InteractiveFly: GeneBrief

teflon: Biological Overview | References

Meiosis in males of higher dipterans is achiasmate. In their spermatocytes, pairing of homologs into bivalent chromosomes does not include synaptonemal complex and crossover formation. While crossovers preserve homolog conjunction until anaphase I during canonical meiosis, an alternative system is used in dipteran males. Mutant screening in Drosophila melanogaster has identified teflon (tef) as being required specifically for alternative homolog conjunction (AHC) of autosomal bivalents. The additional known AHC genes, snm, uno and mnm, are needed for the conjunction of autosomal homologs and of sex chromosomes. This study has analyzed the pattern of TEF protein expression. TEF is present in early spermatocytes but cannot be detected on bivalents at the onset of the first meiotic division, in contrast to SNM, UNO and MNM (SUM). TEF binds to polytene chromosomes in larval salivary glands, recruits MNM by direct interaction and thereby, indirectly, also SNM and UNO. However, chromosomal SUM association is not entirely dependent on TEF, and residual autosome conjunction occurs in tef null mutant spermatocytes. The higher tef requirement for autosomal conjunction is likely linked to the quantitative difference in the amount of SUM protein that provides conjunction of autosomes and sex chromosomes, respectively. During normal meiosis, SUM proteins are far more abundant on sex chromosomes compared to autosomes. Beyond promoting SUM recruitment, TEF has a stabilizing effect on SUM proteins. Increased SUM causes excess conjunction and consequential chromosome missegregation during meiosis I after co-overexpression. Similarly, expression of SUM without TEF, and even more potently with TEF, interferes with chromosome segregation during anaphase of mitotic divisions in somatic cells, suggesting that the known AHC proteins are sufficient for establishment of ectopic chromosome conjunction. Overall, these findings suggest that TEF promotes alternative homolog conjunction during male meiosis without being part of the final physical linkage between chromosomes (Kabakci, 2022).

Meiosis is a key innovation that evolved before the eukaryotic radiation into the extant domain. The canonical program of this conserved process relies on meiotic recombination (MR). MR contributes to the initial pairing of homologous chromosomes and generates crossovers that maintain homologs linked as bivalent chromosomes until the onset of anaphase during the first meiotic division (M I). MR proceeds usually in concert with synapsis, which achieves close homolog pairing all along the chromosomes via formation of the synaptonemal complex (SC). In spite of the eminent significance of MR, diverse meiosis variants have evolved that do not rely on MR. A most thoroughly studied example of such an achiasmate meiosis occurs in Drosophila melanogaster. While meiosis is largely canonical in D. melanogaster females and includes MR, it is achiasmate in the heterogametic males. This sex-specific difference in meiosis is characteristic among higher dipterans. Its evolution is poorly understood, but may be linked to the suppression of recombination between sex chromosomes (Kabakci, 2022).

In D. melanogaster spermatocytes, not only MR but also SC formation does not occur. Nevertheless, soon after the last spermatogonial mitosis, homologous chromosomes are paired all along their length, according to analyses with a lacO/lacI-GFP system and FISH. It remains to be clarified whether the pairing of homologous chromosomes in early spermatocytes during the S1 stage is driven by the same mechanisms that are responsible for the pervasive somatic homolog pairing in D. melanogaster. Importantly, the extensive pairing of homologs in spermatocytes lasts only a few hours. During the S2b/S3 stages, homolog pairing was no longer detectable at any of the analyzed 14 distinct locations with euchromatic lacO array insertions. Moreover, even sister chromatid cohesion appeared to be lost except at centromeres (Kabakci, 2022).

The drastic loss of homolog pairing and sister cohesion in mid-stage spermatocytes starts concomitantly with the process of territory formation, which separates three major chromosome territories apart within the interphase nucleus. One of the major territories contains the chromosome (chr) 2 bivalent, another the chr3 bivalent and the third the chrXY bivalent. The additional bivalent of chr4, a small dot chromosome, is often associated with the chrXY territory. Territory formation breaks up all non-homologous associations between the large chromosomes. Such non-homologous associations are extensive in S1 spermatocytes. They arise from a coalescence of large blocks of pericentromeric heterochromatin into a chromocenter. Similarly, centromeres are clustered initially. Disrupting these non-homologous associations during territory formation at the S2b stage depends on condensin II activity and additional unidentified forces. Failure of territory formation leads to persistence of non-homologous associations until prometaphase I and consequential chromosome segregation errors (Kabakci, 2022).

