Condensin II is enriched at centromeres from late G2 through early G1 phase
To further characterise the function of condensin II, its subnuclear localisation was investigated using GFP fused to CAP-G2, a subunit specific to condensin II in wild-type Arabidopsis nuclei. Because condensin II is strongly expressed in root tips16, we studied cells in the meristematic, elongating, and differentiated zones of the root. In all regions, GFP-tagged condensin II signals were detected in both the nucleoplasm and nucleolus (Fig. 1a). A similar subnuclear localisation was observed in tobacco BY-2 cells expressing Arabidopsis CAP-H2-GFP17. Moreover, we found that some nuclei showed a spot-like localisation of condensin II only in the meristematic zone, in addition to its basic localisation throughout the nucleus (Fig. 1a and Supplementary Fig. S1). Given that the cells in the meristematic zone proliferate, it is possible that the formation of a spot-like localisation of condensin II is cell cycle-dependent. Indeed, using time-lapse imaging analysis of GFP-tagged condensin II during the mitotic cell cycle, the spot-like localisation of condensin II was observed 80 min prior to metaphase and disappeared around 40 min after metaphase, corresponding to late G2 and early G1 phase, respectively (Fig. 1b).
In the primitive red alga Cyanidioschyzon merolae and in the nematode Caenorhabditis elegans, condensin II is specifically localised to centromeres from prophase through anaphase18,19. Thus, we hypothesised that the condensin II spots in A. thaliana nuclei colocalize with centromeres. Ideally, mitotic cells display 10 discrete centromere signals, which can be visualised by fluorescent fusion protein tdTomato-CENH3 (centromeric histone H3). As expected, our observations revealed that each CAP-G2-GFP signal colocalized with a tdTomato-CENH3 signal (Fig. 1c). Collectively, these results suggest that condensin II accumulates at centromeres during this specific cell cycle phase.
Condensin II is required for scattering centromeres in telophase
Fluorescent in situ hybridization (FISH) analysis has shown that condensin II is indispensable for the dissociation of centromeres in interphase nuclei9,10. To determine how condensin II participates in the dynamic regulation of centromeres during the cell cycle, we conducted live imaging of tdTomato-CENH3 in the cap-h2-216 null mutant. Contrary to the scattered centromere distribution in the wild type, the cap-h2-2 mutant showed a biased centromere distribution, in which almost all centromeres were clustered at one side of the nucleus. This occurred not only in meristematic cells, but also in differentiated cells of the root (Fig. 2a). Then, we quantitatively evaluated the degree of bias in centromere distribution. Given that cells above the stem cell niche divide longitudinally into the root apical meristem, we determined an equatorial plane that produces two hemispheres in the nucleus using the centre points of two nuclei neighbouring the target nucleus in the same cell file. We calculated the degree of bias in centromere distribution by counting the number of centromeres in each hemisphere (Fig. 2b, left panel). The cap-h2-2 mutant showed an extremely biased centromere distribution compared to the wild type (Fig. 2b, right panel).
To determine the period during which centromere positioning mediated by condensin II was established, we monitored centromere dynamics from anaphase to early G1 phase (Fig. 2c). In the wild type, all centromere pairs were aligned along the metaphase plate. Then, sister centromeres moved to opposite poles in anaphase, subsequently scattering in telophase (Fig. 2c)7,20. Our imaging analysis revealed that during the transition from anaphase to telophase, the centromeres remained at opposite poles in the cap-h2-2 mutant (2.5 to 7.5 min in Figure 2c). This position persisted after entry into G1 phase (25 and 45 min in Fig. 2c). Given that condensin II was localised to centromeres throughout mitosis (Fig. 1), condensin II function at centromeres during the transition from anaphase to telophase seems indispensable for centromere scattering.
Nuclear envelope-related factors are involved in scattering centromeres in cooperation with condensin II
Nuclear envelope (NE) reassembly occurs in plants during the transition from anaphase to telophase during mitosis20,21. In Arabidopsis, at late anaphase, the NE proteins SUN1 and SUN2 are reassembled at the distal surfaces of the chromosomes, where centromeres are found. These proteins enclose the chromosomes from the distal surface to the proximal surface at telophase20,20. Similar dynamics have been observed for plant nuclear lamina proteins, namely nuclear matrix constituent proteins (NMCPs), in Apium graveolens (celery)21. Because the timing between the reassembling of NE-related factors and centromere scattering is approximately the same, we hypothesised that NE-related factors are involved in centromere scattering.
