InteractiveFly: GeneBrief

Stromalin 2: Biological Overview | References

For meiosis I, homologous chromosomes must be paired into bivalents. Maintenance of homolog conjunction in bivalents until anaphase I depends on crossovers in canonical meiosis. However, instead of crossovers, an alternative system achieves homolog conjunction during the achiasmate male meiosis of Drosophila melanogaster. The proteins SNM, UNO and MNM are likely constituents of a physical linkage that conjoins homologs in D. melanogaster spermatocytes. This study reports that SNM (Stromalin 2) binds tightly to the C-terminal region of UNO. This interaction is homologous to that of the cohesin subunits stromalin/Scc3/STAG and α-kleisin, as revealed by sequence similarities, structure modeling and cross-link mass spectrometry. Importantly, purified SU_C, the heterodimeric complex of SNM and the C-terminal region of UNO, displayed DNA-binding in vitro. DNA-binding was severely impaired by mutational elimination of positively charged residues from the C-terminal helix of UNO. Phenotypic analyses in flies fully confirmed the physiological relevance of this basic helix for chromosome-binding and homolog conjunction during male meiosis. Beyond DNA, SU_C also bound MNM, one of many isoforms expressed from the complex mod(mdg4) locus. This binding of MNM to SU_C was mediated by the MNM-specific C-terminal region, while the purified N-terminal part common to all Mod(mdg4) isoforms multimerized into hexamers in vitro. Similarly, the UNO N-terminal domain formed tetramers in vitro. Thus, it is suggested that multimerization confers to SUM, the assemblies composed of SNM, UNO and MNM, the capacity to conjoin homologous chromosomes stably by the resultant multivalent DNA-binding. Moreover, to permit homolog separation during anaphase I, SUM is dissociated by separase, since UNO, the α-kleisin-related protein, includes a separase cleavage site. In support of this proposal, this study demonstrates that UNO cleavage by tobacco etch virus protease is sufficient to release homolog conjunction in vivo after mutational exchange of the separase cleavage site with that of the bio-orthogonal protease (Kabakci, 2022a).

Drosophila male meiosis is achiasmate and therefore dependent on dedicated proteins (SNM (Stromalin 2), UNO (univalents only) and MNM (modifier of mdg4), together referred to as SUM, that maintain conjunction between homologous chromosomes in replacement for the missing crossovers. The main findings provide insight into the biochemical basis of (1) how the SUM proteins achieve this alternative homolog conjunction (AHC), and (2) how AHC is eliminated in time at the transition from metaphase to anaphase of M I to permit separation of the homologs to opposite spindle poles. In addition, these results are informative concerning the evolution of the AHC system (Kabakci, 2022a).

SNM and the C-terminal domain of UNO form a stable heterodimeric complex (SU_C). Based on sequence comparisons, AlphaFold structural predictions and XL-MS with recombinantly expressed and purified proteins, the SU_C complex is homologous to that formed by stromalin and the stromalin-binding region of α-kleisin. Stromalins and α-kleisins are components of cohesin complexes. While SNM was recognized as highly similar to stromalins early on, the very limited similarity of UNO to α-kleisins has escaped detection until now. The important and conserved N- and C-terminal domains of α-kleisins, which mediate its binding to the SMC heterodimer in cohesin, are not present in UNO. From an α-kleisin precursor, UNO has thus retained only the stromalin-binding region and the previously identified separase cleavage site (Kabakci, 2022a).

Stromalin, via positively charged surface patches, has recently been shown to promote DNA-binding of cohesin in vitro. Purified SU_C also binds DNA. At least one of stromalin's positively charged surface patches [43] is clearly also present in SNM and contributes to the DNA-binding of SU_C, according to in vitro analysis with mutant versions of SU_C. In addition, a conspicuous, positively charged α-helix at the very C-terminus of UNO, which is absent from α-kleisins, makes a contribution to the DNA-binding of SU_C that is even more important than the basic SNM patch. Apart from DNA-binding, the interactions with the other AHC proteins were still normal in case of UNOchm-EGFP, a mutant with acidic or neutral residues in place of the six basic residues in the C-terminal α-helix. In vivo, UNOchm-EGFP displayed strongly reduced chromosome-binding and failed to provide normal AHC during male meiosis. These results strongly argue for the physiological importance of the DNA-binding activity of SU_C. It is speculated that the C-terminal α-helix of UNO might clamp down on a DNA double helix bound to the basic surface patches of SNM and thereby strongly increase the strength of DNA-binding. The binding of SU_C to DNA does not appear to be sequence specific. Clearly, in competition with the scrambled DNA sequence, this study has not detected increased binding to the 240 bp repeat sequence from the rDNA intergenic spacers, which appears to mediate sex chromosome conjunction (Kabakci, 2022a).

Beyond DNA, SU_C binds to MNM. Neither SNM nor UNO interact with MNM individually, indicating that prior association of SNM and UNO is required for MNM binding. These conclusions are based on co-immunoprecipitation experiments after transient expression in S2R+ cells. Of note, this study has not accomplished SUM complex formation with purified proteins in vitro so far. Attempts at expressing and purifying full length MNM were not successful. Moreover, the successfully purified C-terminal region of MNM (MNM_C), which mediates the binding to SU_C according to co-immunoprecipitation experiments, did not bind to SU_C in vitro. It is conceivable, therefore, that binding of MNM to SU depends on prior post-translational processing steps. At present, the inability to generate SUM complexes in vitro precludes a straightforward clarification of the issue whether SU can bind simultaneously to both MNM and DNA. However, the extended contacts between the C-terminal domain of UNO and SNM over long stretches provide ample space with interface potential, thereby increasing the likelihood of simultaneous binding of MNM and DNA to SU (Kabakci, 2022a).

MNM_C mediates binding not only to SU_C but also to TEF, as revealed by the co-immunoprecipitation experiments. The MNM-TEF interaction also remains to be re-constituted with purified proteins in vitro. However, in case of MNM_C, simultaneous binding of both TEF and SU_C is not feasible according to co-immunoprecipitation experiments (Kabakci, 2022a).

