were preferentially associated with the nuclear periphery and did not display specific associations with heterochromatin. The localization of the transgenic Fab-7 element was further analyzed at different states of activity. Activation of the transgenic Fab-7 element resulted in frequent (up to ~50%) association with the boundary of the heterochromatic domain. The percentages of such associations were tissue- and fl y line-dependent. Further investigations of the boundary of heterochromatin showed that this region has a complex organization, where euchromatic sites enriched in the active form of RNA pol II and trimH3K4, sites enriched in dimH3K4 and Pc-binding sites, as well as pericentromeric satellite DNA are exposed and juxtaposed towards each other. The concentration and specific architecture of such sites at the boundary of heterochromatin might help to maintain the equilibrium between activation and repression at this boundary. This specific environment might be favourable for maintaining PREs in the active state. I also investigated in three transgenic lines whether endogenous and transgenic copies of the Fab-7 element interact physically, using 3D distance measurements. In five tissues analyzed, no pairing between endogenous and transgenic copies of the Fab-7 element was observed. Also enhancement of Pc-mediated silencing did not induce pairing. Additionally, the general organization of Pc-binding sites was addressed in six larval tissues. We used different methods of microscopy and image analyses to count the numbers of Pc foci in nuclei from these tissues. Our data did not indicate clustering of Pc-binding sites and formation of so called „Pc bodies“. However, corresponding analyses are not without problems at the current stage of methodology and results must be interpreted carefully. Together with the results of previously published studies, which investigated pairing between PREs (Bantignies et al., 2003; Vazquez et al., 2006), my data demonstrated that such pairing is a highly tissue-specific phenomenon and is likely not involved in the regulation of PREs in various tissues. Activity-related positioning of transgenes was also addressed in transgenic porcine cell lines (Hofmann et al., 2003). Results of 2D erosion analyses showed that the LV-PGK transgene was associated with the nuclear periphery in its inactive state, while it occupied more interior positions in its active state. This corresponds to my results obtained with transgenic Drosophila lines. My data also suggested that the active LV-PGK construct might be associated with heterochromatin in one case. However, further experiments would be necessary to confirm such associations. The results obtained with Drosophila and porcine cells suggested conserved mechanisms for tethering inactive loci to the nuclear periphery. These were further addressed using the human CFTR locus as a model, which is closely associated with the nuclear periphery in its inactive state (Zink et al., 2004). The question was addressed whether Tpr, a protein associated with the nuclear basket, plays a role in the perinuclear localization of the inactive CFTR locus. CFTR showed a high degree of association with the nuclear periphery in control cells in accordance with previous data (Zink et al., 2004). After knock-down of Tpr via RNAi CFTR displayed a more interior positioning. This suggests that Tpr is involved in the organization of inactive gene loci at the nuclear periphery. Moreover, since Drosophila Tpr has a high level of homology to the mammalian Tpr (Zimowska et al., 1997) and as it has been shown that the yeast Tpr homologs Mlp1 and Mlp2 are involved in tethering of inactive loci to the nuclear periphery in yeast cells (Galy et al., 2000), it is possible that Tpr is a part of a conserved mechanism anchoring inactive loci to the nuclear periphery in eukaryotic cells.