InteractiveFly: GeneBrief

Chromosome associated protein D3: Biological Overview | References

Gene name - Chromosome associated protein D3

Synonyms -

Cytological map position - 25E5-25E5

Function - regulation of chromatin condensation

Keywords - Condensin II, chromosome assembly and segregation during mitosis and meiosis, regulation of anti-bacterial immune determinants, fat body, restriction of retrotransposon mobilization

Symbol - Cap-D3

FlyBase ID: FBgn0051989

Genetic map position - chr2L:5525905-5537110

Classification - Chromosome condensation complex Condensin, subunit D2; Cnd1: non-SMC mitotic condensation complex subunit 1

Cellular location - nuclear

NCBI link: EntrezGene

Cap-D3 orthologs: Biolitmine
Recent literature
Klebanow, L. R., Peshel, E. C., Schuster, A. T., De, K., Sarvepalli, K., Lemieux, M. E., Lenoir, J. J., Moore, A. W., McDonald, J. A. and Longworth, M. S. (2016). Drosophila Condensin II subunit, Chromosome Associated Protein-D3, regulates cell fate determination through non-cell autonomous signaling. Development [Epub ahead of print]. PubMed ID: 27317808
The pattern of the Drosophila melanogaster adult wing is heavily influenced by the expression of proteins that dictate cell fate decisions between intervein and vein during development. dSRF (Drosophila Serum Response Factor) expression in specific regions of the larval wing disc promotes intervein cell fate while EGFR (Epidermal Growth Factor Receptor) activity promotes vein cell fate. This study reports that the chromatin organizing protein, dCAP-D3, acts to dampen dSRF levels at the anterior/posterior (A/P) boundary in the larval wing disc, promoting differentiation of cells into the anterior crossvein (ACV). dCAP-D3 represses Knot expression in cells immediately adjacent to the A/P boundary, thus blocking Knot-mediated repression of EGFR activity and preventing cell death. Maintenance of EGFR activity in these cells depresses dSRF levels in the neighboring ACV progenitor cells, allowing them to differentiate into vein cells. These findings uncover a novel transcriptional regulatory network influencing Drosophila wing vein development, and are the first to identify a Condensin II subunit as an important regulator of EGFR activity and cell fate determination in vivo.
King, T. D., Leonard, C. J., Cooper, J. C., Nguyen, S., Joyce, E. F. and Phadnis, N. (2019). Recurrent losses and rapid evolution of the condensin II complex in insects. Mol Biol Evol. PubMed ID: 31270536
Condensins play a crucial role in the organization of genetic material by compacting and disentangling chromosomes. Based on studies in a few model organisms, the condensin I and condensin II complexes are considered to have distinct functions, with the condensin II complex playing a role in meiosis and somatic pairing of homologous chromosomes in Drosophila. Intriguingly, the Cap-G2 subunit of condensin II is absent in Drosophila melanogaster, and this loss may be related to the high levels of chromosome pairing seen in flies. This study finds that all three non-SMC subunits of condensin II (Cap-G2, Cap-D3, and Cap-H2) have been repeatedly and independently lost in taxa representing multiple insect orders, with some taxa lacking all three. All non-Dipteran insects display near-uniform low pairing levels regardless of their condensin II complex composition, suggesting that some key aspects of genome organization are robust to condensin II subunit losses. Finally, consistent signatures of positive selection were found in condensin subunits across flies and mammals. These findings suggest that these ancient complexes are far more evolutionarily labile than previously suspected, and are at the crossroads of several forms of genomic conflicts. These results raise fundamental questions about the specific functions of the two condensin complexes in taxa that have experienced subunit losses, and open the door to further investigations to elucidate the diversity of molecular mechanisms that underlie genome organization across various life forms.
Vernizzi, L. and Lehner, C. F. (2021). Bivalent individualization during chromosome territory formation in Drosophila spermatocytes by controlled condensin II protein activity and additional force generators. PLoS Genet 17(10): e1009870. PubMed ID: 34669718
Chromosome pairing during meiosis must avoid interlocking of non-homologous chromosomes. In spermatocytes of Drosophila, extensive non-homologous associations arise from the coalescence of the large blocks of pericentromeric heterochromatin into a chromocenter and from centromere clustering. Nevertheless, during territory formation, bivalents are moved apart into spatially separate subnuclear regions. The condensin II subunits, Cap-D3 and Cap-H2, have been implicated, but the remarkable separation of bivalents during interphase might require more than just condensin II. For further characterization of this process, time-lapse imaging was applied using fluorescent markers of centromeres, telomeres and DNA satellites in pericentromeric heterochromatin. The dynamics of the disruption of centromere clusters and the chromocenter are described in normal spermatocytes. Mutations in Cap-D3 and Cap-H2 abolish chromocenter disruption, resulting in excessive chromosome missegregation during meiosis I. Chromocenter persistence in the mutants is not mediated by the special system, which conjoins homologs in compensation for the absence of crossovers in Drosophila spermatocytes. However, overexpression of Cap-H2 precluded conjunction between autosomal homologs, resulting in random segregation of univalents. Interestingly, Cap-D3 and Cap-H2 mutant spermatocytes displayed conspicuous stretching of the chromocenter, as well as occasional chromocenter disruption, suggesting that territory formation might involve forces unrelated to condensin II. While the molecular basis of these forces remains to be clarified, they are not destroyed by inhibitors of F actin and microtubules. These results indicate that condensin II activity promotes chromosome territory formation in co-operation with additional force generators and that careful co-ordination with alternative homolog conjunction is crucial.

A conserved interaction between RB proteins and the Condensin II protein CAP-D3 is important for ensuring uniform chromatin condensation during mitotic prophase (Longworth, 2008). The Drosophila melanogaster homologs RBF1 and dCAP-D3 co-localize on non-dividing polytene chromatin, suggesting the existence of a shared, non-mitotic role for these two proteins. This study shows that the absence of RBF1 and dCAP-D3 alters the expression of many of the same genes in larvae and adult flies. Strikingly, most of the genes affected by the loss of RBF1 and dCAP-D3 are not classic cell cycle genes but are developmentally regulated genes with tissue-specific functions and these genes tend to be located in gene clusters. The data reveal that RBF1 and dCAP-D3 are needed in fat body cells to activate transcription of clusters of antimicrobial peptide (AMP) genes. AMPs are important for innate immunity, and loss of either dCAP-D3 or RBF1 regulation results in a decrease in the ability to clear bacteria. Interestingly, in the adult fat body, RBF1 and dCAP-D3 bind to regions flanking an AMP gene cluster both prior to and following bacterial infection. These results describe a novel, non-mitotic role for the RBF1 and dCAP-D3 proteins in activation of the Drosophila immune system and suggest dCAP-D3 has an important role at specific subsets of RBF1-dependent genes (Longworth, 2012).

