InteractiveFly: GeneBrief

Chromosome associated protein H2: Biological Overview | References

BIOLOGICAL OVERVIEW

Several meiotic processes ensure faithful chromosome segregation to create haploid gametes. Errors to any one of these processes can lead to zygotic aneuploidy with the potential for developmental abnormalities. During prophase I of Drosophila male meiosis, each bivalent condenses and becomes sequestered into discrete chromosome territories. This study demonstrates that two predicted condensin II subunits, Cap-H2 and Cap-D3, are required to promote territory formation. In mutants of either subunit, territory formation fails and chromatin is dispersed throughout the nucleus. Anaphase I is also abnormal in Cap-H2 mutants as chromatin bridges are found between segregating heterologous and homologous chromosomes. Aneuploid sperm may be generated from these defects; they occur at an elevated frequency and are genotypically consistent with anaphase I segregation defects. It is proposed that condensin II-mediated prophase I territory formation prevents and/or resolves heterologous chromosomal associations to alleviate their potential interference in anaphase I segregation. Furthermore, condensin II-catalyzed prophase I chromosome condensation may be necessary to resolve associations between paired homologous chromosomes of each bivalent. These persistent chromosome associations likely consist of DNA entanglements, but may be more specific as anaphase I bridging was rescued by mutations in the homolog conjunction factor teflon. It is proposes that the consequence of condensin II mutations is a failure to resolve heterologous and homologous associations mediated by entangled DNA and/or homolog conjunction factors. Furthermore, persistence of homologous and heterologous interchromosomal associations lead to anaphase I chromatin bridging and the generation of aneuploid gametes (Hartl, 2008a).

Several meiotic processes ensure faithful chromosome segregation to create haploid gametes. Errors to any one of these processes can lead to zygotic aneuploidy with the potential for developmental abnormalities. During prophase I of Drosophila male meiosis, each bivalent condenses and becomes sequestered into discrete chromosome territories. This study demonstrates that two predicted condensin II subunits, Cap-H2 and Cap-D3, are required to promote territory formation. In mutants of either subunit, territory formation fails and chromatin is dispersed throughout the nucleus. Anaphase I is also abnormal in Cap-H2 mutants as chromatin bridges are found between segregating heterologous and homologous chromosomes. Aneuploid sperm may be generated from these defects as they occur at an elevated frequency and are genotypically consistent with anaphase I segregation defects. It is proposed that condensin II-mediated prophase I territory formation prevents and/or resolves heterologous chromosomal associations to alleviate their potential interference in anaphase I segregation. Furthermore, condensin II-catalyzed prophase I chromosome condensation may be necessary to resolve associations between paired homologous chromosomes of each bivalent. These persistent chromosome associations likely consist of DNA entanglements, but may be more specific as anaphase I bridging was rescued by mutations in the homolog conjunction factor teflon. It is proposed that the consequence of condensin II mutations is a failure to resolve heterologous and homologous associations mediated by entangled DNA and/or homolog conjunction factors. Furthermore, persistence of homologous and heterologous interchromosomal associations lead to anaphase I chromatin bridging and the generation of aneuploid gametes (Hartl, 2008a).

Some of the processes that ensure proper chromosome segregation take place upon the chromosomes themselves. The chromosomes of Drosophila males undergo an interesting and relatively enigmatic step before entering meiosis, where each paired homologous chromosome becomes clustered into a discrete region of the nucleus. This study provides evidence that improper chromosomal associations are resolved and/or prevented during this 'chromosome territory' formation. This was uncovered through the study of flies mutant for Cap-H2, which have abnormal territory formation and improper chromosomal associations that persist into segregation. Another important process that chromosomes undergo in meiosis is the pairing and physical linking of maternal and paternal homologs to one another. Linkages between homologs are essential to ensure their proper segregation to daughter cells. In contrast to meiosis in most organisms, linkages between homologs in male Drosophila are not recombination mediated. This study provides evidence that Cap-H2 may function to remove Drosophila male specific linkages between homologous chromosomes prior to anaphase I segregation. When chromosomal associations persist during segregation of Cap-H2 mutants, the chromosomes do not detach from one another and chromatin is bridged between daughter nuclei. The likely outcome of this defect is the production of aneuploid sperm (Hartl, 2008a).

There are several critical steps that chromosomes must undergo as they transition from their diffuse interphase state to mobile units that can be faithfully transmitted to daughter cells. In the germline, faulty segregation leading to the creation of aneuploid gametes is likely a leading cause of genetic disease, miscarriages, and infertility in humans (Hartl, 2008a).

Some steps that promote proper segregation are universal to all cell types undergoing cell division. Chromosomal 'individualization' is necessary to remove DNA entanglements that likely become introduced naturally through movements of the threadlike interphase chromatin. Topoisomerase II (top2) contributes to individualization with its ability to pass chromosomes through one another by creating and resealing double strand breaks. The necessity of top2's 'decatenation' activity to chromosome individualization becomes clear from fission yeast top2 mutants and vertebrate cells treated with a top2 inhibitor, where mitotic chromosomes appear associated through DNA threads. Another step that occurs prior to chromosome segregation is chromosome 'condensation,' entailing the longitudinal shortening from the threadlike interphase state into the rod like mitotic chromosome. Condensation is necessary due to the great linear length of interphase chromosomes that would be impossible to completely transmit to daughter cells (Hartl, 2008a).

Because chromosome individualization and condensation appear to occur concurrently, it has been inferred that both are promoted by the same catalytic activity. In support of this idea, the condensin complexes have been implicated in chromosome individualization and condensation, suggesting a molecular coupling of both processes. The condensin I and II complexes are thought to be conserved throughout metazoa, each utilizing Structural Maintenance of Chromosome ATPases SMC2 and SMC4, but carrying different non-SMC subunits Cap-H, Cap-G, Cap-D2 or Cap-H2, Cap-G2, and Cap-D3, respectively (Hirano, 2005; Yeong, 2003). In vitro, condensin I is known to induce and trap positive supercoils into a circular DNA template. Current models to explain condensin I chromosome condensation highlight this activity as supercoiling may promote chromatin gathering into domains that can then be assembled into a higher order structure (Hirano, 2006). Condensin complexes may also promote condensation and individualization through cooperating with other factors, such as chromatin-modifying enzymes. While the effect of condensin mutations or RNAi knockdown on chromosome condensation is variable depending on cell type and organism being studied, in most if not all cases, chromatin bridges are created between chromosomes as they segregate from one another. This likely represents a general role of the condensin complex in the resolution of chromosomal associations prior to segregation (Hartl, 2008a).

While the second cell division of meiosis is conceptually similar to mitotic divisions where sister chromatids segregate from one another, the faithful segregation of homologous chromosomes in meiosis I requires several unique steps. It is essential for homologous chromosomes to become linked to one another for proper anaphase I segregation and most often this occurs through crossing over to form chiasmata. As recombination requires the close juxtaposition of homologous sequences, homologs must first 'identify' one another in the nucleus and then gradually become 'aligned' in a manner that is DNA homology dependent, but not necessarily dictated by the DNA molecule itself. Eventually, the homologous chromosomes become 'paired,' which is defined as the point when intimate and stable associations are established. The paired state is often accompanied by the laying down of a proteinaceous structure called the synaptonemal complex between paired homologous chromosomes, often referred to as 'synapsis'. Importantly, the recombination mediated chiasmata can only provide a linkage between homologs in cooperation with sister chromatid cohesion distal to the crossover (Hartl, 2008a).

Drosophila male meiosis is unconventional in that neither recombination nor synaptonemal complex formation occur, yet homologous chromosomes still faithfully segregate from one another in meiosis I. Two proteins have been identified that act as homolog pairing maintenance factors and may serve as a functional replacement of chiasmata. Mutations to genes encoding these achiasmate conjunction factors, MNM and SNM, cause homologs to prematurely separate and by metaphase I, they can be observed as univalents that then have random segregation patterns. It is likely that MNM and SNM directly provide conjunction of homologs as both localize to the X-Y pairing center (rDNA locus) up until anaphase I and an MNM-GFP fusion parallels this temporal pattern at foci along the 2^nd and 3^rd chromosomes (Thomas, 2005). While MNM and SNM are required for the conjunction of all bivalents, the protein Teflon promotes pairing maintenance specifically for the autosomes (Arya, 2006; Tomkiel, 2001). Teflon is also required for MNM-GFP localization to the 2^nd and 3^rd chromosomes (Thomas, 2005). This suggests that Teflon, MNM, and SNM constitute an autosomal homolog pairing maintenance complex (Hartl, 2008a).

A fascinating aspect of Drosophila male meiosis is that during prophase I, three discrete clusters of chromatin become sequestered to the periphery of the nuclear envelope's interior. Each of these 'chromosome territories' corresponds to one of the major chromosomal bivalents, either the 2^nd, 3^rd or X-Y. A study of chromosomal associations within each prophase I bivalent demonstrated that the four chromatids begin in close alignment. Later in prophase I, all chromatids seemingly separate from one another, but the bivalent remains intact within the territory. It has therefore been proposed that chromosome territories may provide stability to bivalent associations through their sequestration into sub-nuclear compartments (Hartl, 2008a).

