InteractiveFly: GeneBrief

Structural maintenance of chromosomes 1: Biological Overview | References

BIOLOGICAL OVERVIEW

During meiosis, each chromosome must selectively pair and synapse with its own unique homolog to enable crossover formation and subsequent segregation. How homolog pairing is maintained in early meiosis to ensure synapsis occurs exclusively between homologs is unknown. This study aimed to further understand this process by examining the meiotic defects of a unique Drosophila mutant, Mcm5^A7. Mcm5^A7 mutants are proficient in homolog pairing at meiotic onset yet fail to maintain pairing as meiotic synapsis ensues, causing seemingly normal synapsis between non-homologous loci. This pairing defect corresponds with a reduction of SMC1-dependent centromere clustering at meiotic onset. Overexpressing SMC1 in this mutant significantly restores centromere clustering, homolog pairing, and crossover formation. These data indicate that the initial meiotic pairing of homologs is not sufficient to yield synapsis exclusively between homologs and provide a model in which meiotic homolog pairing must be stabilized by centromeric SMC1 to ensure proper synapsis (Hatkevich, 2019).

Accurate segregation of homologous chromosomes during the first meiotic division is essential to reestablish the diploid genome upon sexual fertilization. To ensure faithful meiosis I chromosomal segregation, homologs must become physically connected in part through crossover formation. To enable homolog crossover events, a series of chromosomal and cellular events occur in early meiotic prophase I (Hatkevich, 2019).

During or just prior to the onset of meiosis, homologous chromosomes pair along their entire lengths. Between paired homologs, synapsis, the formation of the synaptonemal complex (SC), ensues. The SC is a tripartite scaffold built between homologs extending the length of the chromosomes and consists of a central region that is nestled between two lateral elements, which are successors of cohesin-based chromosome axes formed between sister chromatids. Coincident with synapsis, DSBs are formed and repaired using a homologous template via homologous recombination (HR), resulting in crossover formation between homologs (Hatkevich, 2019).

Perhaps the most enigmatic event within early meiosis is the mechanism by which a meiotic chromosome selectively pairs and synapses with its unique homologous partner. Initial homolog pairing is believed to be facilitated through early meiotic chromosome movement and telomere or the centromere clustering. However, how homologous pairing is maintained during synapsis to ensure the SC is formed exclusively between homologs is unknown (Hatkevich, 2019).

The model organism Drosophila melanogaster has been used to uncover meiotic mechanisms for over a century. In Drosophila, prior to meiosis, chromosomes enter the germline unpaired; throughout the pre-meiotic region, homologous chromosomes gradually pair. In the nuclei at the last mitotic division prior to meiotic onset (in the 8-cell cyst), centromere-directed chromosomal movements occur, presumably ensuring complete homologous pairing. Also during pre-meiotic mitotic cycles, several proteins, including the cohesin SMC1, are enriched at the centromere. The onset of meiotic prophase I occurs in the 16-cell cyst. At zygotene, the first cytologically resolved stage of prophase, centromeres are clustered into 1 or 2 groups, and the SC nucleates in patches along chromosome arms. As zygotene proceeds into early pachytene, the SC extends between paired chromosomes, yielding full-length SC exclusively between homologs. How these early meiotic events, particularly SMC1 enrichment at the centromere and centromere clustering, contribute to meiotic homologous pairing and synapsis in Drosophila is largely unknown (Hatkevich, 2019).

This study used the Drosophila early meiotic program and a unique genetic mutant to investigate how homolog pairing is maintained during meiotic synapsis. Meiotic homologs in a previously described Drosophila mutant, Mcm5^A7, initially pair, but are unable to maintain pairing during synapsis, suggesting that initial meiotic pairing must be subsequently stabilized by an unknown mechanism to ensure proper synapsis. Using Mcm5^A7 as a genetic tool to interrogate pairing stabilization mechanism(s), it was found that SMC1 localization at the centromere is compromised, correlating with a severe defect in meiotic centromere clustering and a decrease of crossover formation. However, arm cohesion and SC structure appear unperturbed in these mutants. By overexpressing SMC1, this study shows that the defects in centromere clustering, meiotic homolog pairing, homosynapsis, and crossing over in Mcm5^A7 mutants are caused by a lack of centromeric SMC1 localization at meiotic onset. From these results, a model for proper synapsis is suggested in which initial meiotic pairing must be stabilized by centromere clustering, a meiotic event produced by SMC1-enrichment at the centromere and dynamic chromosome movements (Hatkevich, 2019).