Beyond the interaction domains discussed above, analyses demonstrated the presence of multimerization domains in both UNO and MNM. It ia suggested that these domains are likely of crucial importance for the molecular mechanism whereby the SUM proteins generate AHC. In case of UNO, the N-terminal domain (UNO_N), which is highly conserved in UNO homologs, self-associates, forming dimers and tetramers when expressed and purified from bacteria. This UNO_N region has a predicted structure that is very distinct from that of the conserved N-terminal region of α-kleisins, indicating that the evolution of uno involved substitution of N-terminal in addition to deletion of C-terminal coding sequences in an ancestral α-kleisin gene. Multimerization in case of MNM is also mediated by the N-terminal region MNM_N. The primary sequence of MNM_N is identical to that of the N-terminal region present in the considerable number of alternative isoforms expressed from the complex mod(mdg4) locus. This common part of the Mod(mdg4) protein isoforms (thus also designated as Mod(mdg4)_CP) contains a BTB/POZ domain. This protein interaction domain present in many eukaryotic proteins with diverse functions has been shown to mediate homomeric dimerization. In addition, in case of the particular type of BTB domain that is also present in the Mod(mdg4) protein products, heteromeric and higher order multimerization has been reported based on SEC, native gel electrophoresis and crosslinking studies. These results confirm and extend these findings. Purified MNM_N/Mod(mdg4)_CP was found to form stable hexamers according to SEC-MALS. The hexamers were readily modeled by AlphaFold2 as a ring-like complex with three dimers, and negative-stain electron microscopy revealed ring-like complexes of an appropriate dimension. A recent preprint describing similar structural analyses of the same type of BTB domains (i.e. the TTK-type) derived from other Drosophila proteins (Lola and CG6765) and also from Mod(mdg4) provides further confirmation of the ring-shaped hexameric structure (Kabakci, 2022a).

Because of the multimerization domains (UNO_N and MNM_N/Mod(mdg4)_CP), the SUM proteins have presumably the potential to form extended protein assemblies that include many copies of the SU_C DNA-binding site. Thereby, they might be empowered to effectively and stably conjoin distinct double-stranded DNA molecules and function as a chromosome glue. Accordingly, AHC would not rely on a topological ring-like embrace as proposed to be provided by cohesin in case of sister chromatid cohesion. Since the tetramers and hexamers formed in vitro by purified UNO_N and MNM_N/Mod(mdg4)_CP are stable, SUM protein assemblies formed on bivalents in spermatocytes are expected to adopt a more solid rather than a liquid state. Indeed, FRAP analyses confirmed that the SUM proteins in the dots associated with the sex chromosome pairing regions do not undergo dynamic exchange (Kabakci, 2022a).

The proposed extended SUM protein assemblies with their multitude of DNA-binding sites are unlikely to conjoin exclusively homologous DNA strands. Presumably, sister DNA strands are connected as well (and perhaps even neighboring regions on the same strand). Previous characterizations of meiotic mutant phenotypes are consistent with this view. Absence of AHC function results in premature separation of bivalents into univalents in late spermatocytes and early in M I. The SOLO (Sisters on the loose) and SUNN (Sister unbound) proteins, which appear to function similar to the Rec8 cohesin complexes of other eukaryotes, still assure in these univalents a functional unification of sister centromeres for organization of a single kinetochore unit, as well as well as pericentromeric sister chromatid cohesion. In solo and sunn mutants, sister centromeres and pericentromeric regions lack cohesion, but bivalents are still present until the onset of anaphase I. As sister chromatid cohesion within the regions of chromosome arms is normally lost after territory formation already during spermatocyte maturation, the presence of bivalents in solo and sunn mutants during early M I suggests that the SUM proteins conjoin not just homologous chromatids but also sister chromatids. In support of this interpretation, snm solo double mutants display univalents during early M I. It is emphasizes that a chromosomal glue that conjoins DNA strands indiscriminately, as proposed for the SUM protein assemblies (i.e., sister strands and homologous strands in trans and perhaps also neighboring regions in cis) should be perfectly adequate if it is applied at the right time during spermatocyte maturation, i.e., after disruption of non-homologous chromosomal associations by territory formation but before complete disruption of homolog associations (Kabakci, 2022a).

Clearly, the proposal that AHC relies on extended assemblies of SUM proteins providing a high number of DNA-binding sites remains speculative and requires further investigation. For example, understanding how the formation of SUM protein assemblies is controlled and restricted to limited chromosomal regions will be crucial. The mechanism whereby SUM protein assemblies are targeted to the sex chromosome pairing rDNA loci on chromosome X and Y remains unexplained. In case of autosomal bivalents, TEF is likely involved in the initial establishment of SUM protein assemblies. However, after ectopic expression in larval salivary glands, TEF as well as SUM bind to a large number of polytene chromosome bands In contrast, in mature S6 spermatocytes, the autosomal SUM protein assemblies are spatially restricted to one or two dots per chromosome arm. Targeting of SUM protein assemblies to the sex chromosome pairing site and into autosomal dots might involve interactions with additional chromosomal proteins. Mod(mdg4)_T (also designated as 67.2 or 2.2), the most extensively characterized isoform expressed from the complex mod(mdg4) locus, interacts and co-operates with several chromatin architectural proteins (including CP190, HIPP1 and SuHw) at the gypsy insulator. Moreover, Mod(mdg4)_T in combinations with chromatin architectural proteins in various combinations is generally enriched at boundaries between topologically associated chromatin domains and also at button loci that promote the somatic pairing of homologous chromosomes. Recently, Mod(mdg4) function has been implicated in a striking example of chromosome pairing-dependent regulation of physiological gene expression. Thus, multimerization by Mod(mdg4)_CP is likely crucial for chromosomal associations other than AHC during male meiosis. Accordingly, by recruitment of the Mod(mdg4)_H isoform MNM for AHC, evolution might have co-opted a pre-adaption that achieves chromosomal associations by Mod(Mdg4)_CP multimerization (Kabakci, 2022a).

While Mod(mdg4) isoforms other than MNM were found to be unable of binding to SU, these other isoforms clearly have the potential to form heteromeric associations with MNM according to co-immunoprecipitation experiments. Moreover, based on yeast two-hybrid (Y2H) analyses, various other proteins with TTK-type BTB domains might also form heteromeric associations with MNM. Whether such heteromeric interactions are relevant of AHC remains to be clarified. However, phenotypic analyses with various mod(mdg4) alleles have argued against contributions to AHC by Mod(mdg4) isoforms other than MNM. Moreover, while heteromeric associations of Mod(mdg4)_T with other Mod(mdg4) isoforms can readily be detected by Y2H and co-immunoprecipitation after overexpression in S2 cells, their occurrence on chromosomes without overexpression is questionable according to chromatin-immunoprecipitation (Kabakci, 2022a).