This study documents that Drosophila putative condensin II complex subunits, Cap-H2 and Cap-D3, are necessary for normal territory formation. When they are compromised through mutation, chromatin is seemingly dispersed throughout the nucleus. It is proposed that the consequence of this defect is failure to individualize chromosomes from one another leading to the introduction and/or persistence of heterologous chromosomal associations into anaphase I. This underscores the role of chromosome territory formation to prevent ectopic chromosomal associations from interfering with anaphase I segregation. Cap-H2 is also necessary to resolve homologous chromosomal associations, that like heterologous associations, may be mediated by DNA entanglements and/or persistent achiasmate conjunction as anaphase I bridging is rescued by teflon mutations. This highlights condensin II mediated chromosome individualization/disjunction in meiosis I and its necessity to the creation of haploid gametes (Hartl, 2008a).

Faithful chromosome segregation is necessary to organismal viability, therefore it is not surprising that in Drosophila, homozygous lethal alleles exist in the following condensin subunits: SMC4/gluon, SMC2, Cap-H/barren, and Cap-G. It has however been reported that one mutant Cap-D3 allele, Cap-D3^EY00456 is homozygous viable, yet completely male sterile (Savvidou, 2005). This study has confirmed the necessity of Cap-D3 to male fertility; both Cap-D3^EY00456 homozygous and Cap-D3^EY00456/Cap-D3^{Df(2L)Exel6023} males are completely sterile when mated to wild-type females. Furthermore, males trans-heterozygous for strong Cap-H2 mutations are also male sterile; no progeny were derived from crosses of Cap-H2^Z3-0019/Cap-H2^{Df(3R)Exel6159}, Cap-H2^TH1/Cap-H2^{Df(3R)Exel6159}, and Cap-H2^TH1/Cap-H2^Z3-0019 to wild-type females. A third allele, Cap-H2^Z3-5163, is fertile as a homozygote and in trans-combinations with Cap-H2^Z3-0019, Cap-H2^{Df(3R)Exel6159}, and Cap-H2^TH1 alleles (Hartl, 2008a).

To determine whether the primary defect leading to loss of fertility in Cap-H2 mutant males is pre or post copulation, Cap-H2^Z3-0019 homozygous mutant and heterozygous control siblings were engineered to carry a sperm tail marker, don juan-GFP, and aged in the absence of females to allow sperm to accumulate in the seminal vesicles. In contrast to Cap-H2^Z3-0019 heterozygous control males where the seminal vesicles fill with sperm, those from Cap-H2^Z3-0019 homozygous males were seemingly devoid of sperm since no DAPI staining sperm heads or don juan-GFP positive sperm tails were detectable). The lack of mature sperm in the seminal vesicles confirmed that sterility in Cap-H2 mutant males is attributed to a defect in gamete production (Hartl, 2008a).

To test whether a Cap-H2 mutant allelic combination that is male fertile, Cap-H2^Z3-0019/Cap-H2^Z3-5163, has a decreased fertility, males of this genotype and heterozygous controls were mated to wild-type females and the percent of eggs hatched was quantified. There was no significant difference in male fertility between Cap-H2^Z3-0019/Cap-H2^Z3-5163 and Cap-H2^Z3-5163/+ males. However, the introduction of one mutant allele of another condensin subunit, SMC4⁰⁸⁸¹⁹, to the Cap-H2 trans-heterozygote led to a substantial decrease in fertility relative to the SMC4⁰⁸⁸¹⁹/+; Cap-H2^Z3-5163/+ and SMC4⁰⁸⁸¹⁹/+; Cap-H2^Z3-0019/+ double heterozygous controls. This suggests that Cap-H2 is functioning in the Drosophila male germline as a member of a condensin complex along with SMC4 during gametogenesis (Hartl, 2008a).

Given the well-documented roles of condensin subunits in promoting chromosome segregation, it was reasoned that a possible cause of fertility loss in Cap-H2 and Cap-D3 mutants is through chromosome missegregation in the male germline. Male gametogenesis begins with a germline stem cell division. While one daughter maintains stem cell identity, the gonialblast initiates a mitotic program where 4 synchronous cell divisions create a cyst of 16 primary spermatocytes that remain connected due to incomplete cytokinesis. These mature over a period of 3.5 days, undergo DNA replication, and subsequently enter meiosis. To test whether chromosome segregation defects occur during gametogenesis of Cap-H2 mutants, i.e. during the mitotic divisions of the stem cell or gonia or from either meiotic divisions, genetic tests were performed that can detect whether males create an elevated level of aneuploid sperm. In these 'nondisjunction' assays, males are mated to females that have been manipulated to carry a fused, or 'compound', chromosome. Females bearing a compound chromosome and specific genetic markers are often necessary to determine whether eggs had been fertilized by aneuploid sperm. Importantly, in nondisjunction assays, fertilizations from aneuploid sperm generate 'exceptional' progeny that can be phenotypically distinguished from 'normal' progeny that were created from haploid sperm fertilizations (Hartl, 2008a).

Sex chromosome segregation was monitored, with males bred to carry genetic markers on the X and Y chromosomes. These y¹w¹/y+Y; Cap-H2^Z3-0019/Cap-H2^Z3-5163 and corresponding Cap-H2 heterozygous controls males were crossed to females bearing compound X chromosomes [C(1)RM, y² su(w^a)w^a]. No significant amount of exceptional progeny were generated from Cap-H2 mutant males. It is important to point out that the lack of significant sex chromosome segregation defects found in these nondisjunction assays with a likely weak Cap-H2 male fertile mutant may be misleading. In fact, sex chromosome segregation defects are observed cytologically in stronger Cap-H2 mutant backgrounds that could not be tested with nondisjunction assays because of their sterility (Hartl, 2008a).

Fourth chromosome segregation was assayed as described previously for teflon mutants (Tomkiel, 2001), with males carrying one copy of a 4^th chromosome marker mated to females bearing compound 4^th chromosomes (C(4)EN, ci ey). As with the sex chromosome segregation assays, 4^th chromosome segregation did not differ substantially between the Cap-H2^Z3-0019/Cap-H2^Z3-5163 and heterozygous control males. The possibility remains that this hypomorphic Cap-H2 allelic combination is not strong enough to reveal 4^th chromosome segregation defects. Like sex chromosomes, 4^th chromosome segregation abnormalities were observed cytologically in stronger male sterile mutants (Hartl, 2008a).

Effects on second and third chromosome segregation were assayed with the use of females carrying either compound 2 (C(2)EN, b pr) or compound 3 (C(3)EN, st cu e) chromosomes. Interestingly, both the 2^nd and 3^rd chromosomes had a heightened sensitivity to Cap-H2 mutation as Cap-H2^Z3-0019/Cap-H2^Z3-5163 males created an elevated level of exceptional progeny. In both cases, the exceptional class most over represented were those from fertilization events involving sperm that lacked a 2^nd (nullo-2) or 3^rd (nullo-3) chromosome (Hartl, 2008a).

Nullo progeny can be created from defects in either meiotic division. For example, the reciprocal event of incorrect cosegregation of homologs during meiosis I is one daughter cell completely lacking that particular chromosome. Similarly, nullo sperm can be created from meiosis II defects where sister chromatids cosegregate. To address whether meiotic I and or II segregation defects occur, males in the 2^nd chromosome assays were bred to be heterozygous for the 2^nd chromosome marker brown (bw¹). If both 2^nd homologous chromosomes mistakenly cosegregate in meiosis I, then a normal meiosis II will generate diplo-2 sperm that are heterozygous for the paternal male's 2^nd chromosomes (bw¹/+). Additionally, a normal meiosis I followed by a faulty meiosis II where sister chromatids cosegregate would generate diplo-2 sperm homozygous for the paternal male's 2^nd chromosomes (bw¹/bw¹ or +/+). There was a trend toward an elevated level of the bw¹/+ exceptional class from both Cap-H2^Z3-0019/Cap-H2^Z3-5163 and Cap-H2^Z3-0019/+ males. This suggested meiosis I nondisjunction that possibly occurs even in Cap-H2 heterozygous males. Furthermore, there may also be a slight increase in meiosis II nondisjunction as the bw¹/bw¹ class is elevated in the Cap-H2 trans-heterozygous and heterozygous males (Hartl, 2008a).

The Cap-H2 allelic combination utilized in these genetic nondisjunction assays is likely weak in comparison to others where males are completely sterile. Therefore, the elevated frequency of exceptional progeny from 2^nd and 3^rd chromosome assays relative to the sex and 4^th may only represent a heightened sensitivity of these chromosomes rather then a role for Cap-H2 specifically in 2^nd and 3^rd chromosome segregation. In fact, defects in sex and 4^th chromosome segregation were observed in stronger male sterile Cap-H2 mutants. One possible explanation for a major autosome bias in nondisjunction assays may be related to the greater amount of DNA estimated for the 2^nd (60.8 Mb) and 3^rd (68.8 Mb) relative to the X, Y, and 4^th chromosomes (41.8, 40.9, and 4.4 Mb, respectively). Thus, perhaps larger chromosomes require more overall condensin II function to promote their individualization or condensation and are therefore more sensitive to Cap-H2 dosage. While plausible, if sensitivity to Cap-H2 mutation were purely due to chromosome size, it is difficult to explain why a more significant level of XY nondisjunction did not occur given that they are ∼70% the size of the 2^nd and 3^rd (Hartl, 2008a).

An alternative hypothesis involves the fact that 2^nd chromosome conjunction may occur at several sites or along its entire length, whereas XY bivalent pairing is restricted to intergenic repeats of the rDNA locus. This suggests that more total DNA is utilized for conjunction of the 2^nd chromosome relative to the sex bivalent. Assuming the 3^rd and 4^th chromosomes maintain homolog pairing like the 2^nd, then the relative amount of DNA utilized in conjunction is as follows: 3^rd>2^nd>4^th>XY. Given that this closely parallels the trend of sensitivity to Cap-H2 mutation in the nondisjunction assays, it suggests that chromosomes which utilize more overall DNA in pairing/pairing maintenance activities require a greater dose of functional Cap-H2 for their proper anaphase I segregation. This points toward a role for Cap-H2 in the regulation of homolog conjunction/disjunction processes. This hypothesis was addressed through cytological analyses of meiotic chromosome morphology in Cap-H2 mutant backgrounds (Hartl, 2008a).