At the beginning of this study, it was hypothesized that the crossover defect in Mcm5^A7 mutants was due to a homolog pairing deficiency. FISH results support this hypothesis and revealed that homolog pairing can be lost during synapsis, resulting in seemingly normal SC between heterologous sequences. Centromere-directed chromosome movements occur in Mcm5^A7 mutants, presumably to promote initial chromosome arm pairing; however, centromere pairing and clustering are perturbed. SMC1 enrichment at the centromere is decreased in Mcm5^A7 mutants, while arm cohesion appears normal. Overexpression of SMC1 rescues centromeric-SMC1 localization and downstream meiotic defects, including centromere clustering, pairing, crossover formation, and segregation. From these data, it is proposed that centromeric SMC1 stabilizes initial homolog pairing through centromere clustering, securing meiotic pairing, ensuring homosynapsis and promoting crossover formation (Hatkevich, 2019).

Prior to the onset of meiosis, cohesins are loaded onto centromeres, and homologous chromosomes pair, with arm pairing preceding centromere pairing. Initial homolog pairing is achieved in part by centromere-directed movements in the division prior to meiotic onset. A model in which initial homologous chromosomal pairing is stabilized throughout early meiosis by SMC1-dependent centromere clustering (Hatkevich, 2019).

According to this model, the enrichment of SMC1 at the centromere combined with chromosome movements in pre-meiotic stages yield centromere clustering at meiotic onset. While chromosome arms and centromeres enter meiosis paired, heterologous centromere clustering and/or centromere pairing are required to stabilize pairing during SC assembly. As initial euchromatic SC patches extend along the arms of paired homologs, DSBs are formed and subsequently repaired via HR to yield crossovers, which promote accurate disjunction at the end of meiosis (Hatkevich, 2019).

In Mcm5^A7 mutants, coordinated pre-meiotic centromere-directed movements occur, but a sufficient amount of SMC1 is not localized at the centromere to yield centromere clustering. Thus, at meiotic onset, arms are paired, but centromeres are not clustered. As euchromatic SC nucleation occurs, the stabilization provided by centromere clustering is absent, and homologous loci become unpaired. As synapsis extends, the SC is able to form between nearby chromosomes, regardless of homology, yielding heterosynapsis (intrachromosomal and/or interchromomosomal). DSBs made within regions of heterosynapsis are not repaired via HR due to the absence of an available homologous template. Therefore, crossovers are reduced, and nondisjunction occurs at high frequency in Mcm5^A7 mutants (Hatkevich, 2019).

The SMC1-dependent centromere clustering pairing model highlights the finding that initial meiotic pairing is not sufficient to yield complete homosynapsis. Rather, centromeric SMC1-dependent stabilization must occur after pairing and during synapsis. The inherent requirement of pairing stabilization for proper synapsis suggests that there is a force that opposes homolog alignment during synapsis. Perhaps the SC assembly process itself creates an opposing force that can push paired homologs away from one another in the absence of stabilization; a similar hypothesis was previously proposed in C. elegans. An alternative hypothesis is that recombination, which coincides temporally with synapsis assembly, creates a destabilizing force. However, when meiotic DSBs are eliminated in Mcm5^A7 mutants (as shown through mei-P22 Mcm5^A7 double mutants), homologs are unpaired at a frequency similar to Mcm5^A7 mutants, indicating that the opposing force is independent of recombination. Regardless of the origin of the force, it is proposed that SMC1-dependent centromere clusters act as anchors at the nuclear envelope to maintain the proximity of homologous axes (Hatkevich, 2019).

Although meiotic pairing programs vary among organisms, the SMC1-dependent centromere clustering pairing model may be broadly applicable. In Drosophila and C. elegans, meiotic pairing is independent of meiotic recombination. In contrast, meiotic pairing in organisms such as yeast, plants, and mice require DSB formation (although recombination-independent alignment is required for pairing in these organisms). In DSB-dependent pairing programs, homologs are considered paired at ~400 nm, where DSB-mediated interhomolog interactions can be visualized as bridges. However, contemporaneous with DSB formation, centromeres are coupled or clustered. It is speculated that these centromere interactions stabilize the DSB-dependent arm pairing to ensure synapsis exclusively between homologs in many sexually-reproducing organisms (Hatkevich, 2019).

