In this paper we have examined and compared the subnuclear localization of human chromosomes 18 and 19. In different primary and transformed human cells, and in both flattened specimens and in cells fixed to preserve 3D structure, we observed distinct dispositions toward either the interior or periphery of the nucleus for both the p and q arms of chromosomes 19 and 18, respectively (Figs. , , and and Table ).
Specific parts of chromosomes may adopt different orientations during the cell cycle (Ferguson and Ward, 1992
; Vourc'h et al., 1993
; Brown et al., 1997
; Csink and Henikoff, 1998
; Li et al., 1998
), but it was not known whether entire mammalian chromosomes move or change their state of condensation. Measurements of chromatin movements in vivo suggest that significant passive diffusion of chromatin could occur during the course of interphase but predict that whole chromosomes are largely constrained within a limited subregion of the nucleus (Marshall et al., 1997
). Therefore, specific subnuclear localization of DNA segments might be established most effectively as cells exit mitosis and before interphase nuclear architecture is fully formed. The distinctive arrangements of HSA18 and 19, that we have described here, are established early in the cell cycle and are maintained throughout interphase (Fig. ). However, the difference in spatial localization between HSA18 and 19 is at its smallest during late S phase (Table ). This may reflect some movement of HSA18 to a more internal location accompanying the replication of its DNA (Li et al., 1998
In addition to the differences between HSA18 and 19 in subnuclear localization, we have also demonstrated differences in the proportion of nuclear area that these two human chromosomes occupy in 2D preparations. The larger proportion of nuclear area occupied by HSA19, as compared with that of HSA18, is in contrast to the larger physical size (in bp) of the latter chromosome (Morton, 1991
) and its greater size in metaphase preparations (Table ). Since much of HSA18 has the characteristics typical of G-band chromosome regions, whereas HSA19 is more R-band–like in its properties (Dutrillaux et al., 1976
; Korenberg and Rykowski, 1988
; Jeppesen and Turner, 1993
; Craig and Bickmore, 1994
), our data are consistent with the regional differences in chromatin compaction at the 0.1–1.5-Mb level that have been recorded between G- and R-band regions of the human genome in nuclei prepared in similar ways to those described here (Yokota et al., 1997
). The proportion of nuclear area occupied by HSA19 reached a peak in early S phase. This might reflect HSA19 chromatin decondensation preceding its replication (Csink and Henikoff, 1998
) or merely the doubling of DNA content for this gene-rich chromosome before most of the rest of the genome.
The apparently larger area of HSA19 within flattened nuclei is also seen in 3D-preserved nuclei (Fig. b). While sophisticated computational algorithms are necessary to accurately compute chromosome volumes within the nuclear space (Eils et al., 1996
; Visser et al., 1998
), our simplified approach of adding together the chromosome signal area and nuclear area in series of optical sections taken through nuclei suggests that chromosome 19 may occupy a larger volume within the human nucleus than chromosomes 18 and hence be less condensed. Hybridization signals seen with paints for HSA19 also appeared to us to have a more irregular and scattered character than those from HSA18 (e.g., Fig. ). The chromosome territory of the active X (Xa) is similarly more rutted in appearance than that of Xi (Eils et al., 1996
); however, the volumes calculated for the territories occupied by Xa and Xi in optical sections are, in fact, the same despite the more compact appearance of Xi in flattened preparations (Eils et al., 1996
). It remains to be determined whether the actual nuclear volumes occupied by HSA18 and 19 are different from each other.
We have also shown here that transcription affects the topology of chromosome territories since the larger apparent size of HSA19 is only seen in the presence of transcription by RNA polymerase II (Fig. ), i.e., in untreated cells or in cells in which the inhibition of transcription by DRB has been relieved. In the absence of transcription (AMD or DRB treatments) HSA19 occupies a compact territory similar in size, or smaller, than that of HSA18 (Table and Fig. ). We do not see the gross disruption of chromosome territories in the presence of DRB that was reported previously (Haaf and Ward, 1996
), except in a small minority of both treated and untreated cells that we believe are dying cells. We have no explanation for this discrepancy, but different cell types may have different tolerances and responses to the same concentrations of drugs.
The enhanced differences in areas of HSA19 and 18 we recorded when histone deacetylation was inhibited with TSA (Table and Fig. a) suggest that levels of steady-state histone acetylation influence the gross architecture of chromosome territories. However, chromosome position within the nucleus is independent of transcription and histone acetylation activities (Table ).