Importantly, AHC must provide conjunction between homologs in bivalents that is very robust and yet also amenable to rapid and complete elimination after biorientation of all the bivalents in the M I spindle, so that homologs can be separated to opposite poles during anaphase I. Efficient destructibility of AHC was achieved by the evolutionary co-option of the α-kleisin-derived protein UNO. Like α-kleisin, UNO includes a separase cleavage site that is highly conserved among UNO orthologs. This cleavage site was shown to be required for AHC elimination and homolog separation during anaphase I. By exchanging the separase cleavage site in UNO with that cleaved by the bio-orthogonal TEV protease, this study provides evidence that UNO cleavage is indeed sufficient to eliminate AHC. In these experiments, TEV was expressed under control of cis-regulatory sequences from exu or betaTub85D in mid spermatocytes. The presence of normal chromosome territories and of a normal subcellular localization of UNOTEV-EGFP at the onset of TEV expression indicated that this TEV expression occurred after successful AHC establishment, which occurs early during spermatocyte maturation. However, as a consequence of TEV expression, bivalents were prematurely converted into univalents, as clearly indicated by cytological analyses and by time lapse imaging of progression into and through M I. UNO cleavage separates the multimerization domain UNO_N from UNO_C, which mediates DNA-binding in conjunction with SNM. Therefore, it is proposed that UNO cleavage dissociates the chromosomal SUM protein assemblies to an extent where the number of associated DNA-binding sites is no longer sufficient for tight linkage of distinct double-stranded DNA molecules. Clearly, alternative mechanisms of AHC elimination by UNO cleavage remain conceivable, and further work will be required to clarify the mechanistic details of AHC and its elimination (Kabakci, 2022a).

Homologous chromosomes are stably conjoined for Drosophila male meiosis I by SUM, a multimerized protein assembly with modules for DNA-binding and for separase-mediated dissociation co-opted from cohesin

For meiosis I, homologous chromosomes must be paired into bivalents. Maintenance of homolog conjunction in bivalents until anaphase I depends on crossovers in canonical meiosis. However, instead of crossovers, an alternative system achieves homolog conjunction during the achiasmate male meiosis of Drosophila melanogaster. The proteins SNM, UNO and MNM are likely constituents of a physical linkage that conjoins homologs in D. melanogaster spermatocytes. This study reports that SNM binds tightly to the C-terminal region of UNO. This interaction is homologous to that of the cohesin subunits stromalin/Scc3/STAG and α-kleisin, as revealed by sequence similarities, structure modeling and cross-link mass spectrometry. Importantly, purified SU_C, the heterodimeric complex of SNM and the C-terminal region of UNO, displayed DNA-binding in vitro. DNA-binding was severely impaired by mutational elimination of positively charged residues from the C-terminal helix of UNO. Phenotypic analyses in flies fully confirmed the physiological relevance of this basic helix for chromosome-binding and homolog conjunction during male meiosis. Beyond DNA, SU_C also bound MNM, one of many isoforms expressed from the complex mod(mdg4) locus. This binding of MNM to SU_C was mediated by the MNM-specific C-terminal region, while the purified N-terminal part common to all Mod(mdg4) isoforms multimerized into hexamers in vitro. Similarly, the UNO N-terminal domain formed tetramers in vitro. Thus, it is suggested that multimerization confers to SUM, the assemblies composed of SNM, UNO and MNM, the capacity to conjoin homologous chromosomes stably by the resultant multivalent DNA-binding. Moreover, to permit homolog separation during anaphase I, SUM is dissociated by separase, since UNO, the α-kleisin-related protein, includes a separase cleavage site. In support of this proposal, this study demonstrates that UNO cleavage by tobacco etch virus protease is sufficient to release homolog conjunction in vivo after mutational exchange of the separase cleavage site with that of the bio-orthogonal protease (Kabakci, 2022b).

Drosophila male meiosis is achiasmate and therefore dependent on dedicated proteins (SNM (Stromalin 2), UNO (univalents only) and MNM (modifier of mdg4), together referred to as SUM, that maintain conjunction between homologous chromosomes in replacement for the missing crossovers. The main findings (summarized in Fig 7F) provide insight into the biochemical basis of (1) how the SUM proteins achieve this alternative homolog conjunction (AHC), and (2) how AHC is eliminated in time at the transition from metaphase to anaphase of M I to permit separation of the homologs to opposite spindle poles. In addition, these results are informative concerning the evolution of the AHC system (Kabakci, 2022b).

SNM and the C-terminal domain of UNO form a stable heterodimeric complex (SU_C). Based on sequence comparisons, AlphaFold structural predictions and XL-MS with recombinantly expressed and purified proteins, the SU_C complex is homologous to that formed by stromalin and the stromalin-binding region of α-kleisin. Stromalins and α-kleisins are components of cohesin complexes. While SNM was recognized as highly similar to stromalins early on, the very limited similarity of UNO to α-kleisins has escaped detection until now. The important and conserved N- and C-terminal domains of α-kleisins, which mediate its binding to the SMC heterodimer in cohesin, are not present in UNO. From an α-kleisin precursor, UNO has thus retained only the stromalin-binding region and the previously identified separase cleavage site (Kabakci, 2022b).

Stromalin, via positively charged surface patches, has recently been shown to promote DNA-binding of cohesin in vitro. Purified SU_C also binds DNA. At least one of stromalin's positively charged surface patches [43] is clearly also present in SNM and contributes to the DNA-binding of SU_C, according to in vitro analysis with mutant versions of SU_C. In addition, a conspicuous, positively charged α-helix at the very C-terminus of UNO, which is absent from α-kleisins, makes a contribution to the DNA-binding of SU_C that is even more important than the basic SNM patch. Apart from DNA-binding, the interactions with the other AHC proteins were still normal in case of UNOchm-EGFP, a mutant with acidic or neutral residues in place of the six basic residues in the C-terminal α-helix. In vivo, UNOchm-EGFP displayed strongly reduced chromosome-binding and failed to provide normal AHC during male meiosis. These results strongly argue for the physiological importance of the DNA-binding activity of SU_C. It is speculated that the C-terminal α-helix of UNO might clamp down on a DNA double helix bound to the basic surface patches of SNM and thereby strongly increase the strength of DNA-binding. The binding of SU_C to DNA does not appear to be sequence specific. Clearly, in competition with the scrambled DNA sequence, this study has not detected increased binding to the 240 bp repeat sequence from the rDNA intergenic spacers, which appears to mediate sex chromosome conjunction (Kabakci, 2022b).

Beyond DNA, SU_C binds to MNM. Neither SNM nor UNO interact with MNM individually, indicating that prior association of SNM and UNO is required for MNM binding. These conclusions are based on co-immunoprecipitation experiments after transient expression in S2R+ cells. Of note, this study has not accomplished SUM complex formation with purified proteins in vitro so far. Attempts at expressing and purifying full length MNM were not successful. Moreover, the successfully purified C-terminal region of MNM (MNM_C), which mediates the binding to SU_C according to co-immunoprecipitation experiments, did not bind to SU_C in vitro. It is conceivable, therefore, that binding of MNM to SU depends on prior post-translational processing steps. At present, the inability to generate SUM complexes in vitro precludes a straightforward clarification of the issue whether SU can bind simultaneously to both MNM and DNA. However, the extended contacts between the C-terminal domain of UNO and SNM over long stretches provide ample space with interface potential, thereby increasing the likelihood of simultaneous binding of MNM and DNA to SU (Kabakci, 2022b).