This study reveals the interesting phenomenon of stable heterosynapsis in Drosophila. Extensive heterosynapsis has been previously reported in C. elegans and yeast with variable SC integrity. Though SC aberrations in Mcm5^A7 mutants cannot be ruled out, the data reveal no structural defects, supporting the notion that 'normal' synapsis is largely homology-independent in Drosophila, as has been observed in C. elegans (Hatkevich, 2019).

In Drosophila, synapsis along the arms initiates as patches during zygotene. In Mcm5^A7 mutants, synapsis initiation between paired homologs appears normal in zygotene but SC elongation fails to be limited to homologous regions. Thus, initiation of synapsis may require homology, but elongation may not. Because this study examined only specific loci and not whole chromosomes, future studies determining the degree of heterosynapsis in Mcm5^A7 mutants may provide more insight into how synapsis and homology interact in flies (Hatkevich, 2019).

While Mcm5^A7 has proven to be a valuable genetic tool, how this particular mutation affects SMC1 localization at the meiotic centromere is unknown. Mcm5^A7 mutants do not affect centromere clustering and pairing in a manner similar to that of mutants that disrupt centromere integrity, such as cal1 Cenp-C double heterozygotes. However, the results do not exclude a role for MCM5 in centromere function or integrity (Hatkevich, 2019).

The canonical role of MCM5 is to function within the replicative helicase, MCM2-7, unwinding double-stranded DNA ahead of the replication fork during S-phase. Because of its important replication role, Mcm5 is an essential gene in every proliferating cell. Numerous studies have shown that replication is required for cohesion localization and establishment, but examining a direct role for any MCM protein in cohesin deposition is difficult since MCMs are essential for replication, which in turn is required for establishing cohesion (Hatkevich, 2019).

Because MCM5 functions within the MCM2-7 replicative helicase, it is tempting to speculate that the Mcm5^A7 mutation may directly perturb SMC1 localization, either through defects in replication or cohesin deposition. No replication defect in Mcm5^A7 mutants has been detected, in either a mitotic or meiotic context. In the future, when individual pre-meiotic nuclei can be isolated from cysts, higher-resolution replication assays may determine whether replication is subtly disrupted in Mcm5^A7 mutants. At this point, however, it seems more likely that the Mcm5^A7 cleanly separates the replication role of MCM5 from a role in meiotic cohesin deposition (Hatkevich, 2019).

Cohesin occupancy and composition at enhancers and promoters are linked to DNA replication origin proximity in Drosophila

Cohesin consists of the SMC1-SMC3-Rad21 tripartite ring and the SA (Stromalin) protein that interacts with Rad21. The Nipped-B protein loads cohesin topologically around chromosomes to mediate sister chromatid cohesion and facilitate long-range control of gene transcription. It is largely unknown how Nipped-B and cohesin associate specifically with gene promoters and transcriptional enhancers, or how sister chromatid cohesion is established. This study used genome-wide chromatin immunoprecipitation in Drosophila cells to show that Stromalin (SA) and the Fs(1)h (BRD4) BET domain protein help recruit Nipped-B and cohesin to enhancers and DNA replication origins, whereas the MED30 subunit of the Mediator complex directs Nipped-B and Verthandi/Rad21 in Drosophila to promoters. All enhancers and their neighboring promoters are close to DNA replication origins and bind SA with proportional levels of cohesin subunits. Most promoters are far from origins and lack SA but bind Nipped-B and Rad21 with subproportional amounts of SMC1, indicating that they bind cohesin rings only part of the time. Genetic data show that Nipped-B and Rad21 function together with Fs(1)h to facilitate Drosophila development. These findings show that Nipped-B and cohesin are differentially targeted to enhancers and promoters, and suggest models for how SA and DNA replication help establish sister chromatid cohesion and facilitate enhancer-promoter communication. They indicate that SA is not an obligatory cohesin subunit but a factor that controls cohesin location on chromosomes (Pherson, 2019).

Cohesin mediates sister chromatid cohesion to ensure accurate chromosome segregation and also plays roles in DNA repair and gene transcription. In Drosophila, cohesin facilitates enhancer-promoter communication and regulates activity of the Polycomb repressive complex 1 at silenced and active genes (Pherson, 2019).

Cohesin structure and chromosome binding are relatively well understood. The SMC1, SMC3, and Rad21 subunits form a tripartite ring and SA interacts with Rad21. A Nipped-B-Mau2 complex loads cohesin topologically around chromosomes and a Pds5-Wapl complex removes cohesin. SA, Nipped-B, Pds5, and Wapl contain HEAT repeats and interact with cohesin to control its binding and activities. These accessory proteins facilitate ring opening to load and remove cohesin from chromosomes (Pherson, 2019).