In prophase I stage S2, nuclei appear to commence the formation of chromosome territories. By mid-prophase I stage S4, territory formation is more evident and in late prophase I, stage S6 nuclei exhibit three discrete chromosome territories seemingly associated with the nuclear envelope. Each of the three chromosome territories corresponds to the 2^nd, 3^rd, and sex chromosomal bivalents and are thought to have important chromosome organizational roles for meiosis I. In male sterile mutants of the genotype Cap-H2^Z3-0019/Cap-H2^TH1, chromosome organizational steps throughout prophase I are defective, as normal territory formation is never observed in 100% of S2, S4, and S6 stages. Instead, chromatin is seemingly dispersed within the nucleus. Male sterile Cap-D3^EY00456 mutants mimic these defects, suggesting that Cap-D3 and Cap-H2 function together within a condensin II complex to facilitate territory formation. No prophase I defects were observed in Cap-H2^Z3-0019/Cap-H2^Z3-5163 males, although subtle morphological changes may be difficult to detect (Hartl, 2008a).

To establish possible roles for Cap-H2 and Cap-D3 in prophase I chromosome organization, it is important to outline the two general processes that must occur for proper territory formation. One is to gather or condense bivalent chromatin into an individual cluster. The second is to sequester each bivalent into a discrete pocket of the nucleus. Condensin II may perform one or both tasks, for example, perhaps chromatin is dispersed throughout the nucleus in the Cap-H2/Cap-D3 mutants because of faulty condensation. Alternatively, or in addition to, sequestration of chromatin into territories may be a primary defect in Cap-H2/Cap-D3 mutants (Hartl, 2008a).

During late prophase I of wild-type primary spermatocytes, chromosomes from each territory condense further and appear as three dots corresponding to the 2^nd, 3^rd and sex bivalents. This stage, referred to as M1 of meiosis I, may be morphologically abnormal in strong Cap-H2 mutants because it was not detected in these studies. This is likely because these mutants fail to form normal chromosome territories. Proceeding further into meiosis, metaphase I is signified by the congression of the three bivalents into one cluster at the metaphase plate. Despite not forming normal chromosome territories and possibly never reaching normal M1 chromosomal structure, there were no unusual features detected in Cap-H2 male sterile metaphase I figures. Although subtle changes to chromosome morphology would not be detectable, it can be concluded that by metaphase I, gross chromosomal condensation occurs at least somewhat normally in Cap-H2 strong mutant males. This raises the interesting possibility that a gradual prophase I chromosome condensation is catalyzed by condensin II components in the course of chromosomal territory formation and culminates at M1. Next, a second condensation step to form metaphase I chromosomes occurs, which is only partially dependent or completely independent of condensin II components. Perhaps condensin I or some other factor is the major player for metaphase I chromosome assembly or compensates for condensin II loss (Hartl, 2008a).

In contrast to metaphase I, anaphase I is clearly not normal in Cap-H2 mutants, where instead bridges are often found between segregating sets of chromosomes. The frequency of these bridges occurs in a manner that matches other phenotypic trends, found in 30.4% of the anaphase I figures for sterile Cap-H2^Z3-0019/Cap-H2^TH1 males, 11.5% for Cap-H2^Z3-0019/Cap-H2^Z3-5163 males that are fertile yet undergo 2^nd and 3^rd chromosome loss (78), and never in the wild-type. As with territory formation, Cap-H2 is likely functioning along with Cap-D3 because in two cysts observed from Cap-D3^EY00456 homozygous males, 7 of 20 anaphase I figures were bridged. This anaphase I bridging most likely represents a failure to resolve chromosomal associations prior to segregation as chromatin appears to be stretched between chromosomes moving to opposing poles (Hartl, 2008a).

To gain further insight into why anaphase I bridges are created in Cap-H2 and Cap-D3 mutants, a chromosome squashing technique was employed that enables the visualization of individual anaphase I chromosomes. With this method, the 4^th chromosomes are easily identified because of their dot like appearance. Centromere placement enables the identification of the sex chromosomes, where on the X it is located very near the end of the chromosome (acrocentric) and on the Y is about a quarter of the length from one end (submetacentric). The 2^nd and 3^rd chromosomes are indistinguishable from one another because of their similar size and placement of the centromere in the middle of the chromosome (metacentric). Whereas bridged anaphase I figures were never observed in wild-type squashed preparations, bridging occurred in 40.5% of those from Cap-H2^Z3-0019/Cap-H2^TH1 mutant males (Hartl, 2008a).

The chromosome squashing method was utilized to determine the nature of anaphase I bridges, and interestingly, it was concluded that bridging exists between both homologous and heterologous chromosomes. Of the total anaphase I figures from Cap-H2^Z3-0019/Cap-H2^TH1 testes, 21.4% appeared to have anaphase I bridging that existed between homologous chromosomes. A FISH probe that recognizes 2^nd chromosome pericentromeric heterochromatin was used to distinguish 2^nd and 3^rd chromosomes and demonstrates that linkages were between the 3^rd chromosomes, perhaps at regions of shared homology. Furthermore, despite not finding 4^th chromosome segregation defects in nondisjunction assays, the 4^th chromosome was bridged in 4.8% of anaphase I figures. This suggests that chromosome 4 becomes sensitive to further loss of Cap-H2 function in the stronger Cap-H2^Z3-0019/Cap-H2^TH1 mutant background (Hartl, 2008a).

Persistent associations between homologous chromosomes in anaphase I may be explained by a failure to individualize paired homologs from one another prior to anaphase I entry. It is probable that DNA entanglements normally exist between paired homologous chromosomes as they are likely raveled around one another rather then simply aligned side by side in a linear fashion. Therefore, individualization failure in Cap-H2 mutants may allow entanglements to persist into anaphase I. Cap-H2 may mediate homolog individualization in prophase I, where bivalents do not appear to condense properly in Cap-H2 mutants. Another plausible scenario is that Cap-H2 functions to antagonize achiasmate homolog conjunction mediated by teflon, MNM, and SNM at some point prior to anaphase I entry (Hartl, 2008a).

The other 19% of anaphase I figures that were bridged in the Cap-H2^Z3-0019/Cap-H2^TH1 mutant involve heterologous chromosomes and cases where bridging is so substantial that its chromosomal nature could not be determined. The observed X-Y linkage is consistent with the XY pairing site, or 'collochore,' and occurs in wild-type preparations. The other linkage is an atypical heterologous association occurring between the Y and one of the major autosomes (2^nd or 3^rd). It is speculated that the substantially bridged images are comprised of associations between heterologous and/or homologous chromosomes. On example was particularly interesting because the 4^th and sex chromosomes appear to have segregated normally, yet the major autosomes remain in an unresolved chromosomal mass. This pattern fits the trend of the nondisjunction studies, where the 2^nd and 3^rd chromosomes had a heightened sensitivity to Cap-H2 mutation (Hartl, 2008a).

Because the 4^th chromosome naturally tends to be separated from other prometaphase I to anaphase I chromosomes, it was often easily observed to be involved in heterologous chromosomal associations. These appear as threads and occurred in 42.5% of metaphase and anaphase I figures. Interestingly, 4^th-to-heterolog threads were also observed in the wild-type, although at a lower frequency of 19% (Hartl, 2008a).

Persistent associations between heterologous chromosomes may be traced to failed territory formation in Cap-H2 mutant prophase I. Perhaps interphase chromosomes are naturally entangled with one another and the Cap-H2/Cap-D3 mediated nuclear organization steps that occur during territory formation effectively detangle and individualize them into discrete structures. Alternatively, Cap-H2/Cap-D3 mediated chromosome territory formation may act to prevent the establishment of heterologous entanglements. These are plausible scenarios given that failed territory formation in Cap-H2/Cap-D3 mutants seemingly leads to persistent intermingling of all chromosomes. Such an environment could provide a likely source of heterologous chromosomal associations. Heterologous associations involving the 4^th chromosome may also be entanglements that persist and/or were initiated through failure in territory formation. These cannot however be completely attributed to loss of Cap-H2 function because they were observed in the wild-type (Hartl, 2008a).

The anaphase I bridging in Cap-H2 mutant males is one likely source for their elevated amount of nullo-2 and nullo-3 sperm. Chromatin stretched between daughter nuclei may occasionally lead to the creation of sperm lacking whole chromosomes or variable sized chromosomal regions. Bridged anaphase I represent likely scenarios where chromosome loss would occur and furthermore, visualization of the post-meiotic 'onion stage' from Cap-H2 mutants is consistent with chromosome loss. With light microscopy, white appearing nuclei within the onion stage are nearly identical in size to the black appearing nebenkern, which represents clustered mitochondria. In onion stages from Cap-H2^Z3-0019 homozygotes, micronuclei are often observed which may be the manifestation of chromatin lost through anaphase I bridging (Hartl, 2008a).