The SUNs (SUN1-SUN5) are inner nuclear membrane proteins comprising the LINC complex in association with other proteins22. SUNs have the potential to interact with chromatin in the nucleoplasm, along with their interactions with Klarsicht/ANC-1/Syne homology (KASH)-domain proteins, such as WPP domain-interacting proteins (WIPs) and SUN-interacting NE proteins (SINEs) in the outer nuclear membrane. Through interactions between KASH or KASH-associated proteins, such as WPP domain-interacting tail-anchored proteins (WITs) and myosin XI-I, with actin filaments, the SUNs are also connected to the cytoskeleton22. The nuclear lamina comprising Arabidopsis NMCPs, known as CRWN proteins, is located underneath the NE (Fig. 3a)22,23. To evaluate the involvement of NE-related factors in centromere scattering, we analysed centromere distribution in mutants affecting the LINC complex and CRWN proteins (Fig. 3b–e). Mutants affecting KASH (wifi and sine1-1) and SUN proteins (sun1-KOsun2-KD and sun4sun5) showed a significantly more biased centromere distribution than the wild type, although the bias was not as pronounced as previously observed in the condensin II mutant. In contrast, centromere distribution in the crwn1crwn4 and myosin xi-i mutants was comparable to that in the wild type (Fig. 3b,c). Additionally, we found that the inhibition of actin polymerisation by latrunculin B treatment caused a biased centromere distribution, while treatment with oryzalin, resulting in microtubule depolymerisation, did not affect it (Fig. 3d,e). These results suggest that, in addition to condensin II, the LINC complex associated with actin filaments is involved in determining centromere distribution during mitosis.
Next, we investigated whether the LINC complex associates with the centromere and/or condensin II to regulate centromere distribution. Among LINC complex components, only the SUNs have the potential to interact with chromatin as a part of the SUN domain in the nucleoplasm22. Therefore, we evaluated the interaction of SUN proteins with CENH3 and condensin II by co-immunoprecipitation (co-IP). Our co-IP analyses after transient expression of the proteins in tobacco leaves indicated interactions of SUN1 and SUN2 with CENH3 (Fig. 3f). Similarly, we found an interaction between SUN2 and the condensin II component CAP-G2 using co-IP assays (Fig. 3g). The interactions between the SUNs and condensin II components were further supported by yeast two-hybrid assays (Fig. 3h) and bimolecular fluorescence complementation (Bi-FC) assays in cultured tobacco BY-2 cells (Supplementary Fig. 2). Collectively, these results suggest that the LINC complex is essential for correct centromere distribution in interphase through interactions between SUNs, centromeres, and condensin II, most likely during the transition from anaphase to telophase. The corresponding mutants did not show a marked alteration in centromere distribution, as was observed in cap-h2-2, suggesting functional redundancy among SUN and KASH proteins. Hereafter, we termed the interaction of condensin II with the LINC complex involved in the regulation of centromere positioning, CII-LINC.
CII-LINC and CRWNs independently regulate centromere distribution at different stages of mitosis
As mentioned earlier, no bias in centromere distribution was observed in crwn1crwn4 mutants by imaging (Fig. 3b, c). However, when the number of distinguishable centromeres in interphase nuclei was counted, the crwn1crwn4 nuclei had a significantly lower number of centromeres than the wild type, similar to the cap-h2-2 condensin II mutant (Supplementary Fig. S3), consistent with a previous study using FISH9,11. These findings imply that CRWNs contribute to centromere positioning in a manner different from condensin II. To date, CRWNs have been shown to function in tethering chromatin, including pericentromeric and centromeric heterochromatin, to the nuclear periphery15. Consistent with this, we found CRWN1 to interact with CENH3 in co-IP assays in tobacco leaves and an enrichment of CRWN1-GFP at tdTomato-CENH3 positive foci in interphase nuclei (Fig. 4a,b). These findings support that CRWNs directly regulate centromere positioning.