Less is known about how cohesin is targeted to sequences that control gene transcription or how sister chromatid cohesion is established. In Drosophila, cohesin associates with active genes, transcriptional enhancers, and the Polycomb response elements (PREs) that control epigenetic gene silencing. Cohesin occupies all enhancers and PREs, and preferentially those active genes positioned within several kilobases of the early DNA replication origins (Pherson, 2019).

The Pds5 and Wapl cohesin removal factors limit the size of cohesin domains surrounding early origins, whereas Pds5 and the Brca2 DNA repair protein, which form a complex lacking Wapl have opposing effects on SA origin occupancy and sister chromatid cohesion. Pds5 is required for sister chromatid cohesion and facilitates SA binding, whereas Brca2 inhibits SA binding and counters the ability of Pds5 to support sister cohesion when Pds5 levels are low. These findings gave rise to the idea that Pds5 and SA function at replication origins to establish chromatid cohesion (Pherson, 2019).

To gain more insight into how cohesin associates with gene regulatory sequences genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) was used to investigate how multiple cohesin subunits occupy different genomic features in Drosophila cells. The roles were also examined of cohesin subunits, the Mediator complex, and the Fs(1)h (BRD4) BET domain protein in cohesin localization. The results indicate that cohesin associates with enhancers and most promoters by different mechanisms, and that proximity to DNA replication origins influences cohesin occupancy and composition (Pherson, 2019).

The experiments show that SA helps recruit complete cohesin complexes to enhancers, which are all located close to early DNA replication origins and to those promoters that are also close to origins. Nipped-B and Rad21 also occupy origin-distal promoters, which bind cohesin rings only part of the time. The MED30 subunit of the Mediator complex facilitates association of Nipped-B and Rad21 with all promoters and the Fs(1)h (BRD4) mitotic bookmarking protein facilitates cohesin association with enhancers and the origin-proximal promoters. Genetic evidence shows that Fs(1)h functions together with Nipped-B and Rad21 in vivo to support development (Pherson, 2019).

Only those promoters that are close to enhancers and origins are occupied primarily by complete cohesin complexes. It is thus theorized that these are the promoters that are targeted by enhancers. It is envisioned that DNA replication pushes cohesin from enhancers to origin-proximal promoters based on the evidence that replication origins form preferentially at enhancers and prior indications that replication pushes cohesin. It is not known if the Nipped-B and Rad21 that bind origin-distal promoters independently of SA and SMC1 influence gene transcription. This will be challenging to unravel because Nipped-B and Rad21 are essential for complete cohesin rings to bind to chromosomes (Pherson, 2019).

Since it was discovered that sister chromatid cohesion proteins facilitate expression of enhancer-activated genes, it has been proposed that enhancer-promoter looping could be supported by intra-chromosomal cohesion. In the simplest version, a cohesin ring topologically encircles DNA near both the enhancer and the promoter to hold them together. The cohesin at the enhancer and promoter are thus the same molecules. Some of the current findings argue against this idea. In particular, MED30 depletion reduces Nipped-B and Rad21 at origin-proximal promoters but not at the linked enhancers, indicating that different cohesin molecules are present at the enhancers and promoters. It could be that a cohesin ring at a promoter interacts with another at an enhancer to handcuff them together, or that cohesin interacts with Mediator, BRD4, or other proteins to stabilize enhancer-promoter looping (Pherson, 2019).

Cohesin is removed from chromosomes at mitosis and loaded in early G1. Thus, the idea that DNA replication localizes cohesin to facilitate enhancer-promoter communication raises the question of how cohesin supports enhancer function in G1 before replication. One idea is that mitotic bookmarking factors facilitate cohesin loading at enhancers and target promoters. The BRD4 ortholog of Fs(1)h remains bound to mitotic chromosomes and promotes rapid reactivation of transcription after cell division. Thus, the finding that inhibiting Fs(1)h chromosome binding reduces Nipped-B and Rad21 at enhancers and origin-proximal promoters without going through cell division supports the idea that Fs(1)h marks them for cohesin loading (Pherson, 2019).