More genomic sequences partitioning with the operationally defined nuclear matrix (MARs) or nuclear scaffold (SARs) derive from HSA18 than from 19 (Craig et al., 1997
). This, together with the proximity of HSA18 to the lamina (Fig. a), a component of the nuclear matrix, lead us to expect that there might be a tight association of this chromosome with the matrix. However, in Drosophila
, sequences isolated as SARs do not correspond with loci at the nuclear periphery (Marshall et al., 1996
) and so the relationship between MARs-SARs and sequences that visibly remain inside of the residual nucleus after extraction, rather than in the surrounding halo of DNA loops, is not clear. Indeed, we saw very little retention of chromosome 18 DNA within nuclear matrices in contrast to the retention of chromosome 19 within the bounds of residual nuclei (Fig. ). The degree of extension of chromosome 18 sequences varied between nuclei, but within individual nuclei the two homologues behaved similarly (Fig. ). It has been reported that ~16 kb of inactive DNA can decondense to cover ~5 μm in extracted nuclei (Gerdes et al., 1994
); therefore, the 85 Mb of chromosomes 18 extruded from nuclei with high salt retain a substantial degree of higher order structure that is independent of interactions with the nucleus.
RNA is an important component of the nuclear matrix, and active genes associate with residual nuclei and not with the nuclear halo (Gerdes et al., 1994
). However, retention of HSA19 within the residual nucleus and release of HSA18 was seen in the absence of transcription (AMD treatment). The more central location of chromosome 19 in the human nucleus may be mediated by substantive and transcription-independent association with, as yet unidentified, nuclear proteins that resist extraction from the nucleus with high salt. Jackson and Pombo (1998)
have demonstrated that early replicating DNA is retained within the residual nucleus of salt-extracted human cells and, indeed, the bulk of HSA19 replicates earlier in S phase than does HSA18 (Dutrillaux et al., 1976
Subnuclear localization of HSA18 and 19 is not determined by the centromeres of the chromosomes since distinctive localization is retained by regions (<20 Mb) of the chromosome arms of HSA18 and 19 that are translocated to the reciprocal-derived chromosome (Table and Fig. , g–j). We also find no evidence that the telomeres of the chromosomes are attached at the nuclear periphery of human nuclei (Fig. , e and f) as has been observed in simpler eukaryotes (Hiraoka et al., 1990
; Funabiki et al., 1993
; Gotta et al., 1996
). We conclude that differences in the overall composition of bulk chromosome 18 and 19 DNA sequences may play a direct role in the nuclear destiny of these two chromosomes and the genes placed upon them.
It has been argued that lack of phenotypic abnormalities in individuals with balanced translocations is evidence that spatial arrangement of different chromosomes in the nucleus is not functionally important. This was based on the assumption that such translocations disrupt the normal nuclear location of chromosome domains (Haaf and Schmid, 1991
; Qumsiyeh, 1995
). However, we have shown that this is not necessarily the case to any significant degree (Table and Fig. , g–j). The diffusion constraints on chromatin movement in vivo (Marshall et al., 1997
) mean that the physical proximity of different chromosomes in interphase, and their interactions with nuclear substructure, may be important in determining the likelihood of any two chromosomes meeting and exchanging material in a translocation (Qumsiyeh, 1995
). The most recurrent chromosome rearrangements in humans are Robertsonian translocations between the acrocentric rDNA–carrying chromosomes that are known to be physically close to one another in both interphase and metaphase (Kaplan et al., 1993
). We surveyed two large databases cataloguing balanced translocations in humans (http://www.hgmp.mrc
) and found that t(18;19) is indeed very rare in the human population when compared with other translocations among small chromosomes.
The segregation of different chromosomes of the karyotype with different functional characteristics, described here, is reminiscent of the extreme genome separation seen in plant hybrids (Leitch et al., 1991
). What are the biological consequences of this type of compartmentalization? The chromosomal and nuclear position of a gene can influence its activity (Brown et al., 1997
; Andrulis et al., 1998
) and the position of a gene within the nucleus can be dictated by the sequences it is joined to on the chromosome (Csink and Henikoff, 1996
; Dernburg et al., 1996
). The edge of the nucleus is also a place where genes are repressed in many eukaryotes (Bridger and Bickmore, 1998
). Condensed heterochromatin and later replicating DNA tend to concentrate toward the nuclear periphery in many vertebrates (Rae and Franke, 1972
; Fox et al., 1991
; Kill et al., 1991
; Ferreira et al., 1997
), whereas early replicating DNA and poly(A) RNA partition toward the nuclear interior (Carter et al., 1993
). At the level of individual loci some, but not all, active mammalian genes have been found predominantly within the nuclear interior, whereas inactive genes have been located at the nuclear and nucleolar peripheries (Xing et al., 1995
). The number of gene-based markers that has been assigned to HSA18 is small in comparison to those located on HSA19 (Craig and Bickmore, 1994
; Deloukas et al., 1998
). Because of their location within the nuclear space, the relatively small number of genes located on human chromosome 18 might be habituated to very different types of transcriptional regulation to those on HSA19. Flexibility of expression of exogenous genes placed into these two different chromosome environments may also differ. This might impose constraints on the chromosomal position of genes through evolution.