MNM_C mediates binding not only to SU_C but also to TEF, as revealed by the co-immunoprecipitation experiments. The MNM-TEF interaction also remains to be re-constituted with purified proteins in vitro. However, in case of MNM_C, simultaneous binding of both TEF and SU_C is not feasible according to co-immunoprecipitation experiments (Kabakci, 2022b).

Beyond the interaction domains discussed above, analyses demonstrated the presence of multimerization domains in both UNO and MNM. It ia suggested that these domains are likely of crucial importance for the molecular mechanism whereby the SUM proteins generate AHC. In case of UNO, the N-terminal domain (UNO_N), which is highly conserved in UNO homologs, self-associates, forming dimers and tetramers when expressed and purified from bacteria. This UNO_N region has a predicted structure that is very distinct from that of the conserved N-terminal region of α-kleisins, indicating that the evolution of uno involved substitution of N-terminal in addition to deletion of C-terminal coding sequences in an ancestral α-kleisin gene. Multimerization in case of MNM is also mediated by the N-terminal region MNM_N. The primary sequence of MNM_N is identical to that of the N-terminal region present in the considerable number of alternative isoforms expressed from the complex mod(mdg4) locus. This common part of the Mod(mdg4) protein isoforms (thus also designated as Mod(mdg4)_CP) contains a BTB/POZ domain. This protein interaction domain present in many eukaryotic proteins with diverse functions has been shown to mediate homomeric dimerization. In addition, in case of the particular type of BTB domain that is also present in the Mod(mdg4) protein products, heteromeric and higher order multimerization has been reported based on SEC, native gel electrophoresis and crosslinking studies. These results confirm and extend these findings. Purified MNM_N/Mod(mdg4)_CP was found to form stable hexamers according to SEC-MALS. The hexamers were readily modeled by AlphaFold2 as a ring-like complex with three dimers, and negative-stain electron microscopy revealed ring-like complexes of an appropriate dimension. A recent preprint describing similar structural analyses of the same type of BTB domains (i.e. the TTK-type) derived from other Drosophila proteins (Lola and CG6765) and also from Mod(mdg4) provides further confirmation of the ring-shaped hexameric structure (Kabakci, 2022b).

Because of the multimerization domains (UNO_N and MNM_N/Mod(mdg4)_CP), the SUM proteins have presumably the potential to form extended protein assemblies that include many copies of the SU_C DNA-binding site. Thereby, they might be empowered to effectively and stably conjoin distinct double-stranded DNA molecules and function as a chromosome glue. Accordingly, AHC would not rely on a topological ring-like embrace as proposed to be provided by cohesin in case of sister chromatid cohesion. Since the tetramers and hexamers formed in vitro by purified UNO_N and MNM_N/Mod(mdg4)_CP are stable, SUM protein assemblies formed on bivalents in spermatocytes are expected to adopt a more solid rather than a liquid state. Indeed, FRAP analyses confirmed that the SUM proteins in the dots associated with the sex chromosome pairing regions do not undergo dynamic exchange (Kabakci, 2022b).

The proposed extended SUM protein assemblies with their multitude of DNA-binding sites are unlikely to conjoin exclusively homologous DNA strands. Presumably, sister DNA strands are connected as well (and perhaps even neighboring regions on the same strand). Previous characterizations of meiotic mutant phenotypes are consistent with this view. Absence of AHC function results in premature separation of bivalents into univalents in late spermatocytes and early in M I. The SOLO (Sisters on the loose) and SUNN (Sister unbound) proteins, which appear to function similar to the Rec8 cohesin complexes of other eukaryotes, still assure in these univalents a functional unification of sister centromeres for organization of a single kinetochore unit, as well as well as pericentromeric sister chromatid cohesion. In solo and sunn mutants, sister centromeres and pericentromeric regions lack cohesion, but bivalents are still present until the onset of anaphase I. As sister chromatid cohesion within the regions of chromosome arms is normally lost after territory formation already during spermatocyte maturation, the presence of bivalents in solo and sunn mutants during early M I suggests that the SUM proteins conjoin not just homologous chromatids but also sister chromatids. In support of this interpretation, snm solo double mutants display univalents during early M I. It is emphasizes that a chromosomal glue that conjoins DNA strands indiscriminately, as proposed for the SUM protein assemblies (i.e., sister strands and homologous strands in trans and perhaps also neighboring regions in cis) should be perfectly adequate if it is applied at the right time during spermatocyte maturation, i.e., after disruption of non-homologous chromosomal associations by territory formation but before complete disruption of homolog associations (Kabakci, 2022b).

Clearly, the proposal that AHC relies on extended assemblies of SUM proteins providing a high number of DNA-binding sites remains speculative and requires further investigation. For example, understanding how the formation of SUM protein assemblies is controlled and restricted to limited chromosomal regions will be crucial. The mechanism whereby SUM protein assemblies are targeted to the sex chromosome pairing rDNA loci on chromosome X and Y remains unexplained. In case of autosomal bivalents, TEF is likely involved in the initial establishment of SUM protein assemblies. However, after ectopic expression in larval salivary glands, TEF as well as SUM bind to a large number of polytene chromosome bands In contrast, in mature S6 spermatocytes, the autosomal SUM protein assemblies are spatially restricted to one or two dots per chromosome arm. Targeting of SUM protein assemblies to the sex chromosome pairing site and into autosomal dots might involve interactions with additional chromosomal proteins. Mod(mdg4)_T (also designated as 67.2 or 2.2), the most extensively characterized isoform expressed from the complex mod(mdg4) locus, interacts and co-operates with several chromatin architectural proteins (including CP190, HIPP1 and SuHw) at the gypsy insulator. Moreover, Mod(mdg4)_T in combinations with chromatin architectural proteins in various combinations is generally enriched at boundaries between topologically associated chromatin domains and also at button loci that promote the somatic pairing of homologous chromosomes. Recently, Mod(mdg4) function has been implicated in a striking example of chromosome pairing-dependent regulation of physiological gene expression. Thus, multimerization by Mod(mdg4)_CP is likely crucial for chromosomal associations other than AHC during male meiosis. Accordingly, by recruitment of the Mod(mdg4)_H isoform MNM for AHC, evolution might have co-opted a pre-adaption that achieves chromosomal associations by Mod(Mdg4)_CP multimerization (Kabakci, 2022b).