It is hypothesized that origins form at enhancers because enhancers trap the sliding MCM2-7 helicase that will initiate DNA replication. Localization of cohesin to enhancers and origins suggests a simple model for how sister chromatid cohesion is established. Upon initial unwinding of the DNA template by MCM2-7, cohesin behind the nascent replication forks encircles the two single-stranded templates, passively establishing cohesion while cohesin in front of the forks is pushed to origin-proximal promoters (Pherson, 2019).

This model explains why Pds5, a cohesin removal factor, and SA, which is not required for cohesin to bind chromosomes topologically, are required for sister chromatid cohesion. By positioning cohesin at enhancers, they ensure that the nascent sister chromatids will be topologically trapped within cohesin. This does not require that replisomes move through cohesin or new cohesin loading behind the fork as proposed in other models. It is consistent with the finding that cohesin can remain chromosome-bound and establish cohesion during DNA replication in the absence of the Wapl removal factor (Pherson, 2019).

Mammals have two SA orthologs, SA1 (STAG1) and SA2 (STAG2). Only SA2-containing cohesin is present at enhancers in human cells, suggesting that SA2 is the functional ortholog of Drosophila SA. SA2 binds DNA independently of cohesin in vitro with a preference for single-stranded DNA and structures resembling replication forks. This is consistent with the findings that SA is origin-centric and spreads further than cohesin around enhancers (Pherson, 2019).

Mutations in the STAG2 gene encoding SA2 cause intellectual and growth deficits overlapping those seen in cohesinopathies caused by mutations in NIPBL or cohesin subunit genes. Individuals with BRD4 mutations display similar birth defects, and BRD4 and NIPBL colocalize at enhancers. These studies agree with the findings that SA and Fs(1)h facilitate association of Nipped-B and Rad21 with enhancers and that Fs(1)h and Nipped-B function together in development (Pherson, 2019).

The data show parallels with cohesin loading in Xenopus. Cohesin loading in Xenopus oocyte extracts requires assembly of the prereplication complex that licenses replication origins and the Cdc7-Drf1 kinase that activates the prereplication complex interacts with NIPBL. This places cohesin at the site of replication initiation, similar to the role of SA in Drosophila (Pherson, 2019).

Specialized DNA replication factors are needed to establish sister chromatid cohesion in yeast, but it is unclear whether they are required at progressing forks or only upon initiation of replication. A study in human cells showed that NIPBL and cohesin interact with the MCM2-7 helicase. It has been suggested that NIPBL bound to MCM2-7 is transiently held by the replisome and transferred behind the fork to load cohesin and establish sister cohesion, but it is possible that interactions with NIPBL could also trap MCM2-7 at enhancers prior to replication. Whether or not recruiting both MCM2-7 and cohesin to origins is sufficient to establish cohesion or whether cohesion requires new cohesin loading behind the replication fork remains to be resolved (Pherson, 2019).

Stromalin constrains memory acquisition by developmentally limiting synaptic vesicle pool size

Stromalin, a cohesin complex protein, was recently identified as a novel memory suppressor gene, but its mechanism remained unknown. This study shows that Stromalin functions as a negative regulator of synaptic vesicle (SV) pool size in Drosophila neurons. Stromalin knockdown in dopamine neurons during a critical developmental period enhances learning and increases SV pool size without altering the number of dopamine neurons, their axons, or synapses. The developmental effect of Stromalin knockdown persists into adulthood, leading to strengthened synaptic connections and enhanced olfactory memory acquisition in adult flies. Correcting the SV content in dopamine neuron axon terminals by impairing anterograde SV trafficking motor protein Unc104/KIF1A rescues the enhanced-learning phenotype in Stromalin knockdown flies. These results identify a new mechanism for memory suppression and reveal that the size of the SV pool is controlled genetically and independent from other aspects of neuron structure and function through Stromalin (Phan, 2018).

Mutations in the highly conserved cohesin complex genes SMC1, SMC3, Rad21, and stromalin (STAG1/2 in mammals) are known to cause cohesinopathies, such as Cornelia de Lange Syndrome. The current observations prompt the important question of whether alterations in the synaptic vesicle pool and synaptic communication underlie some of the phenotypes associated with the cohesinopathies. The increased memory performance that was observed with Stromalin and SMC1 KD seems at odds with some phenotypes like intellectual disability found in patients. However, an increase in the SV pool across many different types of cells in the human brain resulting from a genomic mutation may produce a more complex and opposite phenotype for learning. Other behavioral phenotypes associated with cohesinopathies, including attention deficit disorder, hyperactivity, repetitive behaviors, and autistic behaviors, might also be explainable by altered synaptic vesicle pools and can interfere with learning and memory processes. Furthermore, the increased SV phenotype may also explain the susceptibility of individuals with cohesinopathies to seizures, since SV depletion following repeated neural stimulation is a common mechanism for synaptic depression, important for limiting synaptic hyperactivity that can otherwise lead to runaway network activity. Thus, cohesin complex gene mutations may attenuate SV depletion, thereby impairing normal synaptic depression and contributing to the development of seizures and behavioral dysfunction in humans (Phan, 2018).