Recent studies have suggested that pRB family members may impact the organization of higher-order chromatin structures, in addition to their local effects on the promoters of individual genes (Longworth, 2010). Mutation of pRB causes defects in pericentric heterochromatin (Isaac, 2006) and RBF1 is necessary for uniform chromatin condensation in proliferating tissues of Drosophila larvae (Longworth, 2008). Part of the explanation for these defects is that RBF1 and pRB promote the localization of the Condensin II complex protein, CAP-D3 to DNA both in Drosophila and human cells (Longworth, 2008). Depletion of pRB from human cells strongly reduces the level of CAP-D3 associated with centromeres during mitosis and causes centromere dysfunction (Longworth, 2012).

Condensin complexes are necessary for the stable and uniform condensation of chromatin in early mitosis. They are conserved from bacteria to humans with at least two types of Condensin complexes (Condensin I and II) present in higher eukaryotes. Both Condensin I and II complexes contain heterodimers of SMC4 and SMC2 proteins that form an ATPase which acts to constrain positive supercoils. Each type of Condensin also contains three specific non-SMC proteins that, upon phosphorylation, stabilize the complex and promote ATPase activity. The kleisin CAPH and two HEAT repeat containing subunits, CAP-G and CAP-D2 are components of Condensin I, while the kleisin CAP-H2 and two HEAT repeat containing subunits, CAP-G2 and CAP-D3, are constituents of Condensin II (Longworth, 2012).

Given the well-established functions of Condensins during mitosis, and of RBF1 in G1 regulation, the convergence of these two proteins was unexpected. Nevertheless, mutant alleles in the non-SMC components of Condensin II suppress RBF1-induced phenotypes, and immunostaining experiments revealed that RBF1 displays an extensive co-localization with dCAP-D3 (but not with dCAP-D2) on the polytene chromatin of Drosophila salivary glands (Longworth, 2008). This co-localization occurs in cells that will never divide, suggesting that Condensin II subunits and RBF1 co-operate in an unidentified process in non-mitotic cells. In various model organisms, the mutation of non-SMC Condensin subunits has been associated with changes in gene expression raising the possibility that dCAP-D3 may affect some aspect of transcriptional regulation by RBF1. However, the types of RBF1-regulated genes that might be affected by dCAP-D3, the contexts in which this regulation becomes important, and the consequences of losing this regulation are all unknown (Longworth, 2012).

This study identified sets of genes that are dependent on both rbf1 and dCap-D3. The majority of genes that show altered expression in both rbf1 and dCap-D3 mutants (larvae or adults) are not genes involved in the cell cycle, DNA repair, proliferation, but are genes with cell type-specific functions and many are spaced within 10 kb of one another in 'gene clusters'. To better understand this mode of regulation, the effects were investigated of RBF1 and dCAP-D3 on one of the most highly misregulated clusters which includes genes coding for antimicrobial peptides (AMPs). AMPs are produced in many organs, and one of the major sites of production is in the fat body. Following production in the fat body, AMPs are subsequently dumped into the hemolymph where they act to destroy pathogens. RBF1 and dCAP-D3 are required for the transcriptional activation of many AMPs in the adult fly. Analysis of one such gene cluster shows that RBF1 and dCAP-D3 bind directly to this region and that they bind, in the fat body, to sites flanking the locus. RBF1 and dCAP-D3 are both necessary in the fat body for maximal and sustained induction of AMPs following bacterial infection, and RBF1 and dCAP-D3 deficient flies have an impaired ability to respond efficiently to bacterial infection. These results identify dCAP-D3 as an important transcriptional regulator in the fly. Together, the findings suggest that RBF1 and dCAP-D3 regulate the expression of clusters of genes in post-mitotic cells, and this regulation has important consequences for the health of the organism (Longworth, 2012).

The idea that dCAP-D3 and RBF1 could cooperate to promote tissue development and differentiation is supported by the fact that both proteins are most highly expressed in the late stages of the fly life cycle, and accumulate at high levels in the nuclei of specific cell types in adult tissues. As an illustration of the cell-type specific nature of RBF1/dCAP-D3-regulation this study shows that dCAP-D3 and RBF1 are both required for the constituive expression of a large set of AMP genes in fat body cells. The loss of this regulation compromises pathogen-induction of gene expression and has functional consequences for innate immunity. Interestingly, different sets of RBF1/dCAP-D3-dependent genes were evident in the gene expression profiles of mutant larvae and adults. Given this, and the fact that the gene ontology classification revealed multiple groups of genes, it is suggested that the targets of RBF1/dCAP-D3-regulation do not represent a single transcriptional program, but diverse sets of cell-type specific programs that need to be activated (or repressed) in specific developmental contexts (Longworth, 2012).

The changes in gene expression seen in the mutant flies suggest that RBF1 has a significant impact on the expression of nearly half of the dCAP-D3-dependent genes. This fraction is consistent with previous data showing partial overlap between RBF1 and dCAP-D3 banding patterns on polytene chromatin, and the finding that chromatin-association by dCAP-D3 is reduced, but not eliminated, in rbf1 mutant animals and RBF1-depeleted cells. Although it has been previously shown that RBF1 and dCAP-D3 physically associate with one another (Longworth, 2008), and the current studies illustrate the fact that they each bind to similar sites at a direct target, the molecular events that mediate the co-operation between RBF1 and dCAP-D3 remain unknown (Longworth, 2012).

These results represent the first published ChIP data for the CAP-D3 protein in any organism. Although only a small number of targets were examined, it is interesting to note that the dCAP-D3 binding patterns are different for activated and repressed genes. More specifically, dCAP-D3 binds to an area within the open reading frame of a gene which it represses. However, dCAP-D3 binds to regions which flank a cluster of genes that it activates. Whether or not this difference in binding is true for all dCAP-D3 regulated genes will require a more global analysis (Longworth, 2012).

Human Condensin non-SMC subunits are capable of forming subcomplexes in vitro that are separate from the SMC protein- containing holocomplex (Kimura, 2000), but currently, the extent to which dCAP-D3 relies on the other members of the Condensin II complex remains unclear. It is noted that fat body cells contain polytene chromatin. Condensin II subunits have been shown to play a role in the organization of polytene chromatin in Drosophila nurse cells (Hartl, 2008a). Given that RB proteins physically interact with other members of the Condensin II complex (Longworth, 2008), it is possible that RBF1 and the entire Condensin II complex, including dCAP-D3, may be especially important for the regulation of transcription on this type of chromatin template (Longworth, 2012).

A potentially significant insight is that the genes that are deregulated in both rbf1 and dCap-D3 mutants tend to be present in clusters located within 10 kb of one another. This clustering effect seems to be a more general feature of regulation by dCAP-D3, which is enhanced by RBF1, since clustering was far more prevalent in the list of dCAP-D3 target genes than in the list of RBF1 target genes (Longworth, 2012).

