Condensin II promotes the formation of chromosome territories by inducing axial compaction of polyploid interphase chromosomes

Abstract

The eukaryotic nucleus is both spatially and functionally partitioned. This organization contributes to the maintenance, expression, and transmission of genetic information. Though our ability to probe the physical structure of the genome within the nucleus has improved substantially in recent years, relatively little is known about the factors that regulate its organization or the mechanisms through which specific organizational states are achieved. Here, we show that Drosophila melanogaster Condensin II induces axial compaction of interphase chromosomes, globally disrupts interchromosomal interactions, and promotes the dispersal of peri-centric heterochromatin. These Condensin II activities compartmentalize the nucleus into discrete chromosome territories and indicate commonalities in the mechanisms that regulate the spatial structure of the genome during mitosis and interphase.

Figure 1. The formation of compact chromosome territories is Condensin II dependent.

3D FISH was performed on nurse cell nuclei to mark the X chromosome in green and the 2^nd chromosome in red. DAPI (DNA) is in blue. Representative nuclei from the following genotypes and developmental stages are depicted: A–C) wild type (y[1] w[67c23]) stages 4, 5, and 10; D–F) Cap-H2^Z3-0019/Df(3R)6159 stages 4, 5, and 10. All scale bars are equal to 5 µm. G) Mean distances between X chromosome and 2^nd chromosome centroids at stages 4, 6, 8, and 10. Error bars correspond to standard error. One asterisk signifies p<.05 and two asterisks signifies p<.01. Within each category, n = 15.

Figure 2. Condensin II inhibits the Rabl conformation.

A) For each X chromosome probe, the radial distance from the center of nurse cell nuclei was measured at stages 6, 8, and 10. For each nucleus, the nuclear radius was estimated based on the volume of DAPI staining and the assumption that each nucleus was a sphere. All radial distances are reported as a fraction of the estimated nuclear radius with 0 corresponding to the center of the nucleus and 1 corresponding to the nuclear periphery. Genotypes are indicated by the legend in the bottom panel. Error bars correspond to standard error. B–E) Images of representative nuclei from wild type and Cap-H2^Z3-0019/Df(3R)6159 mutant nurse cells. Probes label the pericentric heterochromatin of the X (green), 2^nd (red), and 3^rd (white) chromosomes.

Figure 3. Condensin II promotes the dispersal of heterologous centromeres.

The mean distances between the peri-centric microsatellite sequences of the X, 2R, 2L, 3R and 4 chromosome arms at various stages of nurse cell development are shown. The labels below each set of four bars indicate which pair of loci are being compared. The genotypes assayed were Cap-H2^Z3-0019/Df(3R)Exel6159, y[1] w[67c23]; P(w[+mC] = lacW)glu[k08819]/+; Cap-H2^Z3-0019/+, Cap-D3^EY00456/Df(2R)Exel7023, and wild type (y[1] w[67c23]). Error bars correspond to standard errors. One asterisk signifies p<.05, two asterisks signifies p<.01, and three asterisks signifies p<.001. The cartoon in the lower right panel illustrates the distance measurements being made between different heterochromatin FISH signals.

Figure 4. Condensin II promotes chromosome axial compaction.

For developmental stage 6, 8, and 10, distances were measured between pairs of loci on the X chromosome. The X-axis indicates the distance between the loci in megabases and the Y-axis indicates their mean spatial separation in microns. The horizontal line indicates the average nuclear radius which is also a good approximation for the mean distance between two random points within a sphere. The following genotypes were assayed: Cap-H2^Z3-0019/Df(3R)Exel6159, Cap-D3^EY00456/Df(2R)Exel7023, y[1] w[67c23]; P(w[+mC] = lacW)glu[k08819]/+; Cap-H2^Z3-0019/+, wild type (y[1] w[67c23]). Bars represent standard errors. One asterisk signifies p<.05, two asterisks signifies p<.01, and three asterisks signifies p<.001. The cartoon in the lower right panel illustrates the axial length measurements being made between different loci.

Figure 5. Expression of Cap-H2 in salivary glands induces axial shortening of chromosomes.

A) DAPI stained, unspread salivary gland chromosome squash from wild type larva raised at 25°C. B) DAPI stained, unspread salivary gland squash from hsp70>Gal4/UAS>Cap-H2 raised at 25°C. C) Projection of salivary gland nucleus from Hs83>LacI-GFP/+; 50F-LacO/60F-LacO with 1 hr, 37°C heat shock. DAPI is shown in red and GFP signal is shown in green. D) Projection of salivary gland nucleus from Hs83>LacI-GFP/+; 50F-LacO/60F-LacO; hsp70>Gal4/UAS>Cap-H2 with 1 hr, 37°C heat shock. E) A unique example (same treatment as in D) in which the nuclear envelope has ruptured revealing details of chromosome structure. F) Projection of salivary gland nucleus from Hs83>LacI-GFP/+; 50F-LacO/60F-LacO; hsp70>GAL4/UAS>Cap-H2 with 1 hr, 37°C heat shock. Both LacO arrays remain confined within a common territory during the dispersal process. G) A projection of the same nucleus in F through the Y-axis showing the separation between the two LacO arrays. H) Measurements of the distance between LacO insertions in cases similar to C and D. All scale bars correspond to 10 µm.

Figure 6. Model for Condensin II–mediated chromosome territory formation.

A) Condensed mitotic chromosomes are pulled to opposite poles and centromeres cluster at the poles in telophase. Four chromosomes are shown where the ovals represent centromeres and/or peri-centric heterochromatin. The small fourth chromosomes are shown in black. B) In interphase, decondensed chromosomes adopt the Rabl configuration with clustered centromeres simply because of their previous organization in telophase. B') Long and relaxed interphase homologous chromosomes are paired. C) Activation of Condensin II in interphase nuclei has two direct consequences: First it induces axial compaction of the chromosome arms. Second, in order to accommodate the shorting distance between telomeres and centromeres, heterochromatin slides along the nuclear periphery as the axial length of the arms decrease (see red arrows in B). Because heterochromatin proteins interact with the nuclear periphery, we speculate that as euchromatic regions of chromosome arms shorten they are drawn to the nuclear periphery. Heterochromatin and other chromatin interactions with the nuclear periphery combined with the Condensin mediated shortening of chromosome arms forces chromosomes into discrete territories. C') Chromosome arm compaction is most likely accompanied by chromatin folding and coiling that occludes homologous sequences from interacting in trans. D) The model in A–C depicts a diploid cells scenario, but the same mechanical principles can apply to a polyploid nucleus that forms CTs. In the panel on the left, two polytene chromosomes (the X chromosome in red and an autosome in white) are shown where centromeres (cen) are clustered at the North pole and telomeres (tel) are clustered at the South pole of the nucleus. As Condensin II activity increases (left to right) the chromatin fibers within polytenes begin to unpair as the chromosome arms shorten. Note that although euchromatic sequences unpair, homologous heterochromatin blocks remain paired and move in concert. By contrast, heterologous heterochromatin blocks disperse to different parts of the nuclear periphery. As above (B–C), Condensin II mediated compaction drives globular CT formation at the nuclear periphery as a consequence of chromatin interactions with nuclear envelope proteins.