The mechanisms that break up non-homologous chromosome associations during territory formation disrupt also homolog pairing and sister chromatid cohesion, presumably because of inevitable side effects. However, normally, homolog separation does not proceed to completion already during spermatocyte maturation. Complete premature homolog separation is prevented by residual homolog conjunction maintained by a dedicated special system that serves as an alternative to canonical homolog linkage by crossovers. Large-scale mutant screening has led to the identification of three genes (tef, mnm, and snm) that are specifically required for this alternative homolog conjunction (AHC). A proteomic approach has recently uncovered an additional AHC gene (uno). Loss-of-function mutations in these four genes result in chromosome missegregation during M I, but exclusively in males. In mnm, snm and uno mutant males, both sex chromosomes and autosomes are distributed randomly during M I. In contrast, only autosomes are missegregated in tef mutant males during M I (Kabakci, 2022).

The TEF protein includes three C2H2-type zinc fingers and is therefore predicted to bind to DNA. The SNM protein is a distant relative of the stromalins (SCC3/SA/STAG protein family). Stromalins are subunits of cohesins, complexes of crucial importance for chromosome organization during interphase and M phase in somatic and meiotic cells. However, SNM is not co-localized with core components of cohesin, indicating that it does not function as a cohesin subunit. MNM is encoded by one of many differentially spliced mRNAs transcribed from the highly complex mod(mdg4) locus. MNM has an N-terminal BTB/POZ motif that is shared among almost all of the more than 30 distinct protein products expressed from the mod(mdg4) locus. In addition, MNM has a unique C-terminal zinc finger motif of the FLYWCH type. These N- and C-terminal motifs of MNM are predicted to mediate protein-protein interactions. UNO does not have obvious similarities to functionally characterized proteins (Kabakci, 2022).

MNM, SNM and UNO accumulate in early spermatocytes, eventually co-localizing during spermatocyte maturation in multiple subnucleolar foci. At the start of M I, these foci coalesce into a single prominent spot on the chrXY bivalent. In D. melanogaster, chrX and chrY are strongly heteromorphic, lacking extended euchromatic homology that could mediate specific pairing. However, both sex chromosomes harbor rDNA gene clusters in the centromere-proximal heterochromatin and these rDNA clusters function as pairing centers during male M I. The prominent dot formed by MNM, SNM and UNO on the chrXY bivalent at the start of M I is localized on the paired rDNA loci of chrX and chrY. Apart from the prominent dot on the chrXY pairing center, autosomal bivalents, which rely on euchromatic homology for pairing display far weaker dot signals of co-localized MNM, SNM and UNO. These autosomal dot signals were shown to be at least partially dependent on tef function. Strikingly, MNM, SNM and UNO disappear rapidly from all the bivalents within minutes during the onset of anaphase I. Separase, an endoprotease known to eliminate chromosomal cohesin at the metaphase to anaphase transition during mitotic and meiotic divisions, is required for the rapid disappearance of MNM, SNM and UNO from M I bivalents. UNO includes a separase cleavage site. Mutations that abolish this cleavage site prevent the rapid disappearance of MNM, SNM and UNO from M I bivalents and preclude homolog separation (Kabakci, 2022).

The findings summarized above strongly support the notion that SNM, MNM and UNO function as proteinaceous glue that conjoins chromosomes into bivalents. However, it remains to be clarified how these proteins are recruited to chromosomes. SNM, MNM and UNO do not include known bona fide DNA-binding domains. They might therefore be recruited by other chromatin proteins. The zinc finger protein TEF is clearly an attractive candidate factor for chromosomal recruitment of the other AHC proteins. TEF's pattern of expression and its subcellular localization during spermatogenesis have not yet been characterized. This study closes this gap in understanding. Using transgenes encoding tagged functional versions of TEF, it was observed to be only transiently detectable in early spermatocytes. In contrast to the other known AHC proteins (MNM, SNM and UNO), TEF cannot be detected on bivalents at the start of M I, indicating that it is unlikely a stoichiometric component of the homolog-conjoining glue. However, evidence is provided that TEF can recruit MNM to chromosomes by direct protein-protein interaction. Indirectly, TEF can also recruit SNM-UNO, as they bind to MNM. Moreover, presumably by promoting AHC protein interactions, TEF stabilizes these proteins and controls their levels. AHC protein levels need to be controlled, as suggested by the consequences of simultaneous overexpression of all four AHC proteins in spermatocytes, which resulted in ectopic chromosome conjunction, failure of territory formation and segregation errors during M I. Ectopic expression of the four AHC proteins in somatic cells induced aberrant chromosome conjunction during mitosis, suggesting that AHC might not depend on additional spermatocyte-specific proteins beyond those already known (Kabakci, 2022).