These studies focussed on one of the most functionally related families of clustered target genes that were co-dependent on RBF1/dCAP-D3 for activation in the adult fly: the AMP family of genes. AMP loci represent 20% of the gene clusters regulated by RBF1 and dCAP-D3 in adults. ChIP analysis of one such region, a cluster of AMP genes at the diptericin locus, showed this locus to be directly regulated by RBF1 and dCAP-D3 in the fat body and revealed a pattern of RBF1 and dCAP-D3-binding that was very different from the binding sites typically mapped at E2F targets. Unlike the promoter-proximal binding sites typically mapped at E2F-regulated promoters, RBF1 and dCAP-D3 bound to two distant regions, one upstream of the promoter and one downstream of the diptericin B translation termination codon, a pattern that is suggestive of an insulator function. It is hypothesized that RBF1 and dCAP-D3 act to keep the region surrounding AMP loci insulated from chromatin modifiers and accessible to transcription factors needed for basal levels of transcription. The modEncode database shows binding sites for multiple insulator proteins, as well as GATA factor binding sites, at these regions. GATA has been previously implicated in transcriptional regulation of AMPs in the fly, and future studies of dCAP-D3 binding partners in Drosophila fat body tissue may uncover other essential activators. Additionally, the chromatin regulating complex, Cohesin, which exhibits an almost identical structure to Condensin, has been shown to promote looping of chromatin and to bind proteins with insulator functions (Wendt, 2008). Therefore, it remains a possibility that Condensin II, dCAP-D3 may actually possess insulator function, itself. It is proposed that dCAP-D3 may be functioning as an insulator protein, both insulating regions of DNA containing clusters of genes from the spread of histone marks and possibly looping these regions away from the rest of the body of chromatin. This would serve to keep the region in a 'poised state' available for transcription factor binding following exposure to stimuli that would induce activation. In the case of AMP genes, which are made constituitively in specific organs at low levels, dCAP-D3 would bind to regions flanking a cluster, and loop the cluster away from the body of chromatin. Upon systemic infection, these clusters would be more easily accessible to transcription factors like NF-κB. If dCAP-D3 is involved in looping of AMP clusters, then it may also regulate interchromosomal looping which could bring AMP clusters on different chromosomes closer together in 3D space, allowing for a faster and more coordinated activation of all AMPs (Longworth, 2012).

AMP expression is essential for the ability of the fly to recover from bacterial infection. Experiments with bacterial pathogens show that RBF1 and dCAP-D3 are both necessary for induction and maintenance of the AMP gene, drosomycin following infection, but only dCAP-D3 is necessary for the induction of the diptericin AMP gene. Similarly, survival curves indicate, that while dCAP-D3 deficient flies die more quickly in response to both Gram positive and Gram negative bacterial infection, RBF1 deficient flies die faster only in response to Gram positive bacterial infection. The differences seen between RBF1 and dCAP-D3 deficient flies in diptericin induction cannot be attributed to functional compensation by the other Drosophila RB protein family member, RBF2, since results show that loss of RBF2 or both RBF2 and RBF1 do not decrease AMP levels following infection. Since results demonstrate that RBF1 binds most strongly to an AMP cluster prior to infection and regulates basal levels of almost all AMPs tested, it is hypothesized that RBF1 (and possibly RBF2) may be more important for cooperating with dCAP-D3 to regulate basal levels of AMPs. Reports have shown that basal expression levels of various AMPs are regulated in a gene-, sex-, and tissue-specific manner, and it is thought that constitutive AMP expression may help to maintain a proper balance of microbial flora and/or help to prevent the onset of infections. In support of this idea, one study in Drosophila which characterized loss of function mutants for a gene called caspar, showed that caspar mutants increased constitutive transcript levels of diptericin but not transcript levels following infection. This correlated with increased resistance to septic infection with Gram negative bacteria, proving that changes in basal levels of AMPs do have significant effects on the survival of infected flies. Additionally, disruption of Caudal expression, a protein which suppresses NF-κB mediated AMP expression following exposure to commensal bacteria, causes severe defects in the mutualistic interaction between gut and commensal bacteria. It is therefore possible that RBF1 and dCAP-D3 may help to maintain the balance of microbial flora in specific organs of the adult fly and/or be involved in a surveillance-type mechanism to prevent the start of infection. RBF1 deficient flies also exhibit defects in Drosomycin induction following Gram positive bacterial infection. Mutation to Drosophila GNBP-1, an immune recognition protein required to activate the Toll pathway in response to infection with Gram positive bacteria has been show to result in decreased Drosomycin induction and decreased survival rates, without affecting expression of Diptericin. Therefore, it is possible that inefficient levels of Drosomycin, a major downstream effector of the Toll receptor pathway, combined with decreased basal transcription levels of a majority of the other AMPs, would cause RBF1 deficient flies to die faster following infection with Gram positive S. aureus but not Gram negative P. aeruginosa (Longworth, 2012).

Some dCAP-D3 remains localized to DNA in RBF1 deficient flies and it is also possible that other proteins may help to promote the localization of dCAP-D3 to AMP gene clusters following infection. Given that dCAP-D3 regulates many AMPs including some that do not also depend on RBF1 for activation, and given that dCAP-D3 binding to an AMP locus increases with time after infection whereas RBF1 binding is at its highest levels at the start of infection, it may not be too surprising that dCAP-D3 showed a more pronounced biological role in pathogen assays involving two different species of bacteria (Longworth, 2012).

Remarkably, and perhaps unexpectedly, the levels of both RBF1 and dCAP-D3 impact the basal levels of human AMP transcripts, as well. This indicates that the mechanism of RBF1/dCAP-D3 regulation may not be unique to Drosophila. It is striking that many of the human AMP genes (namely, the defensins) are clustered together in a region that spans approximately 1 Mb of DNA. It seems telling that both the clustering of these genes, and a dependence on pRB and CAP-D3, is apparently conserved from flies to humans. The fact that dCAP-D3 and RBF1 dependent activation of Drosomycin was necessary for resistance to Gram positive bacterial infection in flies suggests the same could also be true for the human orthologs in human cells. Human AMPs expressed by epithelial cells, phagocytes and neutrophils are an important component of the human innate immune system. Human AMPs are often downregulated by various microbial pathogenicity mechanisms upon infection. They have also been reported to play roles in the suppression of various diseases and maladies including cancer and Inflammatory Bowel Disease. It is noted that the chronic or acute loss of Rb expression from MEFs resulted in an unexplained decrease in the expression of a large number of genes that are involved in the innate immune system. In humans, the bacterium, Shigella flexneri was recently shown to down regulate the host innate immune response by specifically binding to the LXCXE cleft of pRB, the same site that was previously shown to be necessary for CAP-D3 binding (Zurawski, 2009). An improved understanding of how RB and CAP-D3 regulate AMPs in human cells may provide insight into how these proteins are able to regulate clusters of genes, and may also open up new avenues for therapeutic targeting of infection and disease. Further studies of in differentiated human cells may identify additional sets of genes that are regulated by pRB and CAP-D3 (Longworth, 2012).

