Three-dimensional arrangements of centromeres and telomeres in nucleiof human and murine lymphocytesClaudia Weierich1, Alessandro Brero1, Stefan Stein2,JohannvonHase2, Christoph Cremer2,Thomas Cremer1& Irina Solovei11Department of Biology II, Human Genetics, Ludwig Maximillians University (LMU), RichardWagner Str. 10, 80333 Munich, Germany; Tel: þ 49-89-2180-6713; Fax: þ 49-89-2180-6719;E-mail: [email protected];2Kirchhoff Institute of Physics, University of Heidelberg,Heidelberg, 69120 Germany*CorrespondenceKey words: centromere, chromosome, confocal microscopy, £uorescence in-situ hybridization, interphase,lymphocyte, nuclear architecture, telomereAbstractThe location of centromeres and telomeres was studied in human and mouse lymphocyte nuclei (G0)employing 3D-FISH, confocal microscopy, and quantitative image analysis. In both human and murinelymphocytes, most centromeres were found in clusters at the nuclear periphery. The distribution oftelomere clusters, however, differed: in mouse nuclei, most clusters were detected at the nuclear periphery,while, in human nuclei, most clusters were located in the nuclear interior. In human cell nuclei we furtherstudied the nuclear location of individual centromeres and their respective chromosome territories (CTs)for chromosomes 1, 11, 12, 15, 17, 18, 20, and X. We found a peripheral location of both centromeresand CTs for 1, 11, 12, 18, X. A mostly interior nuclear location was observed for CTs 17 and 20 andthe CTs of the NOR-bearing acrocentric 15 but the corresponding centromeres were still positioned inthe nuclear periphery. Autosomal centromeres, as well as the centromere of the active X, were typicallylocated at the periphery of the respective CTs. In contrast, in about half of the inactive X-CTs, thecentromere was located in the territory interior. While the centromere of the active X often participatedin the formation of centromere clusters, such a participation was never observed for the centromereof the inactive X.IntroductionChromosomes occupy mutually exclusive nucleardomains in mammalian cell nuclei, called chro-mosome territories (CTs). Chromosome armdomains, as well as G- and R-band domains,occupy distinct subregions within these CTs withlittle chromatin intermingling (Dietzel et al.1998a, 1998b, Zink et al. 1999, Chevret et al.2000, Cremer & Cremer 2001). The radial nucleararrangements of CTs and chromosomal sub-regions of di¡erent cell types and di¡erent speciesare non-random and evolutionary conservedsuggesting that they may be important for nuclearfunctions (Cremer et al. 2000, Cremer & Cremer2001, Dundr & Misteli 2001, Parada & Misteli2002, Tanabe et al. 2002a). Knowledge of 3Dlocations of subchromosomal regions, however,Chromosome Research 11: 485^502, 2003. 485# 2003 Kluwer Academic Publishers. Printed in the Netherlandshas remained very limited to date. Some reportsdescribed evidence that actively transcribedsequences are situated at, or close to, the surfaceof interphase CTs, while silent genes and non-transcribed sequences are predominantly found inthe inner portions of CTs (Kurz et al. 1996,Dietzel et al. 1999). It was also shown that theMHC locus is located at the periphery ofthe human CT #6 and that induction ofthe expression of MHC class II genes causes theexpansion of large loops outwards from the CTperiphery (Volpi et al. 2000). Other reports,however, indicate that transcription can also beobserved in the CT interior (Abranches et al.1998, Mahy et al. 2002a, 2002b). In order toexplain these cases one should take into accountthe folding of CTs. This folding could result ininvaginations of the CT surface allowing thedirect contact of chromatin loop domains har-boring the respective genes with the inter-chromatin compartment (Cremer & Cremer,2001). It was recently shown that the associationof genes with heterochromatic chromocenterscontributes to their inactivation (Brown et al.1997, 1999, Schubeler et al. 2000, Baxter et al.2002). This ¢nding suggests that the topology ofa centromere with regard to its respective terri-tory and to the nucleus at large may play a role inthe epigenetic mechanisms that control genesilencing.While a number of publications exist whichdescribe the nuclear locations of centromeres (seebelow), their possible locations with respect totheir CTs have not been analyzed in detail exceptfor two recent publications: one reported that thecentromere of human chromosome #6 occupies aperipheral CT position (Chevret et al. 2000); theother that the centromere of human chromosome#15 is mostly located in the interior of the CT(Nogami et al. 2000).Nuclear positions of centromeres and telomereshave been intensely studied. A tendency to aperipheral location of centromeres at G1/G0wasnoted for both human (Ferguson & Ward 1992,Weimer et al. 1992, Alcobia et al. 2000, Skalnikovaet al. 2000) and mouse cells (Vourc’h et al. 1993).Recent publications have focused on thearrangements of CTs in the interphase nucleus(reviewed by Parada & Misteli, 2002). It has beenshown that CTs have a non-random radial dis-tribution in spherical nuclei of lymphocytes andlymphoblastoid cells (Croft et al. 1999, Boyle et al.2001, Cremer et al. 2001, Tanabe et al. 2002a,Tanabe et al. 2002b). CTs with a high gene densityare located more centrally than gene-poor CTs.The question of how the internal position of gene-poor CTs is compatible with the peripherallocation of centromeres in spherical nuclei has notbeen resolved and requires the analysis of speci¢ccentromere localizations in 3D preserved nuclei(Solovei et al. 2002b).The present study was focused on the spatialarrangements of speci¢c centromeres, includingthe centromeres of human chromosomes 1, 4, 11,12, 13 þ 21, 14 þ 22, 15, 17, 18, 20 and respectivechromosome territories (for most of them) inhuman non-stimulated (G0) lymphocytes fromperipheral blood. Further, we wished to comparethe centromere locations in active and inactiveX-CTs. Since we were not able to identify theBarr body (X inactive) in lymphocyte nuclei, forthis purpose we chose human female ¢broblastswhere the Barr body could easily be identi¢ed.For comparison, the spatial arrangements of allcentromeres, as well as all telomeres, wereanalyzed in nuclei from human and mouse G0lymphocytes. To approach these matters we usedtwo-color 3D-FISH and nucleoli immunostainingin combination with confocal microscopy. Forthe analysis of the distribution of £uorescentlylabeled DNA in
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