While Mod(mdg4) isoforms other than MNM were found to be unable of binding to SU, these other isoforms clearly have the potential to form heteromeric associations with MNM according to co-immunoprecipitation experiments. Moreover, based on yeast two-hybrid (Y2H) analyses, various other proteins with TTK-type BTB domains might also form heteromeric associations with MNM. Whether such heteromeric interactions are relevant of AHC remains to be clarified. However, phenotypic analyses with various mod(mdg4) alleles have argued against contributions to AHC by Mod(mdg4) isoforms other than MNM. Moreover, while heteromeric associations of Mod(mdg4)_T with other Mod(mdg4) isoforms can readily be detected by Y2H and co-immunoprecipitation after overexpression in S2 cells, their occurrence on chromosomes without overexpression is questionable according to chromatin-immunoprecipitation (Kabakci, 2022b).

Importantly, AHC must provide conjunction between homologs in bivalents that is very robust and yet also amenable to rapid and complete elimination after biorientation of all the bivalents in the M I spindle, so that homologs can be separated to opposite poles during anaphase I. Efficient destructibility of AHC was achieved by the evolutionary co-option of the α-kleisin-derived protein UNO. Like α-kleisin, UNO includes a separase cleavage site that is highly conserved among UNO orthologs. This cleavage site was shown to be required for AHC elimination and homolog separation during anaphase I. By exchanging the separase cleavage site in UNO with that cleaved by the bio-orthogonal TEV protease, this study provides evidence that UNO cleavage is indeed sufficient to eliminate AHC. In these experiments, TEV was expressed under control of cis-regulatory sequences from exu or betaTub85D in mid spermatocytes. The presence of normal chromosome territories and of a normal subcellular localization of UNOTEV-EGFP at the onset of TEV expression indicated that this TEV expression occurred after successful AHC establishment, which occurs early during spermatocyte maturation. However, as a consequence of TEV expression, bivalents were prematurely converted into univalents, as clearly indicated by cytological analyses and by time lapse imaging of progression into and through M I. UNO cleavage separates the multimerization domain UNO_N from UNO_C, which mediates DNA-binding in conjunction with SNM. Therefore, it is proposed that UNO cleavage dissociates the chromosomal SUM protein assemblies to an extent where the number of associated DNA-binding sites is no longer sufficient for tight linkage of distinct double-stranded DNA molecules. Clearly, alternative mechanisms of AHC elimination by UNO cleavage remain conceivable, and further work will be required to clarify the mechanistic details of AHC and its elimination (Kabakci, 2022b).

A genome-wide screen identifies genes required for centromeric cohesion

During meiosis, two chromosome segregation phases follow a single round of DNA replication. This study identified factors required to establish this specialized cell cycle by examining meiotic chromosome segregation in a collection of yeast strains lacking all nonessential genes. This analysis revealed Sgo1, Chl4, and Iml3 to be important for retaining centromeric cohesin until the onset of anaphase II. Consistent with this role, Sgo1 localizes to centromeric regions but dissociates at the onset of anaphase II. The screen described in this study provides a comprehensive analysis of the genes required for the meiotic cell cycle and identifies three factors important for the stepwise loss of sister chromatid cohesion (Marston, 2004).

Cohesion between sister chromatids is essential for their bi-orientation on mitotic spindles. It is mediated by a multisubunit complex called cohesin. In yeast, proteolytic cleavage of cohesin's alpha kleisin subunit at the onset of anaphase removes cohesin from both centromeres and chromosome arms and thus triggers sister chromatid separation. In animal cells, most cohesin is removed from chromosome arms during prophase via a separase-independent pathway involving phosphorylation of its Stromalin 1/Stromalin 2 (Scc3-SA1/2) subunits. Cohesin at centromeres is refractory to this process and persists until metaphase, whereupon its alpha kleisin subunit is cleaved by separase, which is thought to trigger anaphase. What protects centromeric cohesin from the prophase pathway? Potential candidates are proteins, known as shugoshins, that are homologous to Drosophila Mei-S332 and yeast Sgo1 proteins, which prevent removal of meiotic cohesin complexes from centromeres at the first meiotic division. A vertebrate shugoshin-like protein associates with centromeres during prophase and disappears at the onset of anaphase. Its depletion by RNA interference causes HeLa cells to arrest in mitosis. Most chromosomes bi-orient on a metaphase plate, but precocious loss of centromeric cohesin from chromosomes is accompanied by loss of all sister chromatid cohesion, the departure of individual chromatids from the metaphase plate, and a permanent cell cycle arrest, presumably due to activation of the spindle checkpoint. Remarkably, expression of a version of Stromalin 1/Stromalin 2 (Scc3-SA2) whose mitotic phosphorylation sites have been mutated to alanine alleviates the precocious loss of sister chromatid cohesion and the mitotic arrest of cells lacking shugoshin. These data suggest that shugoshin, and by inference its Drosophila homolog Mei-S332, prevents phosphorylation of cohesin's Scc3-SA2 subunit at centromeres during mitosis. This ensures that cohesin persists at centromeres until activation of separase causes cleavage of its alpha kleisin subunit. Centromeric cohesion is one of the hallmarks of mitotic chromosomes. These results imply that it is not an intrinsically stable property, because it can easily be destroyed by mitotic kinases, which are kept in check by shugoshin (Marston, 2004).

SOLO: a meiotic protein required for centromere cohesion, coorientation, and SMC1 localization in Drosophila melanogaster

Sister chromatid cohesion is essential to maintain stable connections between homologues and sister chromatids during meiosis and to establish correct centromere orientation patterns on the meiosis I and II spindles. However, the meiotic cohesion apparatus in Drosophila remains largely uncharacterized. Sisters on the loose (SOLO), a novel splice product of Vasa, is essential for meiotic cohesion in Drosophila. In solo mutants, sister centromeres separate before prometaphase I, disrupting meiosis I centromere orientation and causing nondisjunction of both homologous and sister chromatids. Centromeric foci of the cohesin protein SMC1 are absent in solo mutants at all meiotic stages. SOLO and SMC1 colocalize to meiotic centromeres from early prophase I until anaphase II in wild-type males, but both proteins disappear prematurely at anaphase I in mutants for mei-S332, which encodes the Drosophila homologue of the cohesin protector protein Shugoshin. The solo mutant phenotypes and the localization patterns of SOLO and SMC1 indicate that they function together to maintain sister chromatid cohesion in Drosophila meiosis (Yan, 2010).

