In previous studies of chromatin dynamics in mammalian cells, live cell observations were restricted to a limited time window ranging from a few minutes up to five hours. These studies could not exclude the possibility of slow “drifting” movements of chromosome territories during interphase that could result in major changes of the chromosome territory order from early G1 to late G2.
With the long-term in vivo study of Cy3- or Cy5-labeled CTs presented in this work the observation periods could be extended significantly to cover a complete cell cycle of 18 hours or more. Quantitative distance measurements over time showed that CTs were confined within a radius of about 0.5 - 1 µm from mid G1 to late G2, which is in agreement with earlier studies on chromatin dynamics in mammalian cells (Shelby et al., 1996; Abney et al., 1997; Zink and Cremer, 1998; Bornfleth et al., 1999; Chubb et al., 2002; Lucas and Cervantes, 2002). However, more extended positional changes (occasionally exceeding 4 µm) were observed during the first 2 - 3 hours after mitosis. This increased mobility cannot simply be attributed to the rapid volume increase during telophase-G1-transition as the data were corrected for this effect. Moreover, distances between different CTs occasionally became smaller during telophase/early G1, indicating a directed relocalization of CTs. Heterochromatic associations involving large-scale reorganization of chromosomes during G1 were previously described in Drosophila larval nuclei (Csink and Henikoff, 1998). In a study of painted CTs in fixed human diploid fibroblast nuclei Bridger et al. (2000) found that the final locations of CT#18 were established during the first 2 - 4 hours of G1. Evidence for an increased mobility during early G1 compared to later interphase stages was also presented for GFP-tagged centromeres in living HeLa cells (Shelby et al., 1996) and for a GFP-tagged heterochromatic chromosomal site in CHO cells (Tumbar and Belmont, 2001). Together, these results support the idea of a time window of increased chromatin mobility during early G1 until structural components, essential for the stability of CT arrangements are fully established.
More extensive movements during early G1 may also reflect a necessity of CTs to home in to their final nuclear locations and to establish a “polarized” orientation (Sadoni et al., 1999). In all normal and malignant mammalian cell types studied so far, gene-poor chromatin, which replicates in mid S, is preferentially arranged in the nuclear periphery and around the nucleoli, whereas gene-dense, transcriptionally active early replicating chromatin is located in a zone which expands between these two transcriptionally silent compartments (Dimitrova and Berezney, 2002). Interestingly, it was possible to show that replication-timing program is also established in early G1, 1-2 hours after metaphase concomitant with repositioning of chromosomal domains (Dimitrova and Gilbert, 1999; Li et al., 2001). In these studies, CHO cell nuclei isolated at different stages of cell cycle were incubated in a cell-free replication competent Xenopus egg extract. The proper spacio-temporal order of replicated chromatin domains was found when nuclei were isolated in late G1, a random order of replication domains was, however, observed when nuclei were isolated in early G1.
A comparison of the data presented here with other published studies of chromatin movements in nuclei of living cells should be performed with caution: The long-term measurements of constrained movements of whole CTs performed in the present study with sampling intervals ranging from 6 to 30 min for total observation periods of many hours cannot be directly compared with movements of small chromatin sites analyzed in other cell systems using intervals of minutes or seconds for much shorter evaluation periods. For lac-operator repeats integrated into different chromosomal sites of human HT-1080 cells, Chubb et al.
(2002) found that the maximum range of movements depend on the subnuclear localization totaling 0.9 µm for a site located in the nuclear periphery and 1.5 µm for a nucleoplasmic site. Diffusion coefficients were in the range of 10-4 µm2/s or lower. In HeLa cell nuclei initial evaluations of diffusion coefficients for ~1 Mb chromatin domains and entire CTs recorded with sampling intervals of 20 min imply values in the order of 10-5 µm2/s (Bornfleth et al., 1999; Edelmann et al., 2001).
In contrast to mammalian cells, a higher mobility with diffusion coefficients in the order of 10-3 to 10-2 µm2/s was reported for fluorescence tagged chromosomal sites in Drosophila and yeast (for reviews see Gasser, 2002; Marshall, 2002). In Drosophila early embryos, confinement radii ≤ 0.9 µm were reported for a heterochromatic site (Marshall et al., 1997). In Drosophila
spermatocyte nuclei, Vazquez et al. (2001) described long-range motions of integrated lac-operator sites to be confined to a radius of 3 µm in mid G2 and ~0.3 µm in late G2 nuclei. In yeast, the movements of centromeric and telomeric sites were confined to radii ≤ 0.3 µm independent of the cell cycle. In contrast, chromosomal regions close to transcribed genes were found to be less constrained in G1 with a radius of 0.7 µm, showing particularly fast movements of up to 0.5 µm within 10 sec. In S-phase, these movements became constrained to a radius ≤ 0.3 µm due to a replication dependent mechanism (Heun et al., 2001). Notably, in this study on HeLa cells, no changes in the confinement of CTs during S- and G2-phase were observed.
To interpret higher order chromatin dynamics observed in human,
Drosophila and yeast cells, differences in size of their chromosomes (human: 50- 250 Mb; Drosophila: 4.3-70 Mb; yeast: 0.2 - 1.5 Mb) and genome structure have to be considered. For example, the fraction of transcriptionally active chromatin in
yeast amounts to ~90% compared to ~10% in humans. This may correspond to differences in chromatin compaction and rigidity / flexibility of higher order chromatin structures. A “free” yeast chromosome of 1 Mb may behave differently to a human ~1 Mb chromatin domain which is part of a much larger CT.
It was shown that motility of a measured target depends on its chromosomal localization (Heun et al., 2001) and its nuclear position (Chubb et al., 2002). The latter study reported lower chromatin mobility near the nucleoli and the nuclear periphery indicating a function of these structures in maintaining the 3D arrangements of chromatin. While the lamina-coated nuclear envelope is a proven site for chromatin attachments (see below), it is not clear whether the tethering of chromatin to a matrix of branched matrix core filaments plays a major role in constraining chromatin movements (Ma et al., 1999; Cremer et al., 2000). Alternatively, the stability of large-scale chromatin arrangements may also be maintained without the help of an internal nuclear matrix. Movements of a given CT could become constrained mainly by its neighboring CTs.
Major CT movements were observed neither in long term live cell observations after in vivo labeling / segregation in normal diploid fibroblast nor in SH-EP neuroblastoma cells (data not shown) or mouse myoblasts (A. Brero, personal communication) indicating that the large-scale stability of CT order seen in HeLa cell nuclei probably illustrates a general feature of mammalian interphase nuclei. Such stability does not exclude more extended, rapid and possibly directed movements of subchromosomal regions and genes. Initial experiments on the centromere distribution in neuroblastoma cell nuclei at different stages of cell cycle presented in this work also point to an increased mobility of centromeric chromatin. Consistently, this motion is mostly restricted to G1 when most centromeres assume a position at the nuclear periphery.
Functional implications of changes in higher order chromatin architecture are strongly supported by the finding that transcriptional activation / gene silencing is correlated with the repositioning of genes and gene clusters (for review see Baxter et al., 2002, see also Figure 1 and references in the corresponding legend).