Condensin II subunit dCAP-D3 restricts retrotransposon mobilization in Drosophila somatic cells

Retrotransposon sequences are positioned throughout the genome of almost every eukaryote that has been sequenced. As mobilization of these elements can have detrimental effects on the transcriptional regulation and stability of an organism's genome, most organisms have evolved mechanisms to repress their movement. This study has identified a novel role for the Drosophila melanogaster Condensin II subunit, dCAP-D3 in preventing the mobilization of retrotransposons located in somatic cell euchromatin. dCAP-D3 regulates transcription of euchromatic gene clusters which contain or are proximal to retrotransposon sequence. ChIP experiments demonstrate that dCAP-D3 binds to these loci and is important for maintaining a repressed chromatin structure within the boundaries of the retrotransposon and for repressing retrotransposon transcription. dCAP-D3 prevents accumulation of double stranded DNA breaks within retrotransposon sequence, and decreased dCAP-D3 levels leads to a precise loss of retrotransposon sequence at some dCAP-D3 regulated gene clusters and a gain of sequence elsewhere in the genome. Homologous chromosomes exhibit high levels of pairing in Drosophila somatic cells, and FISH analyses demonstrate that retrotransposon-containing euchromatic loci are regions which are actually less paired than euchromatic regions devoid of retrotransposon sequences. Decreased dCAP-D3 expression increases pairing of homologous retrotransposon-containing loci in tissue culture cells. It is proposed that the combined effects of dCAP-D3 deficiency on double strand break levels, chromatin structure, transcription and pairing at retrotransposon-containing loci may lead to (1) higher levels of homologous recombination between repeats flanking retrotransposons in dCAP-D3 deficient cells and (2) increased retrotransposition. These findings identify a novel role for the anti-pairing activities of dCAP-D3/Condensin II and uncover a new way in which dCAP-D3/Condensin II influences local chromatin structure to help maintain genome stability (Schuster, 2013).

This study shows that decreased levels of dCAP-D3/Condensin II lead to retrotransposon mobilization within specific gene clusters shown to be transcriptionally regulated by dCAP-D3. In tissue culture cells, the results demonstrate that homologous retrotransposon containing clusters remain largely unpaired which is in striking contrast to homologous euchromatic loci that do not contain retrotransposon sequences. Interestingly, the mobilization events detected both in vivo and in vitro resulted in either the retention of a single LTR at the locus or a precise loss of retrotransposon sequence in one locus and a small increase in copy number elsewhere in the genome. A model puts forth the hypothesis that dCAP-D3/Condensin II mediated looping of chromatin at homologous, euchromatic, retrotransposon containing loci holds the regions at distances great enough to prevent recombination. In dCAP-D3 deficient cells, this rigid chromatin structure is not maintained, possibly leading to increased double strand breaks within retrotransposon sequence. This in turn would cause an opening of chromatin in the region and would give homologous retrotransposon containing loci more of an opportunity to pair. Repair mechanisms that would lead to a local loss of retrotransposon sequence at one of the loci and a gain of a copy elsewhere in the genome include repair by the single strand annealing pathway or unequal crossover events between the small repeats found before and after the retrotransposon sequence. While these types of recombination repair do explain the local loss of sequence, they do not explain the small increase in copy number seen in dCAP-D3 deficient cells. Therefore, it is also proposed that, as a result of the opening of the chromatin at these loci, transcription increases and allows retrotransposon encoded retrotransposase enzyme to be made and generate additional copies. These new retrotransposition events would allow both original copies to remain in their loci and new copies to be generated and insert elsewhere. Supporting evidence for a role of Condensin II in regulating homologous crossover events comes from a recent study in C. elegans that worms heterozygous for Condensin II subunits exhibit increases in double strand breaks, increases in crossover events, and increases in X chromosome axis length in meiotic tissue (Tsai, 2008). The differential placement and number of double strand breaks in the C. elegans Condensin mutants were hypothesized to be caused by the changes in axial chromatin structure since axis lengths did not change in response to varying numbers of double strand breaks between mutants. Loss of Drosophila Condensin II subunits also lead to axial expansion (Bauer, 2012; Hartl, 2008a; Smith, 2013). Interestingly, the mdg1-1403 retrotransposon locus appears expanded in the dCAP-D3 mutants, and it is possible that this local expansion and change in chromatin structure could be the cause of the repositioning of double stand breaks. Finally, the loss of Condensin II expression results in disorganization of chromosome territories and intermingling of chromosomes in Drosophila cells (Hartl, 2008a). Therefore, it is also possible that the frequency of recombination between retrotransposon sequences on different chromosomes could increase, leading to loss of the remaining retrotransposon copy on one of the homologs in cells deficient for dCAP-D3 (Schuster, 2013).

The minor, but significant increases in retrotransposon transcript levels in somatic tissues and cells expressing lower levels of dCAP-D3 suggest that dCAP-D3 regulates global retrotransposon transcript levels. Previous studies have shown that dCAP-D3 regulates transcription of many genes in Drosophila larvae and adults, but the mechanism remains unclear (Longworth, 2012). Experiments in SG4 cells show that dCAP-D3 binds close to the junction between retrotransposon and neighboring DNA sequence. They also demonstrate that dCAP-D3 is necessary for maintaining basal transcription levels of retrotransposon-containing gene clusters prior to local loss of retrotransposon sequence. If dCAP-D3 acts to set up boundaries between a retrotransposon and neighboring DNA sequence, then binding sites located within the neighboring sequence could confer local specificity. In support of this, the data show an increased spreading of repressive H3K9me3 marks into the area surrounding retrotransposon mdg1-1403 in dCAP-D3 dsRNA treated cells. This data is also consistent with earlier findings that dCAP-D3 is a suppressor of Position Effect Variegation in somatic tissues (Longworth, 2008). Alternatively, the temporary increase in H3K9me3 at the locus prior to loss of retrotransposon sequence could be due to the increase in homolog pairing in dCAP-D3 knock down cells; silencing of extrachromosomal copies of genes proximal to transposons has been shown to increase when these regions pair. Transcription of genes surrounding mdg1-1403 increases above basal levels in dCAP-D3 dsRNA treated cells once the retrotransposon sequence is lost. Interestingly, even when dCAP-D3 expression levels return to normal, the increased transcription and increased levels of active H3K4me3 marks at the locus remain. It is also interesting to note that the band recognized by the mdg1-1403 probe in the dCAP-D3 mutant polytene chromatin squashes appeared longitudinally thicker and less condensed. This supports the model and suggests that the presence of the retrotransposon within the locus elicits a dCAP-D3-dependent structural configuration that is lost when the retrotransposon sequence is lost (Schuster, 2013).