Meiosis is a specialized cell division that functions in sexual reproduction to generate haploid gametes from diploid precursor cells. It consists of two divisions preceded by a single round of DNA replication. Meiosis I is a reductional division in which homologous chromosomes (homologues) segregate to opposite spindle poles. Meiosis II is an equational division in which sister chromatids separate (Yan, 2010).

Two key differences in chromosome behavior underlie the different segregation patterns in meiosis I and II. One is the manner in which segregating chromosomes are connected. Stable connections between segregating chromosomes are essential to prevent them from separating prematurely and to provide the tension required to enable the chromosomes to achieve bipolar alignment on the spindle. In meiosis II, as in mitosis, the critical connections are cohesion between sister centromeres. Cohesion is established during replication and preserved throughout the cell cycle until its removal at the onset of anaphase (anaphase II of meiosis). In meiosis I, stable connections between homologues must be established. In most organisms, these connections take the form of chiasmata, which derive from crossovers between homologous chromatids and which are stabilized by cohesion between sister chromatid arms distal to the crossover sites. Thus, sister chromatid cohesion underlies the connections between segregating chromosomes in both meiotic divisions. However, in some eukaryotes, such as Drosophila males, homologue exchange and chiasmata are absent. In Drosophila, homologue connections are provided by the male meiosis-specific chromosomal proteins stromalin in meiosis (SNM) and mod(mdg4) in meiosis (Yan, 2010).

Cohesion is mediated by a conserved cohesin complex consisting of one member each of the SMC1, SMC3, SCC1/RAD21/REC8, and SCC3/SA families. Proteolytic cleavage of the SCC1 subunit (or its meiotic paralogue REC8) of cohesin at anaphase by separase triggers chromosome segregation during mitosis and at both meiotic divisions. In meiosis, this requires two separate rounds of separase activation: one round at anaphase I to cleave arm cohesins, release chiasmata, and allow homologues to segregate, and a second round at anaphase II to cleave centromere cohesin and allow sister chromatids to segregate. Conserved centromeric proteins called shugoshins function to protect centromeric cohesins from premature cleavage by separase during anaphase I (Yan, 2010).

A second critical difference between meiosis I and II is the orientation adopted by sister centromeres. In meiosis II, as in mitosis, sister centromeres orient back to back and establish separate kinetochores that make independent attachments to spindle poles. In meiosis I, sister centromeres adopt a side by side orientation and collaborate in forming a single functional kinetochore, ensuring that only two functional kinetochores are present per bivalent despite the presence of four chromatids. This enables sister centromeres to coorient (become attached to spindle fibers emanating from the same pole), which in turn enables homologous centromeres to biorient. The mechanism of sister centromere coorientation is not well understood. In Saccharomyces cerevisiae, it depends on a centromeric meiosis I -- specific complex called monopolin. The role of cohesin in centromere orientation is unclear. In Schizosaccharomyces pombe, coorientation requires both meiosis-specific Rec8 cohesin and MoaI, a specialized centromere protein that appears to function primarily by stabilizing occupancy of centromere core sequences by Rec8 cohesin. Recently, it has been shown that provision of an artificial tether between sister centromere core sequences suffices for preferential sister centromere coorientation in the absence of Rec8 or MoaI. The mechanism of coorientation in higher eukaryotes is not known in any detail, but the fact that rec8 mutations disrupt centromere orientation in several model eukaryotes suggests a role for cohesin (Yan, 2010).

Although there is considerable evidence that the aforementioned two-stage, cohesin-based meiotic segregation mechanism is widely conserved, the role of cohesin in meiotic cohesion in Drosophila remains unclear. This is due in large part to the absence of a functional rec8 orthologue and of meiosis-specific cohesin mutations. In addition to the four mitotic cohesins, the fly genome encodes two meiosis-specific cohesin paralogues: C(2)M, an SCC1/RAD21 paralogue required for homologue synapsis and recombination in female meiosis, and SNM, an SCC3/SA paralogue required for stable homologue pairing in male meiosis. However, despite their homology to cohesin proteins, both C(2)M and SNM are dispensable for sister chromatid cohesion in meiosis. Although the orientation disruptor (ord) gene is required for meiotic sister chromatid cohesion, ORD has no homology to cohesins or to any other known proteins, and its subcellular localization pattern differs from that of cohesin. Thus, the relationship between ORD and cohesin and the precise role of ORD in cohesion are unclear (Yan, 2010).

Two lines of evidence support a role for cohesin in Drosophila meiosis. First, immunocytological studies have localized SMC1 to centromeres in both male and female meiosis I and to synaptonemal complexes in female meiosis. Second, mutations in the Drosophila shugoshin homologue mei-S332 cause precocious sister chromatid separation (PSCS) and high frequencies of meiosis II nondisjunction (NDJ), which is consistent with a possible role of MEI-S332 in protection of centromeric cohesin at anaphase I. However, the molecular function of mei-S332 has not been established, and the inviability of cohesin component mutants has thus far prevented their meiotic roles from being characterized. Thus, the molecular basis for meiotic cohesion in Drosophila remains poorly defined (Yan, 2010).

This study describes a novel Drosophila protein, sisters on the loose (SOLO), which is required for sister centromere cohesion and SMC1 localization to centromeres throughout meiosis and colocalizes with SMC1 on centromeres from the onset of meiosis until both proteins disappear at anaphase II. In addition to randomizing chromatid segregation in meiosis II, solo mutations result in a unique 'random 2::2' segregation pattern at meiosis I that reflects complete loss of sister centromere coorientation but partial maintenance of bivalent structure and function. The data indicate that SOLO plays a direct role in sister chromatid cohesion during Drosophila meiosis and suggest that it does so in close association with cohesin (Yan, 2010).

To characterize the solo transcription unit, a nearly full-length cDNA was sequenced as well as several RT-PCR and 5′ and 3′ rapid amplification of cDNA ends (RACE) fragments that include part or all of the intronic exons. Those analyses revealed that in addition to the two intronic exons, solo transcripts also include the three upstream vas exons, which encode several RGG repeats found in RNA-binding proteins but lack the five downstream vas exons, which encode the RNA helicase domain. The three upstream vas exons and the two intronic exons are spliced together to create a continuous ORF that extends from the translation start site of vas in exon 2 to a stop codon in the downstream intronic exon and that could encode a protein 1,031 amino acids in length (Yan, 2010).

