hether chromosomes maintain their nuclear positions during interphase and from one cell cycle to the next has been controversially discussed. To address this question, we performed long-term live-cell studies using a HeLa cell line with GFP-tagged chromatin. Positional changes of the intensity gravity centers of fluorescently labeled chromosome territories (CTs) on the order of several m were observed in early G1, suggesting a role of CT mobility in establishing interphase nuclear architecture. Thereafter, the positions were highly constrained within a range of ‫ف‬ 1 m until the end of G2. To analyze possible changes of chromosome arrangements from one cell cycle to the next, nuclei were photobleached in G2 W maintaining a contiguous zone of unbleached chromatin at one nuclear pole. This zone was stably preserved until the onset of prophase, whereas the contiguity of unbleached chromosome segments was lost to a variable extent, when the metaphase plate was formed. Accordingly, chromatin patterns observed in daughter nuclei differed significantly from the mother cell nucleus. We conclude that CT arrangements were stably maintained from mid G1 to late G2/early prophase, whereas major changes of CT neighborhoods occurred from one cell cycle to the next. The variability of CT neighborhoods during clonal growth was further confirmed by chromosome painting experiments.

In mammalian cells, the replication of genetic and epigenetic information is directly coupled; however, little is known about the maintenance of epigenetic information in DNA repair. Using a laser microirradiation system to introduce DNA lesions at defined subnuclear sites, we tested whether the major DNA methyltransferase (Dnmt1) or one of the two de novo methyltransferases (Dnmt3a, Dnmt3b) are recruited to sites of DNA repair in vivo. Time lapse microscopy of microirradiated mammalian cells expressing GFPtagged Dnmt1, Dnmt3a, or Dnmt3b1 together with red fluorescent protein-tagged proliferating cell nuclear antigen (PCNA) revealed that Dnmt1 and PCNA accumulate at DNA damage sites as early as 1 min after irradiation in S and non-S phase cells, whereas recruitment of Dnmt3a and Dnmt3b was not observed. Deletion analysis showed that Dnmt1 recruitment was mediated by the PCNAbinding domain. These data point to a direct role of Dnmt1 in the restoration of epigenetic information during DNA repair.DNA methylation ͉ Dnmt1 ͉ microirradiation ͉ proliferating cell nuclear antigen I n higher eukaryotes, maintenance and propagation of genetic and epigenetic information is essential for cellular identity and survival. By-products of normal cellular metabolism, spontaneous mutations, and environmental agents can lead to various types of DNA damage. Numerous DNA repair pathways reestablishing the genetic information are known and have been intensively described (1, 2). However, very little is known about enzymes and mechanisms involved in the restoration of the epigenetic information. There are two main epigenetic marks, DNA methylation and histone modifications, which are essential for cell type-specific gene expression and maintained over multiple cell divisions (3-7). Recently, chromatin assembly and remodeling have been linked to DNA repair (8-10).DNA methylation is a postreplicative modification occurring mostly at cytosine residues of CpG dinucleotides and is essential for mammalian development (11), parental imprinting (12), X inactivation (13), and genome stability (14,15). In mammalian cells, DNA methylation is catalyzed by two types of enzymes, maintenance (Dnmt1) and de novo methyltransferases (Dnmt3a, Dnmt3b) (16). The maintenance methyltransferase Dnmt1 has a preference for hemimethylated CpG sites generated during DNA replication and is ubiquitously expressed (16). Dnmt1 associates with replication sites by directly binding to proliferating cell nuclear antigen (PCNA) and thus maintains DNA methylation patterns in the newly synthesized strand after DNA replication (17,18). In contrast to the maintenance methyltransferase Dnmt1, the de novo methyltransferases Dnmt3a and Dnmt3b are responsible for establishing new DNA methylation patterns during development and show a low and tissue-specific expression (19)(20)(21).The importance of maintaining the epigenetic information was recently underscored by knockdown experiments. Lowering Dnmt1 to rate-limiting amounts in transgenic mice lead to a loss of DNA methylation,...

Rad51, a eukaryotic RecA homologue, plays a central role in homologous recombinational repair of DNA double-strand breaks (DSBs) in yeast and is conserved from yeast to human. Rad51 shows punctuate nuclear localization in human cells, called Rad51 foci, typically during the S phase (Tashiro, S., N. Kotomura, A. Shinohara, K. Tanaka, K. Ueda, and N. Kamada. 1996. Oncogene. 12:2165–2170). However, the topological relationships that exist in human S phase nuclei between Rad51 foci and damaged chromatin have not been studied thus far. Here, we report on ultraviolet microirradiation experiments of small nuclear areas and on whole cell ultraviolet C (UVC) irradiation experiments performed with a human fibroblast cell line. Before UV irradiation, nuclear DNA was sensitized by the incorporation of halogenated thymidine analogues. These experiments demonstrate the redistribution of Rad51 to the selectively damaged, labeled chromatin. Rad51 recruitment takes place from Rad51 foci scattered throughout the nucleus of nonirradiated cells in S phase. We also demonstrate the preferential association of Rad51 foci with postreplicative chromatin in contrast to replicating chromatin using a double labeling procedure with halogenated thymidine analogues. This finding supports a role of Rad51 in recombinational repair processes of DNA damage present in postreplicative chromatin.