Results presented in this study show that dCAP-D3 prevents increased γH2AX localization in retrotransposon sequence. Interestingly, human Brd4 isoform B was recently reported to bind to SMC2 and CAP-D3 proteins, and SMC2 was shown to be necessary for Brd4's ability to maintain a more condensed chromatin structure and inhibit DNA damage signaling following gamma irradiation (Floyd, 2013). This suggests 1) that the functions of Condensin II in DNA damage repair may be conserved in human cells, and 2) that Condensin II's role in repair most likely requires its ability to maintain rigid chromosome structure and organization. Recently, a role for Condensins in organizing retrotransposons within the nucleus was reported in yeast. Retrotransposons cluster in yeast and it was demonstrated that the Non-Homologous End Joining (NHEJ) repair associated Ku proteins as well as Condensin were both necessary for the observed clustering (Tanaka, 2012). The reported association between DNA repair proteins and Condensin is intriguing and might suggest, if the interaction was conserved in flies, that Condensins play a role in the actual repair of double strand breaks at retrotransposon sequences. However, no mass clustering is seen of the mdg1-1403 retrotransposon in Drosophila cells and the current studies show in Drosophila that Condensin-associated mechanisms exist to prevent retrotransposons on homologous chromosomes from coming into close contact. Furthermore, sequencing results indicate that either single strand annealing or unequal crossover events have occurred in dCAP-D3 mutants, instead of NHEJ mediated repair. These discrepancies might be attributed to the high degree of homologous chromosome pairing throughout the cell cycle in Drosophila. In fact, single strand annealing (even over NHEJ) has been shown to be the dominant double strand break repair pathway at transposon containing loci in Drosophila when direct repeats flank a double strand break. Additionally, yeast only possess Condensin I and not Condensin II, so it is possible that Condensin II has diverged to have different functions or even to antagonize Condensin I function at retrotransposon sequences (Schuster, 2013).

Interestingly, ChIP for phosphorylated H2AX in human cells expressing SMC2 RNAi showed that double strand breaks occur frequently within LTR sequences and a type of non-LTR retrotransposon, SINES (Samoshkin, 2012). Therefore, the ability of Condensin II to prevent double strand break accumulation and recombination within retrotransposon sequence may not be unique to Drosophila Condensin II. This has important implications for Condensin II as a possible tumor suppressor in human cells. Various types of tumor cells have been found to harbor mutations in Condensin II proteins including CAP-D3 (COSMIC database). While somatic homolog pairing is not as prevalent in human cells as in Drosophila, certain instances of abnormal pairing have been implicated in the generation of tumors. Further studies will be necessary to elucidate whether uncontrolled retrotransposon recombination and/or retrotransposition might play a role in the generation of genomic instability in human cells deficient for or expressing mutant Condensin II proteins (Schuster, 2013).

Condensin II resolves chromosomal associations to enable anaphase I segregation in Drosophila male meiosis

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 2nd and 3rd 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 2nd and 3rd 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 2nd, 3rd 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. However, it has been reported that one mutant Cap-D3 allele, Cap-D3EY00456 is homozygous viable, yet completely male sterile (Savvidou, 2005). This study has confirmed the necessity of Cap-D3 to male fertility; both Cap-D3EY00456 homozygous and Cap-D3EY00456/Cap-D3Df(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-H2Z3-0019/Cap-H2Df(3R)Exel6159, Cap-H2TH1/Cap-H2Df(3R)Exel6159, and Cap-H2TH1/Cap-H2Z3-0019 to wild-type females. A third allele, Cap-H2Z3-5163, is fertile as a homozygote and in trans-combinations with Cap-H2Z3-0019, Cap-H2Df(3R)Exel6159, and Cap-H2TH1 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-H2Z3-0019 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-H2Z3-0019 heterozygous control males where the seminal vesicles fill with sperm, those from Cap-H2Z3-0019 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-H2Z3-0019/Cap-H2Z3-5163, 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-H2Z3-0019/Cap-H2Z3-5163 and Cap-H2Z3-5163/+ 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-H2Z3-5163/+ and SMC408819/+; Cap-H2Z3-0019/+ 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-H2Z3-0019/Cap-H2Z3-5163 and corresponding Cap-H2 heterozygous controls males were crossed to females bearing compound X chromosomes [C(1)RM, y2 su(wa)wa]. 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 4th chromosome marker mated to females bearing compound 4th chromosomes (C(4)EN, ci ey). As with the sex chromosome segregation assays, 4th chromosome segregation did not differ substantially between the Cap-H2Z3-0019/Cap-H2Z3-5163 and heterozygous control males. The possibility remains that this hypomorphic Cap-H2 allelic combination is not strong enough to reveal 4th chromosome segregation defects. Like sex chromosomes, 4th 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 2nd and 3rd chromosomes had a heightened sensitivity to Cap-H2 mutation as Cap-H2Z3-0019/Cap-H2Z3-5163 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 2nd (nullo-2) or 3rd (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 2nd chromosome assays were bred to be heterozygous for the 2nd chromosome marker brown (bw1). If both 2nd 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 2nd 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 2nd chromosomes (bw1/bw1 or +/+). There was a trend toward an elevated level of the bw1/+ exceptional class from both Cap-H2Z3-0019/Cap-H2Z3-5163 and Cap-H2Z3-0019/+ 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 2nd and 3rd chromosome assays relative to the sex and 4th may only represent a heightened sensitivity of these chromosomes rather then a role for Cap-H2 specifically in 2nd and 3rd chromosome segregation. In fact, defects in sex and 4th 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 2nd (60.8 Mb) and 3rd (68.8 Mb) relative to the X, Y, and 4th 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 2nd and 3rd (Hartl, 2008a).

An alternative hypothesis involves the fact that 2nd 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 2nd chromosome relative to the sex bivalent. Assuming the 3rd and 4th chromosomes maintain homolog pairing like the 2nd, then the relative amount of DNA utilized in conjunction is as follows: 3rd>2nd>4th>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 2nd, 3rd, and sex chromosomal bivalents and are thought to have important chromosome organizational roles for meiosis I. In male sterile mutants of the genotype Cap-H2Z3-0019/Cap-H2TH1, 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-D3EY00456 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-H2Z3-0019/Cap-H2Z3-5163 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 2nd, 3rd 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-H2Z3-0019/Cap-H2TH1 males, 11.5% for Cap-H2Z3-0019/Cap-H2Z3-5163 males that are fertile yet undergo 2nd and 3rd chromosome loss, 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-D3EY00456 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 4th 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 2nd and 3rd 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-H2Z3-0019/Cap-H2TH1 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-H2Z3-0019/Cap-H2TH1 testes, 21.4% appeared to have anaphase I bridging that existed between homologous chromosomes. A FISH probe that recognizes 2nd chromosome pericentromeric heterochromatin was used to distinguish 2nd and 3rd chromosomes and demonstrates that linkages were between the 3rd chromosomes, perhaps at regions of shared homology. Furthermore, despite not finding 4th chromosome segregation defects in nondisjunction assays, the 4th 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-H2Z3-0019/Cap-H2TH1 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-H2Z3-0019/Cap-H2TH1 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 (2nd or 3rd). It is speculated that the substantially bridged images are comprised of associations between heterologous and/or homologous chromosomes. One example was particularly interesting because the 4th 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 2nd and 3rd chromosomes had a heightened sensitivity to Cap-H2 mutation (Hartl, 2008a).