To obtain further insights into the function of CRWNs in centromere positioning, we analysed centromere movement in interphase nuclei. Live imaging demonstrated that the centromeres in crwn1crwn4 mutant nuclei are highly dynamic, but static in the wild type and mutants affecting CII-LINC, i.e., cap-h2-2 and sun1-KOsun2-KD, which show a biased centromere distribution (Fig. 4c,d). This result indicates that CRWNs restrain the positions of centromeres during interphase at the nuclear periphery. Next, to evaluate the genetic interaction between CII-LINC and CRWNs in the regulation of centromere positioning, we observed centromere dynamics in the triple cap-h2-2crwn1crwn4 mutant. Intriguingly, we found both types of centromere distribution, biased and scattered, in this triple mutant (Fig. 5a). Subsequently, we monitored centromeres dynamics in the triple mutant (Fig. 5b). First, the triple mutant clearly showed a biased centromere distribution, as observed in cap-h2-2 (Fig. 2c, from 0 to 5 min in Fig. 5b). Over time, however, the centromeres started moving around and, consequently, adopted a distribution similar to that observed in the wild type (from 10 to 45 min in Fig. 5b), contrasting the biased distribution in cap-h2-2 (Fig. 2c and Fig. 4c). Even after centromere scattering, centromere movement in the triple mutant was more dynamic than that in wild type or cap-h2-2 nuclei (Fig 4c,d). These observations suggest that the NE-stabilisation of centromeres by CRWNs occurs continuously during the interphase, and that CII-LINC and CRWNs function independently of each other to regulate centromere positioning.
Considering this, we propose that centromere distribution is determined by a two-step process after the centromere pairs segregate at early anaphase: (i) interactions of CII-LINC with centromeres occur at late anaphase, mediating the scattering of centromeres from late anaphase to telophase, and (ii) CRWNs stabilise the position of scattered centromeres after the entry into interphase (Fig. 5c).
Condensin II and CRWNs make both similar and distinct contributions to chromatin organization
Considering that both CII-LINC and CRWNs have a major impact on centromere distribution, we investigated whether deficiencies in these factors alter higher-order chromatin organization. We performed duplicate Hi-C analysis in Col-0, cap-h2-2, crwn1crwn4, and cap-h2-2crwn1crwn4 plants (Supplementary Table 1). A high correlation between the duplicates was obtained (Supplementary Fig. S4a,b), and the combined data from duplicate experiments were used for subsequent analyses to improve resolution. To compare contact frequencies, we calculated the relative differences between all elements of the two Hi-C matrices of interest, as described previously24 (Fig. 6a). By visual inspection, we found that cap-h2-2 exhibited increased inter-chromosomal pericentromere contacts and increased contacts between the two centromere-flanking halves of the pericentromeres. Additionally, we observed elevated cis-chromosomal inter-arm and trans-chromosomal inter-arm contacts. The cap-h2-2 mutant also showed a conspicuous decrease in pericentromere-arm contacts (both cis- and trans-chromosomal), and a slight decrease in intra-arm contacts and interactions within centromeres (Supplementary Fig. S5). Similar alterations in chromatin contacts were observed in the crwn1crwn4 double and cap-h2-2crwn1crwn4 triple mutants; however, their overall magnitude was greater than that observed in caph2-2. These results suggest that the defects in both condensin II and CRWN mutants affect chromatin organization in a similar way. Additionally, a comparison of crwn1crwn4 and cap-h2-2crwn1crwn4 showed only slightly more enhanced differences in some interacting regions, including inter-chromosomal pericentromere interactions, cis-chromosomal pericentromere-arm interactions, and interactions within each centromere, which are most likely caused by the additional mutation in CAP-H2. These findings suggest that condensin II and CRWNs independently contribute to the organization of chromatin interactions, at least in specific regions.
Next, we analysed the formation of discrete structural domains by calculating the correlation coefficients of the distance-normalised interaction matrix24 (Fig. 6b). The correlation matrix derived from a Hi-C map is closely related to how strong interactions/depletions are among chromatin regions and, thus, helps to highlight the structural separation between different chromosomal regions. Therefore, a weakened correlation matrix indicates a lower degree of spatial separation of different chromatin compartments15. The cap-h2-2 Hi-C map showed a subtly weaker correlation matrix than the wild type, indicating a slightly less well-defined chromatin compartmentalisation. A more strongly weakened chromatin compartmentalisation was observed in the crwn1crwn4 Hi-C map, as previously reported for the individual mutants, crwn1 and crwn415. In addition, the cap-h2-2crwn1crwn4 Hi-C map showed the weakest compartmentalisation.