Oxidative stress in oocytes during midprophase induces premature loss of cohesion and chromosome segregation errors

One hallmark of aging cells is an increase in oxidative damage caused by reactive oxygen species (ROS). Increased oxidative damage in older oocytes may be one of the factors that leads to premature loss of chromosome cohesion and segregation errors. To test this hypothesis, an RNAi strategy was used to induce oxidative stress in Drosophila oocytes, and the fidelity of chromosome segregation was measured during meiosis. Knockdown of either the cytoplasmic SOD or mitochondrial ROS scavenger superoxide dismutase (SOD) caused a significant increase in segregation errors, and heterozygosity for an smc1 deletion enhanced this phenotype. FISH analysis indicated that SOD knockdown moderately increased the percentage of oocytes with arm cohesion defects. Consistent with premature loss of arm cohesion and destabilization of chiasmata, the frequency at which recombinant homologs missegregate during meiosis I is significantly greater in SOD knockdown oocytes than in controls. Together these results provide an in vivo demonstration that oxidative stress during meiotic prophase induces chromosome segregation errors and support the model that accelerated loss of cohesion in aging human oocytes is caused, at least in part, by oxidative damage (Perkins, 2016).

A pathway for synapsis initiation during zygotene in Drosophila oocytes

Formation of the synaptonemal complex (SC), or synapsis, between homologs in meiosis is essential for crossing over and chromosome segregation. How SC assembly initiates is poorly understood but may have a critical role in ensuring synapsis between homologs and regulating double-strand break (DSB) and crossover formation. This study investigated the genetic requirements for synapsis in Drosophila and found that there are three temporally and genetically distinct stages of synapsis initiation. In meiotic prophase 1 'early zygotene' oocytes, synapsis is only observed at the centromeres. It was also found that nonhomologous centromeres are clustered during this process. In 'mid-zygotene' oocytes, SC initiates at several euchromatic sites. The centromeric and first euchromatic SC initiation sites depend on the cohesion protein ORD. In 'late zygotene' oocytes, SC initiates at many more sites that depend on the Kleisin-like protein C(2)M. Surprisingly, late zygotene synapsis initiation events are independent of the earlier mid-zygotene events, whereas both mid and late synapsis initiation events depend on the cohesin subunits SMC1 and SMC3. It is proposed that the enrichment of cohesion proteins at specific sites promotes homolog interactions and the initiation of euchromatic SC assembly independent of DSBs. Furthermore, the early euchromatic SC initiation events at mid-zygotene may be required for DSBs to be repaired as crossovers (Tanneti, 2011).

Drosophila pro-oocytes develop within 16-cell cysts that are arranged in temporal order within the ovary. Each ovary contains several germaria, where pairs of pro-oocytes begin their development and enter prophase in region 2a and a single oocyte is selected by region 3. Oocytes are defined by the presence of the synaptonemal complex (SC), which is detected by antibodies to the transverse element C(3)G, a coiled-coil protein similar to proteins in budding yeast (ZIP1), C. elegans (SYP-1, SYP-2), and mammals (SYCP1). Zygotene pro-oocytes were identified by their patchy C(3)G staining, as opposed to the thread-like staining typical of pachytene. Furthermore, by comparing the amount of synapsis to the relative positions of the pro-oocytes in the wild-type germarium, three stages of zygotene were defined (Tanneti, 2011).

First, early zygotene pro-oocytes have one or two patches of C(3)G that colocalize with CID, a centromere-specific histone H3. These pro-oocytes reside in the earliest (most anterior) part of region 2a, indicating that synapsis initiates at the centromeres before any other sites. These results were confirmed by comparing CID localization to histone modifications specific for the heterochromatin or euchromatin. Because there are four pairs of centromeres, the observation that most wild-type pro-oocytes have one or two CID foci indicates that nonhomologous centromeres cluster in meiotic prophase, confirming previous observations using electron microscopy (Tanneti, 2011).