Genes required specifically for alternative homolog conjunction (AHC) during the achiasmate meiosis of Drosophila males were identified initially by extensive screening of mutants, and the teflon (tef) mutant phenotype was the first to be characterized in detail. The molecular identification of the affected gene revealed that tef encodes a zinc finger protein. This report provides more detailed functional characterization of the TEF protein. Expression pattern and intracellular localization during spermatogenesis were clarified with the help of tagged functional variants. An interaction between TEF and MNM was demonstrated by co-immunoprecipitation, and the responsible binding regions were mapped. Moreover, TEF was shown to bind to chromatin of polytene chromosomes in larval salivary glands. Importantly, TEF recruits MNM to chromatin, and via MNM also the other known AHC proteins SNM and UNO. Moreover, TEF potentiates the chromosome-linking activity of the AHC proteins SNM, UNO and MNM, as revealed by overexpression experiments in spermatocytes and other cell types (Kabakci, 2022).

The TEF expression pattern was characterized with g-tef-sm_myc. This transgene under control of the tef regulatory region results in expression of a TEF version tagged with a spaghetti monster myc epitope tag (sm_myc). According to mutant rescue experiments, TEF-sm_myc is fully functional. Based on the g-tef-sm_myc expression pattern revealed by anti-Myc immunofluorescence, TEF is absent or low in somatic hub cells of testes but present in germline stem cells, spermatogonial cells and spermatocytes. TEF-sm_myc is also abundant in ovaries, where it is not germline-restricted as in testes. TEF's role in ovaries remains unclear, as no aberrant phenotype has been found in tef mutants so far (Kabakci, 2022).

The subcellular localization of TEF-sm_myc was unexpected. During spermatogonial mitoses, a strong enrichment on centrosomes was observed. It is noted that mitotic centrosomal localization is also characteristic of CP190, an architectural chromatin protein, which like TEF has zinc fingers and interacts with a Mod(mdg4) protein. During interphase, TEF-sm_myc was primarily in many intranuclear foci of variable size, and a majority of these did not appear to be chromatin-associated. Intriguingly, the presence of TEF-sm_myc in spermatocytes was transient. TEF-sm_myc levels declined during spermatocyte maturation. It was no longer detectable in late spermatocytes (stages S5 and S6) and during the meiotic divisions. This subcellular localization and transient presence in spermatocytes were also observed in case of bamP-GAL4-VP16 driven UASt-tef-EGFP expression, which also rescues tef mutants. Importantly, in contrast to TEF, the other known AHC proteins (SNM, MNM and UNO, abbreviated as SUM) are all detectable on autosomal bivalents, when expressed analogously (as EGFP fusions from UASt transgenes with bamP-GAL4-VP16). These results argue strongly against the notion that homologous autosomes are conjoined by complexes of AHC proteins containing stoichiometric amounts of TEF. Rather than being an essential component of the glue that keeps homologous autosomes linked until onset of anaphase I, TEF might function only in early spermatocytes in the regulation of AHC establishment. It is noted that a presence of functional TEF in bivalents of late spermatocytes at levels below detectability is not excluded (Kabakci, 2022).