The associations that create anaphase I bridging between chromosomes moving to opposing poles may also be capable of causing improper cosegregation of homologs. In fact, 9.5% of squashed anaphase I figures are of asymmetrically segregating homologs that were never observed in the wild-type. These are consistent with failure in homolog disjunction and subsequent cosegregation to one pole. These may also be the consequence of associations between heterologous chromosomes that lead to one being dragged to the incorrect pole. As an expected outcome of cosegregation in meiosis I, aneuploidy in prophase II and anaphase II figures was also observed. Such events likely explain the slight increase in diplo-2 sperm that were heterozygous for the male's 2^nd chromosomes. They also provide a likely source for the elevated amount of nullo-2 and nullo-3 sperm (Hartl, 2008a).

While the prevalence of meiotic anaphase I bridging is likely a major contributor to the observed 2^nd and 3^rd nondisjunction, it cannot be ruled out that the preceding stem cell and gonial mitotic divisions are also defective and lead to aneuploid sperm. This exists as a formal possibility, yet aneuploid meiotic I cells were not observed in squashed Cap-H2 mutant anaphase I figures where all chromosomes could be distinguished. This suggests that pre-meiotic segregation is unaffected. Similarly, anaphase II defects could have contributed to the elevated nullo-2 and nullo-3 sperm and perhaps the slight increase in bw¹/bw¹ progeny that would have been generated from meiosis II nondisjunction. In fact, anaphase II bridging was observed in 8.7% of Cap-H2^Z3-0019/Cap-H2^TH1 anaphase II figures, 2.1% of those from Cap-H2^Z3-0019/Cap-H2^Z3-5163 males, and never in the wild-type. Anaphase II defects may occur because of a specific role of Cap-H2 in meiosis II, or alternatively, anaphase II bridging could be attributed to faulty chromosome assembly or individualization in meiosis I (Hartl, 2008a).

The protein Teflon is implicated in the maintenance of Drosophila male meiosis I autosome conjunction as teflon mutants lose autosomal associations prior to anaphase I (Arya, 2006). To investigate whether persistent associations between homologous chromosomes in anaphase I of Cap-H2 mutants are Teflon dependent, teflon mutations were crossed into a Cap-H2 mutant background and the frequency of anaphase I bridging was assessed. While 30.4% of anaphase I figures from Cap-H2^Z3-0019/Cap-H2^TH1 males were bridged, bridging existed within only 10.8% of anaphase I figures from tef^Z2-5549/tef^Z2-5864; Cap-H2^Z3-0019/Cap-H2^TH1 males. Furthermore, in squashed preparations anaphase I bridging was decreased from 40.5% in Cap-H2^Z3-0019/Cap-H2^TH1 males to 25.6% in the tef^Z2-5549/tef^Z2-5864; Cap-H2^Z3-0019/Cap-H2^TH1 double mutants (Hartl, 2008a).

The ability of teflon mutations to rescue Cap-H2 mutant anaphase I bridging suggests that Cap-H2 functions to antagonize Teflon mediated autosome conjunction. This may entail deactivation of an achiasmate conjunction complex consisting of MNM, SNM, and perhaps Teflon, at some point prior to the metaphase I to anaphase I transition. Consistent with this hypothesis, the percent of anaphase I figures where homologous chromosomes appeared to be bridged were decreased from 21.4% in the Cap-H2^Z3-0019/Cap-H2^TH1 mutants to 9.3% in tef^Z2-5549/tef^Z2-5864; Cap-H2^Z3-0019/Cap-H2^TH1 males (Hartl, 2008a).

As an important alternative to Cap-H2 functioning to antagonize an achiasmate homolog conjunction complex, it may be that wild-type Teflon exacerbates DNA associations between chromosomes. For example, perhaps Teflon linked homologs are now particularly prone to becoming entangled. Under this scenario, teflon mutations may decrease the opportunity for DNA entanglements to be introduced between homologs because of their spatial distancing from one another during late prophase I to metaphase I. Given the formal possibility of both models, it is concluded that Cap-H2 functions to either remove teflon dependent conjunction and/or to resolve chromosomal entanglements between homologs (Hartl, 2008a).

The remaining bridged anaphase I figures from squashed preparations in tef^Z2-5549/tef^Z2-5864; Cap-H2^Z3-0019/Cap-H2^TH1 males were uninterpretable making it impossible to assess whether Cap-H2 mutant heterologous anaphase I bridging was also rescued by teflon mutation. However, 4^th-to-heterolog threads were greatly suppressed by teflon mutations, decreasing from 42.5% to only 6%. This is a surprising result given that Teflon has been described as a mediator of associations between homologous chromosomes. One plausible explanation is that Teflon can exacerbate heterologous chromosomal associations. This may occur when Teflon establishes autosomal conjunction in a prophase I nucleus where territory formation had failed. Cap-H2 may also antagonize a Teflon mediated autosomal conjunction complex that might mistakenly establish conjunction between heterologs when territories do not form (Hartl, 2008a).

As described above, completely male sterile Cap-D3 and Cap-H2 allelic combinations exist and Cap-H2 mutant males lack mature sperm in their seminal vesicles. One possible explanation for this result is that chromosome damage created during anaphase bridging in the Cap-H2 mutants causes spermatogenesis to abort. This scenario seems less likely because tef^Z2-5549/tef^Z2-5864 rescued Cap-H2^Z3-0019/Cap-H2^TH1 anaphase I bridging to levels near that of fertile Cap-H2 mutants, yet tef^Z2-5549/tef^Z2-5864; Cap-H2^Z3-0019/Cap-H2^TH1 males were still found to be completely sterile. This points toward another function for Cap-H2 in post-meiotic steps of spermatogenesis (Hartl, 2008a).

A working model is presented of how condensin II functions in Drosophila male meiosis to resolve both heterologous and homologous chromosomal associations. It is speculated that these associations likely consist of DNA entanglements that naturally become introduced between interphase chromosomes due to their threadlike nature. The studies herein identified a function for condensin II during prophase I, when paired homologous chromosomes become partitioned into discrete chromosomal territories. It is proposed that condensin II either promotes this partitioning, by actively sequestering bivalents into different regions of the nucleus, or functions to perform prophase I chromosome condensation. It is important to stress that in both scenarios, the role of condensin II mediated territory formation is to ensure the individualization of heterologous chromosomes from one another. When sequestration into territories and/or condensation of the bivalents do not take place, i.e. in the condensin II mutants, individualization does not occur, heterologous entanglements persist into anaphase I, and chromosomes may become stretched to the point where variable sized chromosomal portions become lost. Persistent heterologous entanglements may also lead to one chromosome dragging another to the incorrect pole (Hartl, 2008a).

Despite what appears to be failed chromosome condensation in prophase I of Cap-H2 mutants, by metaphase and anaphase I no obvious defects in chromosome condensation were observed. This suggests that sufficient functional Cap-H2 is present in this mutant background to promote metaphase/anaphase I chromosome condensation. Alternatively, perhaps another factor fulfills this role and/or compensates for condensin II loss. This parallels Cap-G mutants, where embryonic mitotic prophase/prometaphase condensation was abnormal, yet metaphase figures appeared wild-type. In Drosophila, mutant and RNAi knockdown studies of condensin complex subunits in mitosis have shown a range of phenotypes, from complete failure in condensation to seemingly normal axial shortening, but failure in chromatid resolution. The variable phenotypes produced from these studies may reflect differences in cell type specific demand for condensin subunit dosage/activity (Hartl, 2008a).

Anaphase I figures of Cap-H2 mutants also revealed persistent entanglements between homologous chromosomes that may be at regions of shared homology. It is suggested that the paired state of homologs initiates or introduces the opportunity for DNA entangling between homologs and that condensin II functions to resolve these prior to segregation. A likely scenario is that this occurs during prophase I, where chromosome condensation appears abnormal in Cap-H2 and Cap-D3 mutants. Perhaps condensin II mediated prophase I condensation functions to individualize intertwined homologous chromosomes prior to segregation. It is also plausible that condensin II homolog individualization continues up until anaphase I (Hartl, 2008a).

This study has found that mutations in teflon, a gene required for autosomal pairing maintenance, are capable of suppressing anaphase I bridging in Cap-H2 mutant males. Specifically, both homologous and heterologous chromosomal bridging is decreased in the teflon/Cap-H2 double mutant. This may occur because Teflon is capable of exacerbating DNA entanglements, if for example persistent homolog conjunction provides more opportunity for entanglements between homologs to be introduced. Teflon may also exacerbate entanglements between heterologous chromosomes. This might be especially true in a Cap-H2 mutant background with failed territory formation, as Teflon mediated autosomal conjunction may augment the extent of entangling (Hartl, 2008a).

It is also plausible that Cap-H2 acts as an antagonist of Teflon mediated autosomal conjunction. Perhaps autosomal homologous associations persist into anaphase I of Cap-H2 mutants because a homolog conjunction complex was not disabled prior to the metaphase I to anaphase I transition. However, Cap-H2 as an antagonist of Teflon cannot explain persistent heterologous associations into anaphase I, unless Teflon is capable of mistakenly introducing conjunction between heterologous chromosomes. The opportunity for this might exist in a Cap-H2 mutant prophase I nucleus where heterologs continue to intermingle because of failed territory formation (Hartl, 2008a).