Complementation analysis between solo and vas mutations confirmed the proposed exon structure of solo. solo alleles complemented all vas alleles containing mutations in any of the five C-terminal exons, which encode the VASA helicase domain, indicating that the C terminus of VASA is not shared by SOLO. However, vas mutations that map upstream of the SOLO-specific exons, including one nonsense mutation in exon 3, failed to complement the solo alleles, indicating that the 137 amino acids encoded by the upstream exons are present in both proteins. It is unlikely that the SOLO-specific exons are expressed independently of vas in addition to being expressed as a fusion product with the N terminus of VASA, as vas^6356-001 behaves as a null allele of solo, giving X-Y NDJ frequencies of 41%-44% in trans with solo alleles. It is concluded that solo encodes a protein that includes the N-terminal 137 amino acids of VASA fused to 894 amino acids encoded within the third intron of vas (Yan, 2010).

Single homologues of SOLO were identified by BLAST analysis in all 12 sequenced Drosophila genomes. Overall conservation is fairly low; Drosophila SOLO exhibits only ~30% amino acid identity with its homologues in Drosophila virilis and Drosophila pseudoobscura. However, in all of the Drosophila genomes, the solo sequences are nested within a large intron upstream of the exons that encode the helicase domain of VASA, and SOLO appears capable of being expressed by the same alternative splice mechanism used in Drosophila (Yan, 2010).

No homologues of SOLO were identified outside of the genus Drosophila, not even in the genome of the mosquito Anopheles gambiae. Although it is possible that solo exists in A. gambiae but is unrecognizable because of divergence, it would have to be located elsewhere in the genome, as there are no large exons nested within introns of the A. gambiae vas gene. Other than the RGG motifs in the common N terminus, SOLO exhibits no significant homologies with other proteins in the sequence database (Yan, 2010).

solo mutants exhibit premature loss of centromere cohesion and high NDJ at both meiosis I and II. Centromere cohesion is strongly impaired by stage S5 of prophase I long before centromere orientation patterns are established at prometaphase I. Although the premature loss of centromere cohesion is likely the underlying cause of NDJ at both divisions, the mechanisms of meiosis I and meiosis II NDJ nevertheless differ in important ways. During meiosis II, sister chromatids are fully separated at metaphase II, and anaphase II segregation appears to involve random assortment of fully independent chromatids to the two poles. However, during meiosis I, fully separated sister chromatids are rarely observed, and bivalents containing the four chromatids of a homologous pair remain intact throughout the division. Moreover, at least for the X-Y pair, chromatid segregation is not fully random. Although random assortment would lead to numerically unequal segregation (3:1 or 4:0) in 62.5% of meiosis I divisions, in solo males, >95% of anaphase I cells exhibit two chromatids at each pole. This restriction probably applies to autosomes as well because in DAPI-stained preparations, >90% of anaphase I spermatocytes exhibit poles with roughly equal DNA content. Nevertheless, segregation is very abnormal, indeed random in a more limited sense. Unlike WT spermatocytes in which sister chromatids always cosegregate at meiosis I, in solo spermatocytes X and Y chromatids exhibit no preference for or against their sister as a segregation partner. The result is a 2:1 ratio of equational (XY::XY) to reductional (XX::YY) segregations. Thus, bivalents in solo males retain their gross structure and the ability to segregate in an orderly fashion but lose sister-specific connections and with them the ability to distinguish sister from homologous chromatids. The resulting bivalents have four functional kinetochores instead of the normal two, and these orient independently of each other yet are somehow constrained to orient two to each pole (Yan, 2010).

How might SOLO perform its role in sister centromere orientation? One possibility is a role similar to the monopolin complex in S. cerevisiae or MoaI in S. pombe, proteins that function specifically in coorientation. However, mutations in these proteins do not disrupt sister centromere cohesion, whereas solo mutations disrupt both cohesion and coorientation. Therefore, a more parsimonious idea is that the primary role of SOLO is in centromere cohesion and that cohesion is required for coorientation. SOLO would thus be more similar to REC8, a meiotic cohesin component that is also required for both cohesion and coorientation in S. pombe. It remains to be determined whether other proteins analogous to monopolin or MoaI are also required for centromere coorientation in Drosophila (Yan, 2010).

Homologue connections, in the form of recombination-generated chiasmata, have been shown in both S. cerevisiae and S. pombe to promote fidelity of sister centromere coorientation to varying degrees both in WT cells and in cells deficient for other centromere orientation factors. Drosophila males lack chiasmata but use the SNM-MNM complex to maintain homologue pairing until anaphase I. The data indicate that SNM (likely in complex with MNM) serves to coordinate chromatid segregation patterns in the absence of centromere cohesion but has only a minimal effect on sister centromere orientation by itself. The fact that the reductional/equational segregation ratio in solo mutants almost exactly matches the random expectation makes it unlikely that SNM does anything to actively promote reductional segregations. The main effect of the loss of SNM in a SOLO-deficient background is abrogation of the restriction against unequal segregations. More than 40% of anaphase I cells in solo; snm males exhibit numerically unequal segregations compared with <5% in solo males. Although the ratio of equational to reductional 2::2 segregations increases somewhat in solo; snm mutants relative to solo mutants, for reasons that are not clear, reductional segregations are nevertheless preserved and indeed occur at approximately the expected random frequency (12.5%). This stands in sharp contrast to spo13 or Moa1 mutants (in S. cerevisiae and S. pombe, respectively), which exhibit mixed reductional/equational meiosis I segregation patterns similar to solo but which revert to 100% equational segregation when homologue connections are removed by spo11 mutations. The basis for this difference is that spo13 and Moa1 interfere with sister centromere orientation without disrupting cohesion before anaphase I so that loss of homologue connections leaves most chromosomes still connected at sister centromeres. However, solo mutations ablate sister chromatid cohesion so leave no basis for regular equational segregation (Yan, 2010).

How does SNM-MNM promote regular chromatid segregation? A plausible scenario is that SNM-MNM provides nonspecific connections among all four chromatids at homologue-pairing sites such as the rDNA locus of the X-Y pair. Although inadequate to direct centromere orientation, such connections would preserve bivalent stability and could provide the resistance necessary for generation of tension on the meiosis I spindle. The 2::2 segregation bias could reflect a checkpoint mechanism that serves to monitor and balance such tension. Alternatively, it could reflect a rigidity of bivalent structure that tends to discourage unbalanced orientations. Further research will be required to understand the basis for the unique meiosis I segregation pattern in solo (Yan, 2010).

In S. cerevisiae and S. pombe, multiple meiotic cohesion functions are performed by cohesin complexes that include meiosis-specific subunits such as REC8, which replaces the mitotic kleisin subunit RAD21. REC8 is widely conserved among eukaryotes and has been shown in several model plants and animals to be critical for many of the same meiotic functions identified in yeast. However, in Drosophila, no true REC8 homologue has been identified, and the role of cohesin in meiotic cohesion has been unclear (Yan, 2010).