Comparison of the mutant phenotypes caused by loss of tef, on the one hand, and loss of snm or mnm, on the other hand, provides further arguments against the notion that TEF is an essential component of the glue that conjoins autosomal homologs. The two tef alleles present in the transheterozygous mutants that have been analyzed are early non-sense mutations, shown to be amorphic with respect to meiotic chromosome transmission. However, the extent of autosome missegregation was significantly less severe in the transheterozygous tef mutants compared to snm and mnm mutants. This conclusion rests on concurrent findings made by time-lapse imaging of progression through M I and by analyses of meiotic chromosome missegregation with dodeca FISH. In snm and mnm mutants, bivalents are prematurely separated into independent univalents that are segregated randomly during M I. In contrast, in tef mutants there is some residual conjunction of autosomal homologs and their segregation is not completely random. Phenotypic comparisons are therefore consistent with the notion that TEF contributes to AHC establishment in early spermatocytes rather than also to the maintenance of AHC until anaphase I like the SUM protein. According to current observations after ectopic expression of AHC proteins, TEF might contribute to AHC establishment by promoting the recruitment of the SUM proteins to chromatin. TEF is the only AHC protein with a predicted bona fide DNA-binding domain. TEF has three zinc fingers, one in the N-terminal and two in the C-terminal region. Jointly, these N- and C-terminal zinc fingers mediate efficient TEF binding of TEF to polytene chromosomes after ectopic expression in larval salivary glands, as deletion of either the N- or the C-terminal region resulted in a substantial reduction of the chromosome-associated signals. Consistent with the absence of known DNA-binding motifs, none of the other AHC proteins displayed substantial binding to polytene chromosomes when expressed individually. Unexpectedly, however, polytene chromosome binding was clearly observed after co-expression of SNM and UNO. Presumably, these two proteins form a complex (SU) that includes a composite DNA-binding site. The two chromosome-binding entities among the AHC proteins, TEF and SU, have distinct preferences for chromosomal locations. However, both are able to recruit MNM onto polytene chromosomes. TEF and MNM interact directly according to co-immunoprecipitation experiments after transient expression in S2R+ cells, consistent with the previously observed co-purification of TEF with MNM-EGFP from testis extracts. The TEF-MNM interaction is mediated by the N-terminal part of TEF that includes the first zinc finger and by the C-terminal part of MNM. This C-terminal part is uniquely present in MNM. All the many additional isoforms that are generated by differential splicing from the complex mod(mdg4) locus have distinct C-terminal parts, and the three isoforms tested (T, C and P) were unable to bind to TEF. Beyond the TEF-MNM interaction, analyses of polytene chromosome binding and of co-immunoprecipitation suggested that all four AHC proteins (SNM, UNO, MNM and TEF) can co-assemble into SUMT complexes (Kabakci, 2022).

Clearly, in salivary glands, TEF does not just bind to autosomes but also to the X chromosome. Thus, TEF does not appear to have an autosome-specific chromosome-binding ability that would explain why tef is required in spermatocytes for regular M I segregation of autosomes but not of sex chromosomes. It is suggested that the chromosome-specificity of the tef requirement might be linked to an additional effect of TEF on AHC proteins. According to the quantification of expression levels after Sgs3-GAL4-mediated expression of AHC proteins in salivary glands, formation of AHC protein complexes appears to stabilize these proteins. Levels of TEF and MNM were higher after co-expression compared to individual expression. Analogous observations were made with SNM and UNO. Similarly, after bam-GAL4-VP16-driven overexpression of SUM or SUMT in spermatocytes, the levels of the only tagged protein UNO-mCherry were increased by the presence of TEF. Moreover, MNM-EGFP levels were lower in tef mutant spermatocytes. Overall, these observations indicate a positive correlation between TEF and SUM protein levels. In tef mutants, some of the remaining SUM is presumably still recruited to autosomal bivalents due to the chromosome-binding activity of SU. However, as SUM levels during wild-type meiosis are far lower on autosomal bivalents compared to the chrXY bivalent, autosomal bivalents might be more strongly affected when SUM protein levels decrease as a result of a loss of tef function. TEF increases SUM protein levels presumably by promoting the formation of protein associations that are more stable than the individual proteins. Stimulating effects of TEF on SUM gene transcription are not excluded but unlikely as the analyses included experiments where the AHC proteins were expressed with exogenous regulatory sequences (UASGAL4 and hsp70) (Kabakci, 2022).

The proposed explanation for the autosome-specific effect of tef mutations remains speculative, also because of the technical difficulties to detect SUM proteins on autosomal bivalents. Even the normal amounts of autosomal SUM proteins during wild-type meiosis are difficult to detect unequivocally and consistently in each spermatocyte. In this study, by analyzing fluorescent versions of UNO, a quantitative estimate is provided for the striking difference in the amount of SUM proteins on autosomes and sex chromosomes in normal spermatocytes. Around 25-100-fold lower amounts of UNO is found on autosomal bivalents compared to the chrXY bivalents. Without future technical improvements of detection sensitivity, a conclusive demonstration of the postulated residual autosomal SUM complexes in tef mutants is not feasible (Kabakci, 2022).