Because the 4th 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, 4th-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 4th 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-H2Z3-0019 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 2nd 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 2nd and 3rd 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-H2Z3-0019/Cap-H2TH1 anaphase II figures, 2.1% of those from Cap-H2Z3-0019/Cap-H2Z3-5163 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-H2Z3-0019/Cap-H2TH1 males were bridged, bridging existed within only 10.8% of anaphase I figures from tefZ2-5549/tefZ2-5864; Cap-H2Z3-0019/Cap-H2TH1 males. Furthermore, in squashed preparations anaphase I bridging was decreased from 40.5% in Cap-H2Z3-0019/Cap-H2TH1 males to 25.6% in the tefZ2-5549/tefZ2-5864; Cap-H2Z3-0019/Cap-H2TH1 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-H2Z3-0019/Cap-H2TH1 mutants to 9.3% in tefZ2-5549/tefZ2-5864; Cap-H2Z3-0019/Cap-H2TH1 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 tefZ2-5549/tefZ2-5864; Cap-H2Z3-0019/Cap-H2TH1 males were uninterpretable making it impossible to assess whether Cap-H2 mutant heterologous anaphase I bridging was also rescued by teflon mutation. However, 4th-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 tefZ2-5549/tefZ2-5864 rescued Cap-H2Z3-0019/Cap-H2TH1 anaphase I bridging to levels near that of fertile Cap-H2 mutants, yet tefZ2-5549/tefZ2-5864; Cap-H2Z3-0019/Cap-H2TH1 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 4th 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 4th 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 2nd and 3rd require the greatest functional Cap-H2 dose for their proper segregation. This sensitivity of the 2nd and 3rd 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 4th chromosome missegregation, while the other autosomes segregate normally (Arya, 2006). This suggests that the 4th 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-H2Z3-0019/Cap-H2Z3-5163 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 UbxCbx-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 UbxCbx-1 allele is rendered null through the introduction of a second mutation (UbxCbx-1 Ubx1). This UbxCbx-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 UbxCbx-1 Ubx1/++ phenotype was dominantly enhanced by Cap-H2 mutations. Conversely, Cap-H2 overexpression suppressed the UbxCbx-1 Ubx1/++ wing phenotype closer to wild-type (Hartl, 2008b).

Cap-H2 mutant enhancement of the UbxCbx-1 Ubx1 phenotype was suppressed in a chromosomal rearrangement background [R(UbxCbx-1 Ubx1)/++] that is thought to disrupt allelic associations between UbxCbx-1 Ubx1 and wild-type Ubx. The UbxCbx-1 Ubx1/Cap-H2- and R(UbxCbx-1 Ubx1)/Cap-H2- flies only vary by the reciprocal translocation that moves 3R bearing UbxCbx-1 Ubx1 to 2R and vice versa. This suggests that Cap-H2 enhancement of the UbxCbx-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 y82f29/y82f29 and yy1#8/yy1#8 flies, there is minimal cuticle pigmentation, yet when placed in trans to one another (y82f29/yy1#8) complementation occurs with partial restoration of pigment nearer to wild-type levels. The yy1#8 allele is a deletion of the yellow promoter and the y82f29 allele a deletion of upstream enhancer elements. It is thought that partial complementation occurs in y82f29/yy1#8 through the ability of yy1#8's enhancers to act in trans, to associate with the intact promoter of y82f29, and then to activate yellow transcription. As are UbxCbx-1 Ubx1, transvection of y82f29/yy1#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).

RBF1 promotes chromatin condensation through a conserved interaction with the Condensin II protein dCAP-D3

The Drosophila retinoblastoma family of proteins (RBF1 and RBF2) and their mammalian homologs (pRB, p130, and p107) are best known for their regulation of the G1/S transition via the repression of E2F-dependent transcription. However, RB family members also possess additional functions. This study reports that rbf1 mutant larvae have extensive defects in chromatin condensation during mitosis. A novel interaction is described between RBF1 and dCAP-D3, a non-SMC component of the Condensin II complex that links RBF1 to the regulation of chromosome structure. RBF1 physically interacts with dCAP-D3, RBF1 and dCAP-D3 partially colocalize on polytene chromosomes, and RBF1 is required for efficient association of dCAP-D3 with chromatin. dCap-D3 mutants also exhibit chromatin condensation defects, and mutant alleles of dCap-D3 suppress cellular and developmental phenotypes induced by the overexpression of RBF1. Interestingly, this interaction is conserved between flies and humans. The re-expression of pRB into a pRB-deficient human tumor cell line promotes chromatin association of hCAP-D3 in a manner that depends on the LXCXE-binding cleft of pRB. These results uncover an unexpected link between pRB/RBF1 and chromatin condensation, providing a mechanism by which the functional inactivation of RB family members in human tumor cells may contribute to genome instability (Longworth, 2008).

Previous studies have shown that non-SMC proteins are important for the function of Condensin complexes (Hirano, 2005; Nasmyth, 2005). While the SMC proteins contain the ATPase domains that are essential for DNA supercoiling, non-SMC proteins like CAP-D2 and CAP-D3 are thought to help target Condensins to chromatin. Condensin I and Condensin II complexes are known to be selectively recruited to different chromosomal locations (Ono, 2004; Savvidou, 2005), but the mechanisms responsible for specificity have been elusive. The results described in this study show that RB- family members are important for the chromatin association by dCAP-D3, a specific component of the Condensin II complex. One of the most striking features of these data is the specificity of the interactions. RBF1 interacts consistently with dCAP-D3 but not with dCAP-D2. This specificity is illustrated by the polytene staining experiments showing that RBF1 colocalizes with dCAP-D3 but not with dCAP-D2. A similar specificity was observed in the genetic assays, in binding experiments, and in immunostaining experiments and was seen in experiments in both flies and human cells (Longworth, 2008).