Second, mid-zygotene pro-oocytes have the centromeric C(3)G staining plus approximately six additional sites in the euchromatin. Finally, late zygotene pro-oocytes contain many C(3)G foci but lack the continuous threadlike pattern of pachytene. Surprisingly, the mid-zygotene patches do not appear to get longer. Instead, there are more patches in late zygotene, suggesting that the progression from mid- to late zygotene involves the establishment of new SC initiation sites rather than polymerization from the small number of sites in mid-zygotene. It is suggested that the noncentromeric C(3)G sites in mid-zygotene represent the first euchromatic sites to initiate synapsis. This study provides evidence that the mid-zygotene sites have features in common with centromere synapsis sites but are mechanistically distinct and genetically separable from the additional synapsis initiation sites observed in late zygotene (Tanneti, 2011).

C(2)M is a lateral element component and is a member of the Kleisen family that includes Rec8 and Rad21 homologs. In wild-type, C(2)M colocalizes with C(3)G in most locations except at the centromeres. In females lacking C(2)M, the first two stages of zygotene appear to occur normally. Early zygotene pro-oocytes exhibit one or two foci of CID that colocalize with C(3)G, showing that C(2)M is not required for centromere clustering or centromere synapsis. These results confirm previous observations that C(2)M is not required for centromere clustering in pachytene oocytes and are consistent with the observation that C(2)M does not localize to the centromeric regions. Early zygotene in c(2)M mutants is followed by cysts with several patches of euchromatic C(3)G staining that resemble wildtype cells in mid-zygotene. Synapsis in a c(2)M mutant does not, however, progress beyond this point. Examination of histone modifications in c(2)M mutants confirmed that synapsis is blocked in mid-zygotene with a small number of euchromatin initiation sites. Based on the similarities between wild-type mid-zygotene and c(2)M mutants, it is suggested that synapsis initiates in a c(2)M-independent manner at a small number of specialized sites on the chromosomes, which include approximately six euchromatic sites and the centromeres, and that C(2)M is required for additional initiation sites typical of late zygotene (Tanneti, 2011).

There is a striking similarity between the number of euchromatic synapsis initiation sites (~6) during mid-zygotene and the number of crossovers in Drosophila females. In order to determine the relationship between SC initiation sites and double-strand break (DSB) formation, c(2)M mutant oocytes were stained for C(3)G and γ-H2AV. DSBs in a c(2)M mutant are usually associated with a patch of C(3)G staining (55/56 γ-H2AV foci were touching or overlapped a patch of C(3)G). This experiment was also performed in an okr mutant background (okr encodes the Drosophila homolog of Rad54) where the DSBs are not repaired and γ-H2AV staining accumulates, allowing all DSBs to be counted. Most of the γ-H2AV foci in okr c(2)M mutant germaria colocalized with a patch of C(3)G, suggesting that the initiation of SC and recombination usually occur within the same region in c(2)M mutants. Indeed, MEI-P22, a protein required for DSB formation, also colocalizes with the SC in c(2)M mutant oocytes. It should be noted that previous observations showed that DSB formation is partially dependent on the SC. Indeed, the number of γ-H2AV foci in the okr c(2)M double mutant in region 3 oocytes was reduced compared to a okr single mutant. Overall, these results suggest that the SC, or a factor which stimulates SC formation, promotes recruitment of proteins required for DSB formation (Tanneti, 2011).

To investigate whether there is a connection between early SC initiation events and meiotic recombination, double mutants with c(2)M were constructed. Unlike wild-type, where γ-H2AV foci are not observed until pachytene, the block in synapsis observed in c(2)M mutants allowed examination of the relationship between SC initiation and DSB formation. By double staining with CID, it was found that eliminating meiotic DSBs with a mei-W68 mutation did not prevent formation of either the centromere and euchromatic SC in a c(2)M mutant. The small decrease in the number of euchromatic SC sites in the c(2)M mei-W68 double mutant may indicate that the number of initiation sites is sensitive to DSB formation. Furthermore, SC initiation is not grossly affected by a reduction in crossing over (mei-218), an increase in crossing over (TM6), or a defect in DSB repair (okr). DSBs do not occur in the heterochromatin; thus, it is not surprising that centromere SC is independent of DSB formation. However, these results show that the initiation of euchromatic synapsis at mid zygotene does not depend on DSBs or crossovers (Tanneti, 2011).