An interesting result in this course of studies was the heightened amount of chromosome 2 and 3 nondisjunction in weaker male fertile Cap-H2 allelic combinations, whereas the sex and 4^th chromosomes were unaffected. This is reminiscent of mutants from several other genetic screens that only affected the segregation of specific chromosomes or subsets. However, given that sex and 4^th chromosome segregation defects are observed in the stronger male sterile Cap-H2 mutant background, it is proposed that condensin II functions upon all chromosomes, yet the 2^nd and 3^rd require the greatest functional Cap-H2 dose for their proper segregation. This sensitivity of the 2^nd and 3^rd chromosomes may be due to their greater total amount of DNA utilized in homolog pairing and pairing maintenance activities. For example, perhaps longer stretches of paired DNA are more prone to entanglements or require more achiasmate conjunction factors and therefore necessitate higher levels of Cap-H2 individualization or disengagement activity. As an interesting corollary to support this theory, weak teflon mutations only lead to 4^th chromosome missegregation, while the other autosomes segregate normally (Arya, 2006). This suggests that the 4^th chromosomes are more sensitive to Teflon dosage because of their fewer sites of conjunction (Hartl, 2008a).

The majority of the data provided in this manuscript were on studies of mutant Cap-H2 alleles, however, a homozygous viable Cap-D3 mutant also failed to form normal chromosomal territories and exhibited anaphase I chromosome bridging. This provides support that these two proteins are functioning together within a condensin II complex. It is important to point out however, that to date there is no data in Drosophila to support that these proteins physically associate with each other or with other condensin subunits, namely SMC2 and SMC4 (a Drosophila Cap-G2 has yet to be identified with computational attempts) (Hartl, 2008a).

At this point in studies of putative condensin II subunits in disjunction of achiasmate male homologous chromosomes, it is not possible to distinguish between possible scenarios that Cap-H2 and Cap-D3 act to disentangle chromosomes through individualization activity, that they function as antagonists of Teflon dependent achiasmate associations, or a combination of both activities. The fact that Teflon mutations do rescue Cap-H2 anaphase I bridging defects is an especially intriguing result as it points toward a molecular mechanism for Cap-H2 as an antagonist of achiasmate associations. While three genes have been found to promote achiasmate conjunction (teflon, MNM, and SNM), no factors have been identified that act to negatively regulate conjunction and allow homologs to disengage at the time of segregation. Interestingly, one conjunction factor, SNM, is orthologous to the cohesin subunit Scc3/SA that appears to be specialized to engage achiasmate homologs (Thomas, 2005). Condensin has been shown to antagonize cohesins in budding yeast meiosis and mitotic human tissue culture cells. This raises the possibility that a conserved molecular mechanism exists for condensin II as a negative regulator of SNM in Drosophila male meiosis. The investigation of Teflon, MNM, and SNM protein dynamics in a Cap-H2 mutant background will be an important set of future studies to help decipher the function of Cap-H2 in achiasmate segregation mechanisms (Hartl, 2008a).

Homologous chromosomal individualization in meiosis I has been previously documented as a condensin complex catalyzed activity in C. elegans; homologs remained associated in hcp-6/Cap-D3 mutants even in the absence of recombination and sister chromatid cohesion. This study has demonstrated that condensin subunits are also required to individualize heterologous chromosomes from one another prior to anaphase I. This is likely through condensin II mediated chromosome organizational steps that occur during prophase I territory formation. This suggests that Drosophila males carry out territory formation to disfavor associations between heterologs, while also enriching for interactions between homologs. This model is particularly interesting as it may point toward an adaptation of Drosophila males to ensure meiotic I segregation in a system lacking a synaptonemal complex and recombination (Hartl, 2008a).

Chromosome alignment and transvection are antagonized by condensin II

Polytene chromosome structure is a characteristic of some polyploid cells where several to thousands of chromatids are closely associated with perfect alignment of homologous DNA sequences. This study shows that Drosophila condensin II promotes disassembly of polytene structure into chromosomal components. Condensin II also negatively regulates transvection, a process whereby certain alleles are influenced transcriptionally via interallelic physical associations. It is proposed that condensin II restricts trans-chromosomal interactions that affect transcription through its ability to spatially separate aligned interphase chromosomes (Hartl, 2008b).

Interphase chromosomal trans-interactions occur in many species and impact chromosome structure and gene expression. As evidenced in Drosophila, trans-interactions can lead to polytene chromosomes, where all maternal and paternal chromatids are aligned in precise register. The Drosophila ovarian nurse cells disassemble their polytene chromosomes into unpaired homologs and chromatid fibers during mid-oogenesis. This system was used to isolate two noncomplementing mutations in a predicted condensin II subunit, Cap-H2, that cause failure in nurse cell polytene disassembly. Polyteny instead persists in the trans-heterozygous combinations of Cap-H2^Z3-0019/Cap-H2^Z3-5163 and when either allele is in trans to a deletion of its genomic locus. This was corroborated through fluorescence in situ hybridization (FISH) labeling to a specific locus in stage 7 egg chambers, where wild-type polytenes disassembled, yet 92.9% of mutant nuclei had all maternal and paternal chromatids aligned in register. Polytene persistence in Cap-H2 mutants likely does not occur indirectly through altered cell cycle progression or DNA replication patterns because neither the length of S phase nor ploidy were detectably different in homozygous polytene mutants versus heterozygous controls. This result instead suggests that Cap-H2 function is necessary to disassemble nurse cell polytene chromosomes (Hartl, 2008b).

Metazoa have two condensin complexes that are referred to as condensin I and II. Each uses the adenosine triphosphatases SMC2 and SMC4, but forms complexes with different non-SMC subunits Cap-H, Cap-G, and Cap-D2 or Cap-H2, Cap-G2, and Cap-D3, respectively. Condensins function in the condensation of chromosomes, facilitate proper anaphase segregation, and in vitro induce and trap DNA positive supercoiling. Supercoiling has been proposed to gather chromatin into domains that are then further ordered to assemble metaphase chromosomes. Cap-H2 likely acts within a condensin II complex, as other predicted condensin II subunits also regulate nurse cell polytene dispersal. Cap-D3 mutants exhibited nurse cell polytene persistence that was enhanced through the introduction of one mutant Cap-H2 copy. Furthermore, SMC4/Cap-H2 double-heterozygotes had a loosened, but clear, polytene morphology. Consistent with a polytene disassembly function, Cap-H2 protein first becomes enriched within posterior stage 5 and 6 egg chambers, where disassembly is initiated, and Cap-H2 is detected in all stage 7 to 10 nuclei (Hartl, 2008b).

Unlike nurse cells, polyteny is persistent in the nuclei of the larval salivary glands. Cap-H2 overexpression induced drastic separation of salivary gland polytene chromosomal components, as visualized through green fluorescent protein (GFP) labeling of a second chromosome locus. In the wild-type, the GFP locus had a width 15.9 ± 1.6% (SEM) of the nuclear radius, yet individual foci reached distances 110.8 ± 9.1% (SEM) of the nuclear radius after Cap-H2 induction (Hartl, 2008b).

In the salivary gland, Cap-H2-induced polytene disassembly occurs only 6 hours after Cap-H2 overexpression in fully developed late larvae, which makes it unlikely that disassembly is an indirect consequence of altered larval development. It is also improbable that disassembly occurs through the creation of large-scale chromosomal breaks, because this was not detected after Cap-H2 overexpression, and that the creation of DNA breaks with {gamma}-radiation did not alter polytene alignment. Rather, the ability of Cap-H2 overexpression to induce polytene disassembly indicates that polytene alignment of chromatids is constrained with wild-type Cap-H2 levels. Providing excess Cap-H2 may induce polytene disassembly because its dosage is limiting to other condensin II subunits in salivary glands and/or it acts as a catalytic subunit that promotes condensin II activity. Cap-H2 does rely on Cap-D3 to induce polytene disassembly; all salivary gland nuclei from a Cap-D3 mutant background overexpressing Cap-H2 had polytenes that appeared like the wild-type. This contrasted to Cap-D3 heterozygous controls, where only 24.1 ± 8.5% (SEM) nuclei per gland contained wild-type polytenes (Hartl, 2008b).

Because of Cap-H2's ability to transform aligned polytene structure into chromosomal components, it was predicted to function in a similar manner to disrupt aligned loci within diploid somatic cells. Therefore whether it regulates diploid trans-chromosomal interactions was investigated by studying its role in transvection, a phenomenon whereby certain mutant alleles are influenced transcriptionally via association with their homologous locus. It is inferred from transvection phenomena that somatic homolog pairing also plays a role in regulating wild-type loci (Hartl, 2008b).

The first transvection system that was utilized involves the gain-of-function mutation Ubx^Cbx-1, which causes misexpression of Ubx in the imaginal wing disc and leads to a partial wing-to-haltere transformation. A wing transformation occurs even in flies where Ubx of the Ubx^Cbx-1 allele is rendered null through the introduction of a second mutation (Ubx^Cbx-1 Ubx¹). This Ubx^Cbx-1 Ubx¹/++ phenotype suggested that the Cbx1 lesion is capable of transcriptionally activating the wild-type Ubx on the homologous chromosome through a trans physical association. This was supported by the ability of chromosomal rearrangements (R) that disrupt homolog pairing at Ubx to suppress transvection. Consistent with a role for Cap-H2 in antagonizing homolog pairing, the Ubx^Cbx-1 Ubx¹/++ phenotype was dominantly enhanced by Cap-H2 mutations. Conversely, Cap-H2 overexpression suppressed the Ubx^Cbx-1 Ubx¹/++ wing phenotype closer to wild-type (Hartl, 2008b).