While the lack of interaction with dCAP-D2 provides a valuable negative control, it also suggests that there is an important distinction between the two Condensin subunits and, by inference, between the two Condensin complexes. When tested in Xenopus extracts, the depletion of Condensin I resulted in decondensation of sperm chromatin with no chromosome formation while depletion of Condensin II primarily affected the morphology of chromosomes, resulting in kinked chromosomes that lacked the normal degree of rigidity. These differences indicate that Condensin I and Condensin II play different roles in the organization of chromosome structure, but precisely how the functions of these two complexes differ, or how their functions are integrated, is not well understood. One idea, suggested by studies in mammalian cells showing that Condensin II associates with chromatin prior to nuclear envelope breakdown (NEB), whereas Condensin I complexes are excluded from the nucleus and only gain access to chromatin following NEB, is that Condensin II complexes perform the initial compaction of the chromosome in prophase, and that Condensin I helps to complete the process. However, in Drosophila, both Condensin I and II proteins have been found to localize with chromatin prior to NEB and this temporal distinction is less certain. The fact that the two Condensin complexes localize to distinct regions of euchromatin suggests that they determine the architecture of different subdomains. If this model is correct, then this implies that RB family members are important for the structural organization of particular regions of the chromosome. This may explain why the abnormally condensed chromosomes seen in rbf1 mutant animals contain interspersed segments of condensed and hypocondensed chromatin. In a more general sense, this link between RB proteins and chromosome organization suggests that RB family members are poised to control global changes in chromatin architecture, a position that may help them to act as global regulators of proliferation and differentiation (Longworth, 2008).

It is noted that several studies have described roles for Condensins outside of mitosis. The Caenorhabditis elegans SMC4 homolog DPY-27 is required for the regulation of the dosage compensation complex and the transcriptional repression of the autosomal male sex determination gene, her-1. Non-SMC subunits, which have been shown to form complexes independent of the SMC heterodimer, can also effect gene expression. dCAP-G, (a member of both Condensin I and II) has been shown to effect position effect variegation (PEV), suggesting that it may have a role in maintenance of heterochromatin. An interaction between murine CAP-G2 (MTB) and the SCL and E12 transcription factors allows MTB to repress transcription during erythoid cell development. Recently, a kleisin β mutation in mice was shown to be responsible for defects in T-lymphocyte differentiation. It is possible, therefore, that the interaction between RB family members and CAP-D3 protein may be used, in part, to control gene expression, and that the changes in chromosome condensation seen when RBF1 levels are altered may be a consequence of too little/too much chromatin-associated CAP-D3 that was initially recruited for a different purpose, such as transcriptional regulation (Longworth, 2008).

Interestingly, this study found that mutant alleles of dCap-D3 suppress PEV, supporting the idea that this complex can affect chromatin states. dCAP-D3 and RBF1 do not colocalize with bands of constitutive heterochromatin, which stain strongly with DAPI, but are found in the lighter DAPI-stained or non-DAPI-stained regions, raising the possibility that they act together in areas of repressed euchromatin or facultative heterochromatin. Clearly, further studies are needed to identify the regions of dCAP-D3 that are important for this interaction and to identify the chromatin elements that are bound by dCAP-D3/RBF1 (Longworth, 2008).

Several of the current results raise the intriguing possibility that RBF1 may target a subpopulation of dCAP-D3 that acts independently of dSMC proteins. It is striking that the UAS-RBF1 phenotype was not modified by mutant alleles of dsmc2 or dsmc4. Moreover, antibodies to dSMC4 failed to stain several bands on polytene chromosomes that were costained with antibodies to dCAP-D3 and RBF1, and, while a clear decrease was seen in the levels of dCAP-D3 associated with chromatin in the absence of RBF1, no clear reduction was seen in the level of dSMC4. Possibly, dCAP-D3 may exist in several different complexes, some of which contain dSMC proteins and some that do not, and the activity that is targeted by RBF1 may be different from the traditional view of Condensin complexes. Alternatively, since dSMC2 and dSMC4 are components of both Condensin I and Condensin II, one could argue that the lack of interaction with dsmc2 or dsmc4 and the failure to see a clear reduction in dSMC4 levels when RBF1 was removed are simply due to redundancy between the two Condensin complexes. The overlap between dSMC4 and dCAP-D3 staining on polytene chromosomes was surprisingly high and it is possible that the dSMC4 antibody marks some Condensin complexes better than others. Since dSMC4 coimmunoprecipitates with RBF1, since SMC4 binds to Rb, p107, and p130 fusion proteins in vitro, since many of the dCAP-D3/RBF bands on the polytene chromosomes also costain with dSMC4, and mutant alleles of dCap-H2 and dCap-G also modified the UAS-rbf1 phenotype, it seems likely that this activity of RBF1 involves its interaction with multiple components of the Condensin II complex. However, further studies are needed to fully characterize the RBF1/dCAP-D3 complexes and to determine which of the Condensin II proteins are important for this activity (Longworth, 2008).

Analysis of polytene chromosomes shows that RBF1 is needed for chromatin localization of CAP-D3 in interphase cells. In experiments carried out in human cells, the earliest point in the cell cycle when the ability of pRB to promote chromatin association by dCAP-D3 was late anaphase, the point in the cell cycle where pRB becomes dephosphorylated and associates tightly with chromatin. This, together with the observation that recombinant RB polypeptides associate with hCAP-D3 and the finding that the interaction between RB and hCAP-D3 was disrupted by mutations that mimic phosphorylation, leads to a proposal that RBF1/RB proteins recruit CAP-D3 to DNA at the times when they are traditionally thought to act. While the possibility that a subpopulation of RBF1 interacts with dCAP-D3 later in the cell cycle cannot be excluded, the changes in chromatin condensation in rbf1 mutants are most likely a consequence of reduced dCAP-D3 localization earlier in the cell cycle (Longworth, 2008).

The best-known functions of the RB family proteins in both mammalian cells and Drosophila involve interactions with E2F transcription factors. However neuroblast squashes of rbf1/dDP mutants and either dE2F2 or dDP-null larvae show that the hypocondensation caused by the loss of RBF1 does not require dE2F/dDP activity and cannot be generated by simply inactivating dE2F/dDP complexes. In agreement with this, it was found that the striking colocalization of RBF1 and dCAP-D3 is maintained on polytene squashes from dDP-null larvae. This does not preclude the possibility that dCAP-D3 and RBF1 complex may interact in the vicinity of dE2F-regulated promoters, or that the dCAP-D3/RBF1 interaction may be influenced by changes in E2F activity. Clearly though, these results show that RBF1 recruits dCAP-D3 to chromatin in a manner that does not depend on the conventional model of E2F/DP DNA-binding activity. Many different transcription factors have been shown to interact with RB family members, and there is no shortage of possibilities for such an E2F-independent function. Intriguingly, DNMT3b and HDAC1, which have been linked previously to pRB, have also been found to associate with both SMC4 and SMC2 (Geiman, 2004). This raises the possibility that the interaction between Condensin II proteins and RBF1 may involve one or more of the chromatin-associated complexes that have been connected previously to pRB (Longworth, 2008).