If detectable, the autosomal SUM proteins appear to be confined to 1-2 dots per bivalent at NEBD I in normal spermatocytes. Do these dots mark the location of autosomal homolog conjunction, or might there be additional SUM complexes at other locations below the limit of detection that contribute to conjunction as well? Recent cytological analyses of meiotic quadrivalents in spermatocytes heterozygous for autosomal translocations have indicated that autosomal homolog conjunction is spatially constrained to dot-like chromosomal locations. The spatial control of autosomal homolog conjunction in spermatocytes appears to be analogous to that of canonical crossovers. As a rule, a single restricted region within the euchromatic portion of each autosomal chromosome arm is linked by AHC protein assemblies to its homologous region. Thus, AHC positions might be controlled by processes analogous to crossover interference. The particularly strong crossover interference in C. elegans has recently been proposed to involve spatially restricted biomolecular condensation of recombination nodule proteins in combination with a regulated coarsening process. It is tempting, therefore, to speculate about the significance of MNM's apparent liquid phase separation potential. Both MNM and TEF include substantial portions that are predicted to be intrinsically disordered. Such regions are thought to favor liquid-liquid unmixing when they confer multivalent interactions. At high levels of expression, MNM-EGFP formed droplets in salivary gland nuclei, while this was hardly observed with MNM-mCherry. The known weak dimerization of EGFP might reinforce multivalent associations. With TEF-EGFP droplets were not obtained when expressed alone but when co-expressed with MNM-mCherry, which was co-localized with TEF-EGFP in the droplets. Thus, droplet formation was stimulated by the EGFP tag when present on either MNM or its binding partner TEF. In case of untagged endogenous proteins, droplet formation might remain restricted to chromosomally recruited AHC assemblies. Accordingly, liquid phase separation of AHC proteins might be involved in the control of establishment or maintenance of alternative homolog conjunction in Drosophila spermatocytes (Kabakci, 2022).

Experiments with spermatocytes revealed that an excess of AHC proteins is detrimental to regular chromosome segregation during male meiosis. Overexpression of SUMT severely inhibited chromosome territory formation most likely because it results in increased and more widespread conjunction between not only homologous but also non-homologous chromosomes. As a consequence, presumably, chromosomes fail to separate normally, often forming prominent bridges during anaphase and telophase of M I. The meiotic defects observed after SUMT overexpression are highly reminiscent of those caused by a loss of condensin II function. Conversely, absence of SUMT, as in mutants, has very similar phenotypic consequences as overexpression of the limiting condensin II subunit Cap-H2. Evidently, AHC proteins and condensin II have opposing activities that need to be in proper balance (Kabakci, 2022).

The detrimental effects on meiotic chromosome segregation were much stronger after overexpression of SUMT compared to SUM. Overexpression of individual AHC proteins had barely any effect. These results provide further support for the proposal that in normal male meiosis, TEF assists in the chromosomal recruitment of SUM, the actual glue that maintains homolog linkage in bivalents until anaphase I onset. Accordingly, overexpression of TEF alone might not have severe detrimental effects because potentially low levels of endogenous SUM proteins might not allow excess assembly. Similarly, previous overexpression of individual SUM proteins was not observed to cause severe detrimental effects, perhaps also because low levels of other SUM subunits might limit excess assembly. The finding that ectopic SUMT expression in mitotically proliferating wing imaginal disc cells results in mitotic defects that resemble closely to the meiotic defects observed after SUMT overexpression in spermatocytes might indicate that AHC during male meiosis does not depend on additional spermatocyte-specific proteins beyond the known AHC proteins. The three proteins SNM, UNO, and MNM appear to be sufficient to induce conjunction between mitotic chromosomes and thus interfere with their normal segregation during anaphase. In combination with TEF, SUM had even more detrimental effects on mitotic chromosome segregation. However, much remains to be learned about the regulation that controls the appropriate chromosomal positioning of AHC during male meiosis (Kabakci, 2022).