Cap-H2 mutant enhancement of the Ubx^Cbx-1 Ubx¹ phenotype was suppressed in a chromosomal rearrangement background [R(Ubx^Cbx-1 Ubx¹)/++] that is thought to disrupt allelic associations between Ubx^Cbx-1 Ubx¹ and wild-type Ubx. The Ubx^Cbx-1 Ubx¹/Cap-H2- and R(Ubx^Cbx-1 Ubx¹)/Cap-H2- flies only vary by the reciprocal translocation that moves 3R bearing Ubx^Cbx-1 Ubx¹ to 2R and vice versa. This suggests that Cap-H2 enhancement of the Ubx^Cbx-1 Ubx¹ phenotype is through increasing the association of homologous loci. Alternatively, Cap-H2 function may follow trans-chromosomal interactions, for example, acting locally to enable enhancer interactions in trans or as a general transcriptional repressor. Although either is formally possible, Cap-H2's ability to globally disrupt aligned polytene structure suggests it carries out a related function in diploid cells to antagonize trans-chromosomal interactions (Hartl, 2008b).

Cap-H2 was tested in a second transvection system involving mutant alleles of the gene yellow (y). In y^82f29/y^82f29 and y^y1#8/y^y1#8 flies, there is minimal cuticle pigmentation, yet when placed in trans to one another (y^82f29/y^y1#8) complementation occurs with partial restoration of pigment nearer to wild-type levels. The y^y1#8 allele is a deletion of the yellow promoter and the y^82f29 allele a deletion of upstream enhancer elements. It is thought that partial complementation occurs in y^82f29/y^y1#8 through the ability of y^y1#8's enhancers to act in trans, to associate with the intact promoter of y^82f29, and then to activate yellow transcription. As are Ubx^Cbx-1 Ubx¹, transvection of y^82f29/y^y1#8 is enhanced in a Cap-H2 mutant background, which leads to darker pigmentation of the abdominal stripes relative to controls (Hartl, 2008b).

Transvection can be enhanced by slowing the rate of cell division. The percent of Cap-H2 homozygous mutant cells specifically in mitosis was cytologically found to be greater relative to heterozygous controls, but this was statistically insignificant. Furthermore, with flow cytometry, homozygotes and heterozygotes did not vary significantly in the percentage of cells in G1, S, and G2/M. Although these data do not rule out a cell cycle delay leading to enhanced transvection, they also do not support a major regulatory role for Cap-H2 in cell cycle progression. Cap-H2's ability to disassemble the aligned structure of polytene chromosomes instead suggests that it antagonizes transvection by inhibiting homology-dependent chromosomal interactions in diploid somatic cells (Hartl, 2008b).

Just as condensin-mediated supercoiling has been proposed to initiate chromosome condensation, it is speculated that supercoiling activity also exists in interphase nuclei and can disrupt chromosome alignment. This may be through providing a force that physically disrupts interchromosomal associations and/or favors intrachromosomal higher-order structures that make inaccessible regions prone to trans-associate. This condensin activity may be a crucial aspect of gene regulation by disrupting trans-communication of allelic regulatory elements (Hartl, 2008b).

The relative ratio of condensin I to II determines chromosome shapes

To understand how chromosome shapes are determined by actions of condensins and cohesin, a series of protocols was devised in which their levels are precisely changed in Xenopus egg extracts. When the relative ratio of condensin I to II is forced to be smaller, embryonic chromosomes become shorter and thicker, being reminiscent of somatic chromosomes. Further depletion of condensin II unveils its contribution to axial shortening of chromosomes. Cohesin helps juxtapose sister chromatid arms by collaborating with condensin I and counteracting condensin II. Thus, chromosome shaping is achieved by an exquisite balance among condensin I and II and cohesin (Shintomi, 2011).

While the unique shape of metaphase chromosomes had been observed and described by cytogeneticists and cell biologists for more than a century, addressing the molecular basis of chromosome shaping became possible only recently after the discovery of condensins and cohesin. The current study represents the first systematic attempt to address this question by precisely manipulating the level of these essential components for chromosome morphogenesis. From a mechanistic point of view, the assembly of a metaphase chromosome would involve at least three different processes: axial shortening, lateral compaction of each chromatid, and cohesion between sister chromatids. Given the fixed volume of a chromatin fiber whose density within a chromosome has been shown to be constant from prometaphase through metaphase, axial shortening would create shorter and thicker chromatids, whereas lateral compaction would make chromatids longer and thinner. In fact, it has been demonstrated that chromosomes progressively get shorter and thicker during animal development. On the basis of the ratio manipulation experiments reported in this study, it is proposed that the primary actions of condensin I and II are aimed at lateral compaction and axial shortening, respectively, and that their intricate balance acts as one of the critical determinants in shaping chromatids. While this is clearly an oversimplified model, it is consistent with the recent finding that caspase-mediated cleavage of a condensin I subunit results in the formation of short and thick chromosomes in cells arrested at metaphase for a long time. On the other hand, the current results suggest that cohesin contributes to chromosome shaping in two ways. First, release of cohesin itself from chromosome arms facilitates condensin II-mediated axis formation. Second, residual cohesin holds sister chromatid arms together, whose juxtaposition could further be reinforced by condensin I-mediated chromatid rigidity. In the future, it will be of great interest to combine the sophisticated cell-free protocols reported here with other assays such as micromanipulation of chromosomes. Furthermore, it will be important to examine exactly how expression of condensins and cohesin might be under the control of developmental cues, and how their intricate balance might be established and fine-tuned among different organisms. Such considerations would undoubtedly help to link chromosome biology to other neighboring areas such as developmental biology and evolutionary biology (Shintomi, 2011).

The initial phase of chromosome condensation requires Cdk1-mediated phosphorylation of the CAP-D3 subunit of condensin II

The cell cycle transition from interphase into mitosis is best characterized by the appearance of condensed chromosomes that become microscopically visible as thread-like structures in nuclei. Biochemically, launching the mitotic program requires the activation of the mitotic cyclin-dependent kinase Cdk1 (cyclin-dependent kinase 1), but whether and how Cdk1 triggers chromosome assembly at mitotic entry are not well understood. This study reports that mitotic chromosome assembly in prophase depends on Cdk1-mediated phosphorylation of the condensin II complex. Thr 1415 of the CAP-D3 subunit was identified as a Cdk1 phosphorylation site, which proved crucial as it was required for the Polo kinase Plk1 (Polo-like kinase 1) to localize to chromosome axes through binding to CAP-D3 and thereby hyperphosphorylate the condensin II complex. Live-cell imaging analysis of cells carrying nonphosphorylatable CAP-D3 mutants in place of endogenous protein suggested that phosphorylation of Thr 1415 is required for timely chromosome condensation during prophase, and the Plk1-mediated phosphorylation of condensin II facilitates its ability to assemble chromosomes properly. These observations provide an explanation for how Cdk1 induces chromosome assembly in cells entering mitosis, and underscore the significance of the cooperative action of Plk1 with Cdk1 (Abe, 2011).

Drosophila Casein Kinase I alpha regulates homolog pairing and genome organization by modulating Condensin II subunit Cap-H2 levels

The spatial organization of chromosomes within interphase nuclei is important for gene expression and epigenetic inheritance. Although the extent of physical interaction between chromosomes and their degree of compaction varies during development and between different cell-types, it is unclear how regulation of chromosome interactions and compaction relate to spatial organization of genomes. Drosophila is an excellent model system for studying chromosomal interactions including homolog pairing. Recent work has shown that condensin II governs both interphase chromosome compaction and homolog pairing and condensin II activity is controlled by the turnover of its regulatory subunit Cap-H2. Specifically, Cap-H2 is a target of the SCFSlimb E3 ubiquitin-ligase which down-regulates Cap-H2 in order to maintain homologous chromosome pairing, chromosome length and proper nuclear organization. This study identifies Casein Kinase I α (CK1α) as an additional negative-regulator of Cap-H2. CK1α-depletion stabilizes Cap-H2 protein and results in an accumulation of Cap-H2 on chromosomes. Similar to Slimb mutation, CK1α depletion in cultured cells, larval salivary gland, and nurse cells results in several condensin II-dependent phenotypes including dispersal of centromeres, interphase chromosome compaction, and chromosome unpairing. Moreover, CK1alpha loss-of-function mutations dominantly suppress condensin II mutant phenotypes in vivo. Thus, CK1alpha facilitates Cap-H2 destruction and modulates nuclear organization by attenuating chromatin localized Cap-H2 protein (Nuyen, 2015).

Condensin II drives large-scale folding and spatial partitioning of interphase chromosomes in Drosophila nuclei

Metazoan chromosomes are folded into discrete sub-nuclear domains, referred to as chromosome territories (CTs). The molecular mechanisms that underlie the formation and maintenance of CTs during the cell cycle remain largely unknown. This paper reports the development of high-resolution chromosome paints to investigate CT organization in Drosophila cycling cells. Large-scale chromosome folding patterns and levels of chromosome intermixing are shown to be remarkably stable across various cell types. The data also suggest that the nucleus scales to accommodate fluctuations in chromosome size throughout the cell cycle, which limits the degree of intermixing between neighboring CTs. Finally, this study shows that the cohesin and condensin complexes are required for different scales of chromosome folding, with condensin II being especially important for the size, shape, and level of intermixing between CTs in interphase. These findings suggest that large-scale chromosome folding driven by condensin II influences the extent to which chromosomes interact, which may have direct consequences for cell-type specific genome stability (Rosin, 2018).

Metazoan genomes are arranged into a nested hierarchy of structural features, ranging from small chromatin loops to larger insulated neighborhoods or topologically associated domains (TADs). TADs are believed to direct and insulate gene regulatory networks, which can engage in long-range interactions with each other, ultimately packaging chromosomes into sub-nuclear compartments termed chromosome territories (CTs) (Rosin, 2018).