Reversible acetylation of nucleosomal histones H3 and H4 generally is believed to be correlated with potential transcriptional activity of eukaryotic chromatin domains. Here, we report that the extent of H4 acetylation within euchromatin and heterochromatic domains is linked with DNA replication rather than with transcriptional activity, whereas H3 acetylation remains fairly constant throughout the cell cycle. Compared with euchromatin, plant nucleolus organizers were more strongly acetylated at H4 during mitosis but less acetylated during S phase, when the nucleolus appeared to be (at least transiently) devoid of nucleosomes. Deposition-related acetylation of lysines 5 and 12 of H4 seems to be conserved in animals and plants and extended to K16 in plants. A possibly species-specific above-average acetylation at lysines 9/18 and 14 of H3 appeared in 4 ,6-diamidino-2-phenylindole (DAPI)-stained heterochromatin fractions. These results were obtained by combining immunodetection of all acetylatable isoforms of H3 and H4 on mitotic chromosomes and nuclei in G1, early S, mid-S, late S, and G2 phases of the field bean with identification of specific chromatin domains by fluorescence in situ hybridization or DAPI staining. In addition, the histone acetylation patterns of distinct domains were compared with their replication and transcription patterns. INTRODUCTIONThe histone proteins H2A, H2B, H3, and H4 form octamers that constitute the nucleosome core particles in all eukaryotes. Their N-terminal tails are subject to post-translational modifications such as acetylation, phosphorylation, methylation, ubiquitination, glycosylation, and ADP ribosylation (reviewed in Smith et al., 1995;Spencer and Davie, 1999).The reversible acetylation of N-terminal lysine residues at positions 5, 8, 12, and 16 of H4 and 9, 14, 18, and 23 of H3 mediates decondensation of the nucleosome structure (Loidl, 1988(Loidl, , 1994Garcia-Ramirez et al., 1995), alters histone-DNA interactions (Hong et al., 1993), and facilitates access and binding of transcription factors to genes transcribed by RNA polymerases II or III (Lee et al., 1993;Vettese-Dadey et al., 1996).A correlation between histone acetylation and potential transcriptional activity, initially proposed by Allfrey et al. (1964), has been proved in several cases (reviewed in Csordas, 1990;Turner, 1991Turner, , 1993Loidl, 1994;Grunstein, 1997;Struhl, 1998). According to one attractive recent hypothesis, histone modifications may constitute a concerted code to "specify unique downstream functions" (Strahl and Allis, 2000;Turner, 2000).After indirect immunolabeling with antibodies raised against acetylated isoforms of histone H4 (Turner and Fellows, 1989;, mammalian metaphase chromosomes show intense acetylation of euchromatic R-bands and less intense acetylation of constitutive and facultative heterochromatin (Jeppesen and Turner, 1993). The patterns of histone H4 acetylation described for plant chromosomes (Houben et al., 1996Belyaev et al., 1997;Vyskot et al., 1999) also reveal a below...

A quantitative comparison of higher-order chromatin arrangements was performed in human cell types with three-dimensionally (3D) preserved, differently shaped nuclei. These cell types included flat-ellipsoid nuclei of diploid amniotic fluid cells and fibroblasts and spherical nuclei of B and T lymphocytes from peripheral human blood. Fluorescence in-situ hybridization (FISH) was performed with chromosome paint probes for large (#1-5) and small (#17-20) autosomes, and for the two sex chromosomes. Other probes delineated heterochromatin blocks of numerous larger and smaller human chromosomes. Shape differences correlated with distinct differences in higher order chromatin arrangements: in the spherically shaped lymphocyte nuclei we noted the preferential positioning of the small, gene dense #17, 19 and 20 chromosome territories (CTs) in the 3D nuclear interior--typically without any apparent connection to the nuclear envelope. In contrast, CTs of the gene-poor small chromosomes #18 and Y were apparently attached at the nuclear envelope. CTs of large chromosomes were also preferentially located towards the nuclear periphery. In the ellipsoid nuclei of amniotic fluid cells and fibroblasts, all tested CTs showed attachments to the upper and/or lower part of the nuclear envelope: CTs of small chromosomes, including #18 and Y, were located towards the centre of the nuclear projection (CNP), while the large chromosomes were positioned towards the 2D nuclear rim. In contrast to these highly reproducible radial arrangements, 2D distances measured between heterochromatin blocks of homologous and heterologous CTs were strikingly variable. These results as well as CT painting let us conclude that nuclear functions in the studied cell types may not require reproducible side-by-side arrangements of specific homologous or non-homologous CTs. 3D-modelling of statistical arrangements of 46 human CTs in spherical nuclei was performed under the assumption of a linear correlation between DNA content of each chromosome and its CT volume. In a set of modelled nuclei, we noted the preferential localization of smaller CTs towards the 3D periphery and of larger CTs towards the 3D centre. This distribution is in clear contrast to the experimentally observed distribution in lymphocyte nuclei. We conclude that presently unknown factors (other than topological constraints) may play a decisive role to enforce the different radial arrangements of large and small CTs observed in ellipsoid and spherical human cell nuclei.