The results described in this study provide a very simple explanation for the aneuploidy and anaphase defects observed in Rb-/- and RbΔLXCXE MEFs. Since the inactivation of Condensin II complexes causes global changes in chromatin structure, these findings may also explain the balloon-like, butterfly chromosomes described in TKO MEFs or the changes in chromatin structure previously noted in Rb-/- MEFs (Longworth, 2008 and references therein).

pRB is one of the most well-studied tumor suppressor proteins and is mutated or inactivated in many types of human cancer. The idea that RB family members are required for normal Condensin II function provides a simple route through which the functional inactivation of RB family proteins, either by deletion, phosphorylation, or by viral proteins that bind to the LXCXE-binding cleft, will promote genomic instability. Indeed, there are several independent lines of evidence implicating Condensin activity in tumorigenesis. Mutations in Condensin subunits SMC2 and SMC4 have been identified in cases of pyothorax-associated lymphoma. Strikingly, a recent study of 102 early-onset breast cancer patients showed that 53% of cases had loss of heterozygosity (LOH) occurring in the region that includes the hCAP-D3 locus. This LOH also correlated with higher tumor grade and a more unfavorable prognosis. These observations seem particularly significant when one considers that heterozygosity for dCAP-D3 was not only sufficient to suppress RBF1-induced phenotypes but also caused visible defects in chromatin condensation and segregation. Given the evidence that RB family members promote CAP-D3 association with chromatin and the evidence that LOH at the CAP-D3 locus occurs frequently in breast cancer, it is suggested that Condensin II function is likely to be compromised in many different human tumor cells (Longworth, 2008).


Search PubMed for articles about Drosophila Cap-D3

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

Bauer, C. R., Hartl, T. A. and Bosco, G. (2012). Condensin II promotes the formation of chromosome territories by inducing axial compaction of polyploid interphase chromosomes. PLoS Genet 8: e1002873. PubMed ID: 22956908

Floyd, S. R., et al. (2013). The bromodomain protein Brd4 insulates chromatin from DNA damage signalling. Nature 498: 246-250. PubMed ID: 23728299

Geiman, T. M., Sankpal, U. T., Robertson, A. K., Chen, Y., Mazumdar, M., Heale, J. T., Schmiesing, J. A., Kim, W., Yokomori, K., Zhao, Y. and Robertson, K. D. (2004). Isolation and characterization of a novel DNA methyltransferase complex linking DNMT3B with components of the mitotic chromosome condensation machinery. Nucleic Acids Res 32: 2716-2729. PubMed ID: 15148359

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

Isaac, C. E., Francis, S. M., Martens, A. L., Julian, L. M., Seifried, L. A., Erdmann, N., Binne, U. K., Harrington, L., Sicinski, P., Berube, N. G., Dyson, N. J. and Dick, F. A. (2006). The retinoblastoma protein regulates pericentric heterochromatin. Mol Cell Biol 26: 3659-3671. PubMed ID: 16612004

Kimura, K. and Hirano, T. (2000). Dual roles of the 11S regulatory subcomplex in condensin functions. Proc Natl Acad Sci U S A 97: 11972-11977. PubMed ID: 11027308

Longworth, M. S., Herr, A., Ji, J. Y. and Dyson, N. J. (2008). RBF1 promotes chromatin condensation through a conserved interaction with the Condensin II protein dCAP-D3. Genes Dev 22: 1011-1024. PubMed ID: 18367646

Longworth, M. S. and Dyson, N. J. (2010). pRb, a local chromatin organizer with global possibilities. Chromosoma 119: 1-11. PubMed ID: 19714354

Longworth, M. S., Walker, J. A., Anderssen, E., Moon, N. S., Gladden, A., Heck, M. M., Ramaswamy, S. and Dyson, N. J. (2012). A shared role for RBF1 and dCAP-D3 in the regulation of transcription with consequences for innate immunity. PLoS Genet 8: e1002618. PubMed ID: 22496667

Nasmyth, K. and Haering, C. H. (2005). The structure and function of SMC and kleisin complexes. Annu Rev Biochem 74: 595-648. PubMed ID: 15952899

Ono, T., Fang, Y., Spector, D. L. and Hirano, T. (2004). Spatial and temporal regulation of Condensins I and II in mitotic chromosome assembly in human cells. Mol Biol Cell 15: 3296-3308. PubMed ID: 15146063

Samoshkin, A., Dulev, S., Loukinov, D., Rosenfeld, J. A. and Strunnikov, A. V. (2012). Condensin dysfunction in human cells induces nonrandom chromosomal breaks in anaphase, with distinct patterns for both unique and repeated genomic regions. Chromosoma 121: 191-199. PubMed ID: 22179743

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

Schuster, A. T., Sarvepalli, K., Murphy, E. A. and Longworth, M. S. (2013). Condensin II Subunit dCAP-D3 Restricts Retrotransposon Mobilization in Drosophila Somatic Cells. PLoS Genet 9: e1003879. PubMed ID: 24204294

Smith, H. F., Roberts, M. A., Nguyen, H. Q., Peterson, M., Hartl, T. A., Wang, X. J., Klebba, J. E., Rogers, G. C. and Bosco, G. (2013). Maintenance of interphase chromosome compaction and homolog pairing in Drosophila is regulated by the condensin cap-h2 and its partner Mrg15. Genetics 195: 127-146. PubMed ID: 23821596

Tanaka, A., Tanizawa, H., Sriswasdi, S., Iwasaki, O., Chatterjee, A. G., Speicher, D. W., Levin, H. L., Noguchi, E. and Noma, K. (2012). Epigenetic regulation of condensin-mediated genome organization during the cell cycle and upon DNA damage through histone H3 lysine 56 acetylation. Mol Cell 48: 532-546. PubMed ID: 23084836

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

Tsai, C. J., Mets, D. G., Albrecht, M. R., Nix, P., Chan, A. and Meyer, B. J. (2008). Meiotic crossover number and distribution are regulated by a dosage compensation protein that resembles a condensin subunit. Genes Dev 22: 194-211. PubMed ID: 18198337

Wendt, K. S., Yoshida, K., Itoh, T., Bando, M., Koch, B., Schirghuber, E., Tsutsumi, S., Nagae, G., Ishihara, K., Mishiro, T., Yahata, K., Imamoto, F., Aburatani, H., Nakao, M., Imamoto, N., Maeshima, K., Shirahige, K. and Peters, J. M. (2008). Cohesin mediates transcriptional insulation by CCCTC-binding factor. Nature 451: 796-801. PubMed ID: 18235444

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

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Biological Overview

date revised: 11 January 2014

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