CTs are a widespread feature of nuclear organization across a variety of cell types and species, as revealed by both fluorescence in situ hybridization (FISH) and chromosome-conformation-capture (3C)-based studies. Recently, several studies have implicated the ring-shaped SMC (structural maintenance of chromosomes) complexes-cohesin and condensin-in the regulation of large-scale chromatin folding and CT formation. However, the contribution of each complex to local topology, large-scale chromatin folding, and chromosome individualization at single-cell resolution has been hindered by technical limitations. The consequence of CT loss during interphase also remains unclear. This is due, in part, to both the paucity of factors known to directly influence this level of organization and the difficulty in visualizing their effects at single cell resolution. However, CT intermixing has been theorized to influence the location and frequency of translocations and the position of a gene within and between CTs seems to influence its access to the machinery responsible for specific nuclear functions, such as transcription, splicing, and DNA repair (Rosin, 2018).

This study leveraged the flexible, scalable Oligopaint FISH technology to generate high-resolution chromosome paints to the entire Drosophila genome. Combined with a custom 3D segmentation pipeline, a comprehensive picture of chromosome size, shape, and position at single-cell resolution. The results show that various cell types in Drosophila harbor spatially partitioned CTs. Interestingly, widespread somatic homolog pairing in Drosophila results in homologs sharing a single CT, suggesting that homologous and heterologous chromosomes are distinguished at the cellular level in this species. Further, this study characterize the differential roles of cohesin and condensin complexes in local chromatin compaction, large-scale chromatin folding, and CT formation. Cohesin and condensin II were shown to drive different scales of chromatin folding during interphase, with condensin II being especially important for large-scale interactions and the spatial partitioning of chromosomes. These findings indicate that condensin II-driven large-scale chromatin conformations during interphase influence the extent to which chromosomes interact, which has the potential to affect gene regulation and genome stability (Rosin, 2018). In this study, we demonstrate that Drosophila cells harbor spatially distinct CTs and found remarkably consistent levels of intermixing in a variety of cell types and throughout the cell cycle. While the vast majority of cells showed contact between all three major chromosomes, it was possible to measure that, on average, only 40% of the Drosophila genome is intermixed (not accounting for homologous chromosomes). This is strikingly similar to the estimate of 40-46% CT intermixing in human lymphocytes, possibly indicating a widespread and conserved restraint on inter-chromosomal interactions. However, it is noted that a small population of cells do exhibit >90% overlap between neighboring CTs. The fate of these cells will be important to explore in the future (Rosin, 2018).

Further, the condensin II complex was identified as an essential factor for CT formation in cycling cells. These results are consistent with those reported on condensin in yeast, tetrahymena, and post-mitotic polytene cells of Drosophila. These data are also in line with previous work showing that condensin II serves as an 'anti-pairing' factor that disrupts pairing interactions and separates homologous loci. Additionally, it was shown that condensin II overexpression can further compact chromosomes and reduce the level of CT intermixing. Together, these data highlight the highly conserved role of the condensin II complex in controlling the level of inter-chromosomal associations in eukaryotic cells (Rosin, 2018).

If condensin II has the capacity to spatially separate homologous and heterologous chromosomes, how does somatic pairing persist in Drosophila cells that have CTs? One possibility is that pairing interactions are established prior to CT formation and thus, homologous chromosomes would be folded in concert. This would be consistent with some persistence of homolog pairing through mitosis and suggests a model in which chromosomes are folded into CTs through post-mitotic condensin II activity. In addition, pairing interactions may require additional condensin activity to separate homologous versus heterologous interactions. Indeed, these studies showed that condensin II overexpression increases whole-chromosome unpairing in Kc167 and BG3 cells. It is speculated that interphase condensin II levels and thus inter-chromosomal associations are tightly regulated, and could be modified in a cell-type-specific manner. For instance, in contrast to virtually all other cell types in Drosophila, homologous chromosomes in germline stem cells remain unpaired throughout development. This separation between homologs could potentially reflect increased levels of condensin II activity and may indicate that inter-chromosomal associations are reduced to protect the stem-cell population from potentially deleterious rearrangements. Indeed, previous work has shown that different extents of chromosome intermixing correlate with translocation frequencies-both those occurring naturally in the human population and those induced experimentally in human and mouse lymphocytes. Therefore, an alteration in condensin II activity and subsequent CT intermixing levels has the potential to influence the location and frequency with which translocations occur. Intriguingly, mice carrying a hypomorphic allele of cap-H2 were recently shown to frequently develop T-cell lymphomas with highly rearranged chromosomes in the transformed cells. It will be important to determine whether this increased genome instability is associated with increased CT contact prior to the rearrangement event (Rosin, 2018).

When accounting for the popular model of loop extrusion and the stabilizing function of SMC complexes, condensin II activity could potentially fold whole chromosomes into a configuration that limit their interactions with the rest of the genome. While the nature of these interactions remains unknown, they are clearly distinct from cohesin-driven interactions given that cohesin depletion does not significantly change intermixing levels in Drosophila or yeast. Consistent with this hypothesis, a recent study demonstrated that depletion of the cohesin complex in mammals eliminates chromatin looping and TAD formation but does not disrupt long-range interactions between similar chromatin states, highlighting the notion that local insulation and higher-order folding must rely on distinct molecular determinants. Combined with the current findings that large-scale configurations are stable throughout the cell cycle and require condensin II activity, it is proposed that condensin II drives long-range interactions that are established early in interphase. In this model, condensin II may act as an 'organizational bookmark' by prioritizing intra-chromosomal folding immediately following mitotic exit. As condensin II is enriched at highly active regions of the genome marked by H3K4me3, its activity could potentially allow gene regulatory networks and chromatin compartments to favor intra- versus inter-chromosomal interactions. Further studies identifying the interactions driven by condensin II in relation to cohesin will be critical for understanding how these molecular machines cooperatively guide the genome through the cell cycle and development (Rosin, 2018).

Finally, this report describes an efficient and scalable method of high-resolution chromosome painting using Oligopaint FISH technology. Combined with a custom 3D-segmentation pipeline, quantitative measurements of chromosome size, shape, position, and overlap can be analyzed in a systematic and potentially high-throughput fashion. Moreover, the ability to conduct sequential rounds of hybridization with Oligopaints permits 3D analysis of many, if not all, CTs simultaneously. It is anticipated that this technology will lead to an enhanced ability to visualize and karyotype chromosomes in a number of systems, providing a novel battery of assays to better characterize how chromatin is packaged and spatially partitioned in the nucleus (Rosin, 2018).

Evolutionarily conserved principles predict 3D chromatin organization

Interaction domains in Drosophila chromosomes form by segregation of active and inactive chromatin in the absence of CTCF loops, but the role of transcription versus other architectural proteins in chromatin organization is unclear. This study finds that positioning of RNAPII via transcription elongation is essential in the formation of gene loops, which in turn interact to form compartmental domains. Inhibition of transcription elongation or depletion of cohesin decreases gene looping and formation of active compartmental domains. In contrast, depletion of condensin II, which also localizes to active chromatin, causes increased gene looping, formation of compartmental domains, and stronger intra-chromosomal compartmental interactions. Condensin II has a similar role in maintaining inter-chromosomal interactions responsible for pairing between homologous chromosomes, whereas inhibition of transcription elongation or cohesin depletion has little effect on homolog pairing. The results suggest distinct roles for cohesin and condensin II in the establishment of 3D nuclear organization in Drosophila (Rowley, 2019).

Inter- and intra-chromosomal interactions among DNA-bound proteins establish patterns of chromatin organization detectable by Hi-C. The original low-resolution genome-wide Hi-C maps described the segregation of active and inactive chromatin into A and B compartments. Later, higher-resolution maps identified domains characterized by preferential intra- versus inter-domain contacts. Interaction domains have been described in different organisms and are commonly referred to as topologically associating domains (TADs). In addition to these features, intense point-to-point loops have been detected by high-resolution Hi-C in mammals. The anchors of these loops are enriched in CTCF and cohesin, and predominantly contain CTCF motifs in convergent orientation (Rowley, 2019).

CTCF loops are an important component of chromatin organization in vertebrates, yet plants and invertebrates either lack a homolog or CTCF does not appear to form stable loops. Instead, chromosomal domains in these organisms, including Drosophila, correspond to the transcriptional state of specific sequences in the genome. Borders between these domains form at discontinuities between active and inactive regions containing proteins and histone modifications characteristic of their transcriptional state. This pattern of 3D organization is similar to that observed in mammals after depletion of CTCF or Rad21 and has been studied in detail in Drosophila, where analyses of high-resolution Hi-C data show that chromatin is predominately organized by the fine-scale segregation of active and inactive chromatin into A and B compartmental domains (Rowley, 2017). Indeed, transcriptional state alone can be used to computationally simulate the experimental Hi-C interaction pattern at 1-kb resolution with great accuracy (Rowley, 2017). In further support for a role of transcription or factors associated with the transcriptional state of genes in chromatin organization, inhibition of transcription initiation and subsequent degradation of RNA polymerase II (RNAPII) using triptolide disrupts Drosophila compartmental domains and their interactions. Interestingly, the extent of disruption of 3D organization correlates with the levels of RNAPII after triptolide treatment. Drosophila Hi-C maps also show a few hundred punctate signals corresponding to specific point-to-point interactions, but these loops are not associated with CTCF. Instead, the loop anchors are enriched for developmental enhancers, Pc, and Rad21. It is unclear whether these Pc loops are formed by cohesin-mediated loop extrusion as it has been proposed for CTCF loops in mammals (Rowley, 2019).