Dynamics and control of sister kinetochore behavior during the meiotic divisions in Drosophila spermatocytes

Sister kinetochores are connected to the same spindle pole during meiosis I and to opposite poles during meiosis II. The molecular mechanisms controlling the distinct behavior of sister kinetochores during the two meiotic divisions are poorly understood. To study kinetochore behavior during meiosis, time lapse imaging was optimized with Drosophila spermatocytes, enabling kinetochore tracking with high temporal and spatial resolution through both meiotic divisions. The correct bipolar orientation of chromosomes within the spindle proceeds rapidly during both divisions. Stable bi-orientation of the last chromosome is achieved within ten minutes after the onset of kinetochore-microtubule interactions. Analyses of something that sticks like glue (snama or mini-me) and teflon tef mutants, where univalents instead of bivalents are present during meiosis I, indicate that the high efficiency of normal bi-orientation depends on pronounced stabilization of kinetochore attachments to spindle microtubules by the mechanical tension generated by spindle forces upon bi-orientation. Except for occasional brief separation episodes, sister kinetochores are so closely associated that they cannot be resolved individually by light microscopy during meiosis I, interkinesis and at the start of meiosis II. Permanent evident separation of sister kinetochores during M II depends on spindle forces resulting from bi-orientation. In mnm and tef mutants, sister kinetochore separation can be observed already during meiosis I in bi-oriented univalents. Interestingly, however, this sister kinetochore separation is delayed until the metaphase to anaphase transition and depends on the Fzy/Cdc20 activator of the anaphase-promoting complex/cyclosome. It is proposed that univalent bi-orientation in mnm and tef mutants exposes a release of sister kinetochore conjunction that occurs also during normal meiosis I in preparation for bi-orientation of dyads during meiosis II (Chaurasia, 2018).

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 (Hirano, 2005; Yeong, 2003). In vitro, condensin I is known to induce and trap positive supercoils into a circular DNA template. Current models to explain condensin I chromosome condensation highlight this activity as supercoiling may promote chromatin gathering into domains that can then be assembled into a higher order structure (Hirano, 2006). Condensin complexes may also promote condensation and individualization through cooperating with other factors, such as chromatin-modifying enzymes. While the effect of condensin mutations or RNAi knockdown on chromosome condensation is variable depending on cell type and organism being studied, in most if not all cases, chromatin bridges are created between chromosomes as they segregate from one another. This likely represents a general role of the condensin complex in the resolution of chromosomal associations prior to segregation (Hartl, 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 (Thomas, 2005). While MNM and SNM are required for the conjunction of all bivalents, the protein Teflon promotes pairing maintenance specifically for the autosomes (Arya, 2006; Tomkiel, 2001). Teflon is also required for MNM-GFP localization to the 2^nd and 3^rd chromosomes (Thomas, 2005). This suggests that Teflon, MNM, and SNM constitute an autosomal homolog pairing maintenance complex (Hartl, 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 (Savvidou, 2005). This study has confirmed the necessity of Cap-D3 to male fertility; both Cap-D3^EY00456 homozygous and Cap-D3^EY00456/Cap-D3^{Df(2L)Exel6023} males are completely sterile when mated to wild-type females. Furthermore, males trans-heterozygous for strong Cap-H2 mutations are also male sterile; no progeny were derived from crosses of Cap-H2^Z3-0019/Cap-H2^{Df(3R)Exel6159}, Cap-H2^TH1/Cap-H2^{Df(3R)Exel6159}, and Cap-H2^TH1/Cap-H2^Z3-0019 to wild-type females. A third allele, Cap-H2^Z3-5163, is fertile as a homozygote and in trans-combinations with Cap-H2^Z3-0019, Cap-H2^{Df(3R)Exel6159}, and Cap-H2^TH1 alleles (Hartl, 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 (Arya, 2006). To investigate whether persistent associations between homologous chromosomes in anaphase I of Cap-H2 mutants are Teflon dependent, teflon mutations were crossed into a Cap-H2 mutant background and the frequency of anaphase I bridging was assessed. While 30.4% of anaphase I figures from Cap-H2^Z3-0019/Cap-H2^TH1 males were bridged, bridging existed within only 10.8% of anaphase I figures from tef^Z2-5549/tef^Z2-5864; Cap-H2^Z3-0019/Cap-H2^TH1 males. Furthermore, in squashed preparations anaphase I bridging was decreased from 40.5% in Cap-H2^Z3-0019/Cap-H2^TH1 males to 25.6% in the tef^Z2-5549/tef^Z2-5864; Cap-H2^Z3-0019/Cap-H2^TH1 double mutants (Hartl, 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 (Arya, 2006). 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 (Thomas, 2005). Condensin has been shown to antagonize cohesins in budding yeast meiosis and mitotic human tissue culture cells. This raises the possibility that a conserved molecular mechanism exists for condensin II as a negative regulator of SNM in Drosophila male meiosis. The investigation of Teflon, MNM, and SNM protein dynamics in a Cap-H2 mutant background will be an important set of future studies to help decipher the function of Cap-H2 in achiasmate segregation mechanisms (Hartl, 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).