These data strongly suggest that SOLO and SMC1 function as partners in mediating centromere cohesion in Drosophila meiosis. First, anti-SMC1 and Venus::SOLO foci overlap extensively on centromeres throughout meiosis until anaphase II when both proteins disappear. Second, both Venus::SOLO and anti-SMC1 foci disappear prematurely at anaphase I in mei-S332 mutants, which is consistent with a role of MEI-S332 to protect meiotic cohesin from proteolytic cleavage by separase. Third, centromere localization of SMC1 is abolished at all meiotic stages in solo spermatocytes. Finally, evidence has been obtained for a physical interaction between SMC1 and SOLO in ovaries (Yan, 2010).

Another protein with an essential role in Drosophila meiotic cohesion is ORD. The phenotypes of solo and ord mutations are very similar, including missegregation of both homologous and sister chromatids and ablation of centromeric SMC1 foci. Like SOLO, ORD is a centromere protein, but there are significant differences in the localization patterns of the two proteins. SOLO localizes to centromeres from the earliest stages of prophase I and remains on the centromeres until anaphase II. ORD has been reported to localize predominantly to interchromosomal spaces in early prophase I nuclei in male meiosis, then to the chromosome arms in late prophase I, finally accumulating on centromeres at prometaphase I where it remains until anaphase II. Nevertheless, the striking phenotypic similarity of solo and ord mutants strongly suggests that both ORD and SOLO are intimately involved in establishing and maintaining cohesion in Drosophila meiosis (Yan, 2010).

The exact role of SOLO (and ORD) in meiotic cohesion remains to be determined. One possibility is that SOLO is a regulatory protein required for stable localization of cohesin to centromeres. Several known cohesin cofactors are required for specific aspects of cohesin function, such as chromosomal loading, establishment of cohesion, removal of cohesin during prophase, protection of centromeric cohesin, etc.. SOLO appears to play a more general role than most of these cofactors: it is involved both in stable chromosome association of cohesin and in the establishment and maintenance of cohesion throughout meiosis. Moreover, unlike the known cofactors that associate with cohesin during certain stages of the cell cycle, SOLO colocalizes with SMC1 throughout meiosis. Thus, except for the lack of homology to any of the four families of cohesin proteins, the data are consistent with the possibility that SOLO is a novel component of a meiosis-specific cohesin complex. It will be of considerable interest to determine the composition of the meiotic cohesin complexes in Drosophila (Yan, 2010).

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; they occur at an elevated frequency and are genotypically consistent with anaphase I segregation defects. It is proposed that condensin II-mediated prophase I territory formation prevents and/or resolves heterologous chromosomal associations to alleviate their potential interference in anaphase I segregation. Furthermore, condensin II-catalyzed prophase I chromosome condensation may be necessary to resolve associations between paired homologous chromosomes of each bivalent. These persistent chromosome associations likely consist of DNA entanglements, but may be more specific as anaphase I bridging was rescued by mutations in the homolog conjunction factor teflon. It is proposes that the consequence of condensin II mutations is a failure to resolve heterologous and homologous associations mediated by entangled DNA and/or homolog conjunction factors. Furthermore, persistence of homologous and heterologous interchromosomal associations lead to anaphase I chromatin bridging and the generation of aneuploid gametes (Hartl, 2008).

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, 2008).

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, 2008).

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, 2008).

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, 2008).

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. 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. 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, 2008).

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, 2008).

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

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

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, 2008).

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

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

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

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, 2008).

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

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

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

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

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

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

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

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, 2008).

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

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

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

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

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, 2008).

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

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

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

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

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

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

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

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

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, 2008).

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

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

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. These studies 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, 2008).

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, 2008).

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, 2008).

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, 2008).

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, 2008).

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

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, 2008).

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. 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, 2008).

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, 2008).

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

In Drosophila males, homologous chromosomes segregate by an unusual process involving physical connections not dependent on recombination. This study identified two meiotic proteins specifically required for this process. Stromalin in Meiosis (SNM) is a divergent member of the SCC3/SA/STAG family of cohesin proteins, and Modifier of Mdg4 in Meiosis (MNM) is one of many BTB-domain proteins expressed from the mod(mdg4) locus. SNM and MNM colocalize along with a repetitive rDNA sequence known to function as an X-Y pairing site to nucleolar foci during meiotic prophase and to a compact structure associated with the X-Y bivalent during prometaphase I and metaphase I. Additionally, MNM localizes to autosomal foci throughout meiosis I. These proteins are mutually dependent for their colocalization, and at least MNM requires the function of teflon, another meiotic gene. SNM and MNM do not colocalize with SMC1, suggesting that the homolog conjunction mechanism is independent of cohesin (Thomas, 2005).

Search PubMed for articles about Drosophila Stromalin 2

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

Kabakci, Z., Yamada, H., Vernizzi, L., Gupta, S., Weber, J., Sun, M. S. and Lehner, C. F. (2022a). Teflon promotes chromosomal recruitment of homolog conjunction proteins during Drosophila male meiosis. PLoS Genet 18(10): e1010469. PubMed ID: 36251690

Kabakci, Z., Reichle, H. E., Lemke, B., Rousova, D., Gupta, S., Weber, J., Schleiffer, A., Weir, J. R. and Lehner, C. F. (2022b). Homologous chromosomes are stably conjoined for Drosophila male meiosis I by SUM, a multimerized protein assembly with modules for DNA-binding and for separase-mediated dissociation co-opted from cohesin. PLoS Genet 18(12): e1010547. PubMed ID: 36480577

Marston, A. L., Tham, W. H., Shah, H., Amon, A. (2004). A genome-wide screen identifies genes required for centromeric cohesion. Science, 303(5662):1367-1370 PubMed ID: 14752166

Mengoli, V., Bucciarelli, E., Lattao, R., Piergentili, R., Gatti, M. and Bonaccorsi, S. (2014). The analysis of mutant alleles of different strength reveals multiple functions of topoisomerase 2 in regulation of Drosophila chromosome structure. PLoS Genet 10: e1004739. PubMed ID: 25340516

Thomas, S. E., Soltani-Bejnood, M., Roth, P., Dorn, R., Logsdon, J. M., Jr., McKee, B. D. (2005). Identification of two proteins required for conjunction and regular segregation of achiasmate homologs in Drosophila male meiosis. Cell, 123(4):555-568 PubMed ID: 16286005

Yan, R., Thomas, S. E., Tsai, J. H., Yamada, Y. and McKee, B. D. (2010). SOLO: a meiotic protein required for centromere cohesion, coorientation, and SMC1 localization in Drosophila melanogaster. J. Cell Biol. 188(3): 335-49. PubMed ID: 20142422

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