In addition to inter- and intra-chromosomal interactions, Drosophila chromosomes participate in extensive pairing with their homologs. Pairing between homologs is responsible for the transvection phenomenon, which involves interactions between enhancers and promoters of genes located in two homologous chromosomes. Analysis of the extent of this pairing typically makes use of fluorescence in situ hybridization (FISH) probes hundreds of kilobases long, making it difficult to determine whether pairing occurs at discrete loci or in large regions. Several proteins have been shown to affect homolog pairing including condensin II, the levels of which are regulated by the SCFSlimb ubiquitin ligase. Depletion of Slimb increases levels of condensin II and decreases homolog pairing, while depletion of condensin II increases homolog pairing, suggesting that condensin II antagonizes chromosome pairing. While the role of condensin II in this aspect of nuclear organization is well known, its relationship to other aspects of chromosome organization is largely unexplored (Rowley, 2019).

This study examined the contribution of condensin II, cohesin, and the distribution of RNAPII to the establishment of various features of Drosophila 3D chromatin organization. Furthermore, analysis of homologous pairing interactions using Hi-C data suggests that pairing occurs at discrete loci with an average length of 6.4 kb enriched for architectural proteins. The results highlight the importance and distinct roles of RNAPII or other components of the transcription complex, cohesin, and condensin II in the establishment of nuclear organization (Rowley, 2019).

These results support a model of chromatin organization where RNAPII and cohesin promote interactions within genes to create small gene domains. Interactions between adjacent gene domains result in the formation of active compartmental domains, and interactions among these domains give rise to the characteristic plaid pattern of Hi-C heatmaps often referred to as the A compartment. The frequency of interactions within and between genes and A compartmental domains correlates with the amount of RNAPII and cohesin, which co-localize extensively in the genome. Because of this, the allocation of a specific sequence to the A compartment should not be done in absolute terms. Rather, sequences in the A compartment have different positive eigenvector values that correlate with the amount of RNAPII and cohesin. Contiguous sequences lacking RNAPII and cohesin have a negative eigenvector value and form B compartmental domains. Interactions among B compartmental domains in Drosophila are more infrequent compared to those among A compartmental domains, that is, the plaid pattern of Hi-C heatmaps in Drosophila arises in large part due to interactions between A compartments. However, sequences within B compartmental domains interact as frequently as those located in A domains. These interactions may arise as a consequence of proteins present in silenced genes. Alternatively, or in addition, interactions within B compartmental domains may result from interactions between adjacent A domains, which enclose B domains within loops similar to those formed by CTCF/cohesin in vertebrates. This is supported by results showing that inhibition of transcription initiation with triptolide or using the heat shock response, which result in the loss of A compartmental domains, also result in decreased interaction frequencies within B domains (Rowley, 2019).

These findings suggest that, whereas interaction frequency of sequences in active genes correlates with transcription elongation, it is likely that the presence of RNAPII, or other components of the transcription/elongation complexes, is a better candidate to explain the correlation between transcription and 3D organization. Inhibition of transcription results in dramatic changes to chromatin domains in Drosophila, yet transcription inhibition was reported to have little effect in mammalian embryonic nuclei. It is speculated that transcription inhibition studies in mammalian cells could be affected by the prevalence of CTCF loop domains. These loops may tether chromatin together such that inhibition of transcription for short periods of time is insufficient to disrupt chromatin organization. Meanwhile, in organisms that lack CTCF loops, such as Drosophila and prokaryotes, the larger effect of transcription inhibition may be due to the lack of point-to-point chromatin tethering by CTCF loops. It would be interesting to analyze whether absence of transcription or depletion of RNAPII with inhibitors such as triptolide have a stronger effect in cells depleted of CTCF (Rowley, 2019).

Previous results have shown a role for condensin II in chromatin structure during interphase. Condensin II colocalizes extensively with Drosophila architectural proteins, but in spite of the similar distribution, some observations suggest a distinct role for Cap-H2 in chromatin biology with respect to other architectural proteins. For example, all architectural proteins, including Rad21, are re-distributed during the heat shock response and they accumulate at enhancer sequences. However, the amount of enhancer-bound Cap-H2 and the number of occupied enhancers decreases after temperature stress. These observations may be explained by the opposing roles that condensin II and cohesin play in mediating intra-chromosomal interactions. Condensin II is present in active chromatin but it antagonizes the formation of gene domains and A compartmental domains, and condensin II depletion results in an increase to long-range A-A compartmental interactions. These results are in line with recent observations indicating that chromosome volume, as detected by Oligopaint, increases in Cap-H2 knockdown Drosophila cells (Rosin, 2018). The mechanisms by which these two SMC motors play opposing role in chromatin interactions is unclear. Presumably, their function in chromatin 3D organization is related to their ability to extrude loops, as was proposed for cohesin in mammals. Condensin has also been shown to extrude loops in vitro (Ganji, 2018), and it would be interesting to understand whether its role, opposite to that of cohesin, is based on different potential extrusion mechanisms between these two complexes. Thus, condensin II could antagonize cohesin interactions by directly inhibiting these same interactions or by promoting different interactions (Rowley, 2019).

Drosophila chromosomes participate in extensive homologous chromosome pairing, but the details of the mechanisms underlying this phenomenon are not well understood. Analysis of Hi-C data support a button model of pairing, where the buttons are short pairing sites likely corresponding to binding sites for specific proteins, rather than large domains. These pairing sites are enriched in architectural proteins, including Rad21 and Cap-H2. Although depletion of Rad21 only has no effect on pairing, it is possible that some architectural proteins may promote pairing while others act as anti-pairers, as is the case for Cap-H2. The general antagonistic role of condensin II in the establishment of interactions between homologs as well as short- and long-range intra-chromosomal contacts suggests common mechanisms responsible for these apparently different phenomena (Rowley, 2019).

Search PubMed for articles about Drosophila Cap-H2

Abe, S., et al. (2011). The initial phase of chromosome condensation requires Cdk1-mediated phosphorylation of the CAP-D3 subunit of condensin II. Genes Dev. 25(8): 863-74. PubMed ID: 21498573

Arya, G. H., Lodico, M. J., Ahmad, O. I., Amin, R. and Tomkiel, J. E. (2006). Molecular characterization of teflon, a gene required for meiotic autosome segregation in male Drosophila melanogaster. Genetics 174: 125-134. PubMed ID: 16816414

Hartl, T. A., Sweeney, S. J., Knepler, P. J. and Bosco, G. (2008a). Condensin II resolves chromosomal associations to enable anaphase I segregation in Drosophila male meiosis. PLoS Genet. 4(10): e1000228. PubMed ID: 18927632

Hartl, T. A., Smith, H. F. and Bosco, G. (2008b). Chromosome alignment and transvection are antagonized by condensin II. Science 322(5906): 1384-7. PubMed ID: 19039137

Hirano, T. (2005). Condensins: organizing and segregating the genome. Curr. Biol. 15: R265-R275. PubMed ID: 15823530

Hirano, T. (2006)/ At the heart of the chromosome: SMC proteins in action. Nat. Rev. Mol. Cell Biol. 311-322. PubMed ID: 16633335

Nguyen, H. Q., Nye, J., Buster, D. W., Klebba, J. E., Rogers, G. C. and Bosco, G. (2015). Drosophila Casein Kinase I alpha regulates homolog pairing and genome organization by modulating Condensin II subunit Cap-H2 levels. PLoS Genet 11: e1005014. PubMed ID: 25723539

Rosin, L. F., Nguyen, S. C. and Joyce, E. F. (2018). Condensin II drives large-scale folding and spatial partitioning of interphase chromosomes in Drosophila nuclei. PLoS Genet 14(7): e1007393. PubMed ID: 30001329

Rowley, M. J., Nichols, M. H., Lyu, X., Ando-Kuri, M., Rivera, I. S. M., Hermetz, K., Wang, P., Ruan, Y. and Corces, V. G. (2017). Evolutionarily conserved principles predict 3D chromatin organization. Mol Cell 67(5): 837-852. PubMed ID: 28826674

Rowley, M. J., Lyu, X., Rana, V., Ando-Kuri, M., Karns, R., Bosco, G. and Corces, V. G. (2019). Condensin II counteracts Cohesin and RNA Polymerase II in the establishment of 3D chromatin organization. Cell Rep 26(11): 2890-2903. PubMed ID: 30865881

Savvidou, E., Cobbe, N., Steffensen, S., Cotterill, S. and Heck, M. M. (2005). Drosophila CAP-D2 is required for condensin complex stability and resolution of sister chromatids. J. Cell Sci. 118: 2529-2543. PubMed ID: 15923665

Shintomi, K. and Hirano, T. (2011). The relative ratio of condensin I to II determines chromosome shapes. Genes Dev. 25(14): 1464-9. PubMed ID: 21715560

Thomas, S. E., et al. (2005). Identification of two proteins required for conjunction and regular segregation of achiasmate homologs in Drosophila male meiosis. Cell 123: 555-568. PubMed ID: 16286005

Tomkiel, J. E., Wakimoto, B. T. and Briscoe, A. (2001). The teflon gene is required for maintenance of autosomal homolog pairing at meiosis I in male Drosophila melanogaster. Genetics 157: 273-281. PubMed ID: 11139508

Yeong, F. M., et al. (2003). Identification of a subunit of a novel Kleisin-beta/SMC complex as a potential substrate of protein phosphatase 2A. Curr. Biol. 13: 2058-2064. PubMed ID: 14653995

date revised: 7 August 2019

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