Molecular characterization of teflon, a gene required for meiotic autosome segregation in male Drosophila melanogaster

Drosophila melanogaster males lack recombination and have evolved a mechanism of meiotic chromosome segregation that is independent of both the chiasmatic and achiasmatic segregation systems of females. The teflon (tef) gene is specifically required in males for proper segregation of autosomes and provides a genetic tool for understanding recombination-independent mechanisms of pairing and segregation as well as differences in sex chromosome vs. autosome segregation. This study reports on the cloning of the tef gene and the molecular characterization of tef mutations. Rescue experiments using a GAL4-driven pUAS transgene demonstrate that tef corresponds to predicted Berkeley Drosophila Genome Project (BDGP) gene CG8961 and that tef expression is required in the male germ line prior to spermatocyte stage S4. Consistent with this early prophase requirement, expression of tef was found to be independent of regulators of meiotic M phase initiation or progression. The predicted Tef protein contains three C2H2 zinc-finger motifs, one at the amino terminus and two in tandem at the carboxyl terminus. In addition to the zinc-finger motifs, a 44- to 45-bp repeat is conserved in three related Drosophila species. On the basis of these findings, a role is proposed for Tef as a bridging molecule that holds autosome bivalents together via heterochromatic connections (Arya, 2006).

Identification of two proteins required for conjunction and regular segregation of achiasmate homologs in Drosophila male meiosis

In Drosophila males, homologous chromosomes segregate by an unusual process involving physical connections not dependent on recombination. This study has 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).

Isolation and cytogenetic characterization of male meiotic mutants of Drosophila melanogaster

Proper segregation of homologous chromosomes in meiosis I is ensured by pairing of homologs and maintenance of sister chromatid cohesion. In male Drosophila melanogaster, meiosis is achiasmatic and homologs pair at limited chromosome regions called pairing sites. This study screened for male meiotic mutants to identify genes required for normal pairing and disjunction of homologs. Nondisjunction of the sex and the fourth chromosomes in male meiosis was scored as a mutant phenotype. 2306 mutagenized and 226 natural population-derived second and third chromosomes were screened and seven mutants were obtained representing different loci on the second chromosome and one on the third. Five mutants showed relatively mild effects (<10% nondisjunction). mei(2)yh149 and mei(2)yoh7134 affected both the sex and the fourth chromosomes, mei(2)yh217 produced possible sex chromosome-specific nondisjunction, and mei(2)yh15 and mei(2)yh137 produced fourth chromosome-specific nondisjunction. mei(2)yh137 was allelic to the teflon gene required for autosomal pairing. Three mutants exhibited severe defects, producing >10% nondisjunction of the sex and/or the fourth chromosomes. mei(2)ys91 (a new allele of the orientation disruptor gene) and mei(3)M20 induced precocious separation of sister chromatids as early as prometa-phase I. mei(2)yh92 predominantly induced nondisjunction at meiosis I that appeared to be the consequence of failure of the separation of paired homologous chromosomes (Hirai, 2004).

Search PubMed for articles about Drosophila Teflon

Arya, G. H., Lodico, M. J., Ahmad, O. I., Amin, R. and Tomkiel, J. E. (2006). Molecular characterization of teflon, a gene required for meiotic autosome segregation in male Drosophila melanogaster. Genetics 174(1): 125-134. PubMed ID: 16816414

Chaurasia, S. and Lehner, C. F. (2018). Dynamics and control of sister kinetochore behavior during the meiotic divisions in Drosophila spermatocytes. PLoS Genet 14(5): e1007372. PubMed ID: 29734336

Hirai, K., Toyohira, S., Ohsako, T. and Yamamoto, M. T. (2004). Isolation and cytogenetic characterization of male meiotic mutants of Drosophila melanogaster. Genetics166(4): 1795-1806. PubMed ID: 15126399

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. (2022). Teflon promotes chromosomal recruitment of homolog conjunction proteins during Drosophila male meiosis. PLoS Genet 18(10): e1010469. PubMed ID: 36251690

Thomas. S. E., Soltani-Bejnood, M., Roth, P., Dorn, R., Logsdon, J. M. and 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

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

date revised: 11 December 2023

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