Next, we performed a principal component analysis on the correlation matrix of each chromosome, including centromeric regions (Fig. 6c). In this analysis, regions exhibiting negative eigenvalues corresponded to B compartments, which, in Arabidopsis, mainly cover the constitutive heterochromatin of pericentromeres. We found that the eigenvalue distributions along each of the chromosome arms were similar among the plants analysed. However, both crwn1crwn4 and cap-h2-2crwn1crwn4 exhibited a similarly altered eigenvalue distribution pattern around the pericentromeric regions of all chromosomes, with negative eigenvalues expanding into more distal regions of the pericentromeres. Consequently, the eigenvalue boundaries between pericentromeres and chromosome arms became blurred (Fig. 6c). To further evaluate chromatin structure, we determined the interaction decay exponents (IDEs) of entire chromosomes, pericentromeres, and chromosome arms (Fig. 6d). The IDE is indicative of the chromatin structure model, i.e., whether a region behaves according to the fractal- or the equilibrium-globule model25. Therefore, no differences in the IDEs of the entire chromosomes and chromosome arms were observed between the mutants and the wild type. However, significantly higher pericentromeric IDEs were observed in crwn1crwn4 and cap-h2-2crwn1crwn4 mutants, with IDEs close to those of chromosome arms (Fig. 6d). This suggests a euchromatinisation of parts of the pericentromeres in these mutants. It is worth noting that the eigenvalue distribution and the IDEs of pericentromeres were comparable between cap-h2-2 and the wild type (Fig. 6c,d). Collectively, these results suggest that CRWNs are involved in the organization of chromatin structure, especially at pericentromeres, establishing a defined compartmentalisation of heterochromatin. In contrast, the contribution of condensin II to chromatin structure was not evident, despite its involvement in chromatin interactions at pericentromeres.
Previous studies reported that the transcriptome is not significantly affected in a crwn1-single mutant, which shows weakened chromatin compartmentalisation and higher IDEs at pericentromeres15,24, similar to the crwn1crwn4 double mutant used in this study. Our transcriptome analysis of the crwn1crwn4 double mutant revealed only 83 differentially expressed genes (DEGs). Additionally, the extent of the difference was less than 2-fold in 60% of the DEGs (Supplementary Fig. S6). Similarly, the cap-2h-2 mutation had little effect on the transcriptome: there were only 164 DEGs, 129 of which showed less than a 2-fold difference (Supplementary Fig. S6). These results suggest that, in addition to alterations in chromatin organization, abnormal centromere distribution does not strongly influence local gene regulation in mutants affecting CRWNs or condensin II.
Centromere distribution is involved in the maintenance of genome integrity
After evaluating the biological significance of CII-LINC- and CRWN-mediated centromere distribution, we focused on investigating its potential role in maintaining genome integrity. Condensin II ameliorates DNA damage caused by abiotic stress and genotoxic chemical treatments16. CRWNs, including CRWN1 and CRWN4, are involved in protecting genomic DNA against oxidative stress caused by methyl methanesulfonate26,27. Accordingly, we evaluated the levels of DNA double-strand breaks (DSBs) in the root tips of mutants showing an abnormal centromere distribution under normal conditions using comet assays (Fig. 7a). We confirmed that cap-h2-2, crwn1crwn4, and sun1sun2 (a double knockout mutant whose background is Col-0/Ws heterozygote) mutants had increased DSBs compared to the wild type. Additionally, primary root growth was severely delayed in the mutants after 2 and 4 days of treatment with the DSB-inducing reagent zeocin (Fig. 7b). Moreover, sun1sun2 exhibited a highly deformed root tip, and all mutants showed a reduction in meristem size after 2 days of zeocin treatment (Fig. 7c,d). These results confirm that genome integrity is impaired in mutants with abnormal centromere distribution. Additionally, we found that the cap-h2-2crwn1crwn4 mutant displayed an additive phenotype compared to cap-h2-2 and crwn1crwn4 with respect to DSBs and hypersensitivity to zeocin in terms of primary root growth and root morphology (Fig. 7a,b,c), suggesting that condensin II and CRWNs contribute to the maintenance of genome integrity via different mechanisms.
To assess whether the hypersensitivity of the mutants to DNA damage could be attributed to impaired gene regulation in response to the induction of DNA damage, we performed transcriptome analysis at 0, 1, and 3 h after zeocin treatment (Supplementary Fig. S7). We confirmed that 1h and 3h treatments with zeocin markedly altered the transcriptome in the wild type (Supplementary Fig. S7a). Under these conditions, we compared the transcriptomes of the wild type, cap-h2-2, and crwn1crwn4 and found that all three transcriptomes were similar (Supplementary Fig. S7b). These results suggest that the regulation of gene expression upon DNA damage is normal in both the cap-h2-2 and crwn1crwn4 mutants.