Because DSBs or recombination are not required for synapsis in wild-type or c(2)M mutants, tests were performed to see whether structural components of the meiotic chromosomes regulate SC initiation. ORD is a meiosis-specific protein required for cohesion and crossover formation that may be a component of the SC lateral elements. Although previous studies have shown that ord mutant oocytes generate threads of C(3)G staining that resemble pachytene, the effect of ord on zygotene progression has not been previously examined (Tanneti, 2011).

Consistent with previous results, this study found that centromere clustering is defective and the association of SC proteins with the centromeres is disrupted in ord mutant oocytes. Furthermore, zygotene appeared abnormal; rather than observing centromeric and euchromatic SC initiation sites typical of mid-zygotene in early region 2a, it was found that many ord mutant pro-oocytes with C(3)G staining only around the nuclear DNA. Of the 108 pro-oocytes examined in five germaria, 36 (33%) had no nuclear C(3)G. The remaining pro-oocytes [72, (67%)] either had a number of C(3)G patches that was more typical of late zygotene, usually in region 2a, or were in pachytene. It is concluded that the centromeric and euchromatic synapsis sites typical of early and mid zygotene are absent in ord mutants, suggesting that, in the absence of ORD, synapsis does not initiate normally (Tanneti, 2011).

Because ord mutants do eventually form threads of SC, it was difficult to be sure that SC initiation was defective. To test whether ord has a role in mid-zygotene synapsis, tests were performed to see whether the euchromatic patches of C(3)G in a c(2)M mutant depend on ord. Even though both single mutants exhibit at least some SC formation, most of the C(3)G staining in the c(2)M ord double mutant surrounded the DNA and within the nucleus. This nonchromosomal C(3)G localization in the c(2)M ord double mutant was much more pronounced than in the ord single mutant. In addition, C(3)G-staining ring-like structures were observed similar to what has been reported in some c(3)G missense mutants. All the nonchromosomal C(3)G staining may be due to polycomplex formation. c(2)M ord double mutant pro-oocytes were identified by the prominent C(3)G around the DNA, and the number of C(3)G patches on the chromosomes was found to be drastically reduced compared to wild-type zygotene or either single mutant (Tanneti, 2011).

These results demonstrate that ord is required for the centromeric and euchromatic synapsis sites observed in c(2)M mutants. Conversely, C(2)M is required for the threadlike synapsis observed in ord mutants. The synergistic phenotype of the double mutant suggests that there are two types of synapsis initiation -- one depends on ORD (early and mid-zygotene) and the other depends on C(2)M (late zygotene) -- and that these are independent events. In the absence of both types of synapsis initiation, C(3)G cannot load onto the chromosomes and accumulates in polycomplexes (Tanneti, 2011).

Like other Kleisin family members, C(2)M has been shown to physically interact with the cohesin subunit SMC3. To determine whether C(2)M localization depends on an interaction with cohesin, oocytes lacking SMC1 and SMC3 were examined. To examine oocytes lacking SMC3 (encoded by cap), the recently developed short hairpin RNA (shRNA) resource, which allows RNA interference (RNAi) knockdown of gene expression in the Drosophila female germline, was used. Both the chromosomal localization of C(3)G and C(2)M were absent when cap shRNA was expressed in the germline. Furthermore, SMC1 staining was eliminated, suggesting that the RNAi was effective at knocking out SMC3 function. Like the c(2)M ord double mutant, most C(3)G staining accumulated around the periphery of the DNA, suggesting that the function of SMC3 in synapsis occurs through at least two independent interactions with C(2)M and ORD. Unlike the c(2)M ord double mutant, however, it was not possible to distinguish the pro-oocytes from the nurse cells because C(3)G staining was evenly distributed among the cells in each germarium cyst. Importantly, oocyte selection was not perturbed because one cell in each cyst accumulated ORB protein, a cytoplasmic marker for the oocyte. Thus, the loss of SMC3 may have a more severe phenotype than the c(2)M ord double mutant (Tanneti, 2011).

These results were confirmed with the analysis of SMC1 mutant germline clones. As with cap RNAi, there was an absence of nuclear C(2)M and C(3)G threads in oocytes lacking SMC1, indicating a complete block in synapsis. Also similar to cap RNAi, the accumulation of ORB in one cell indicated that an oocyte was established. The only difference compared to cap RNAi was that there was much less C(3)G staining around the periphery of the DNA. It is not known whether this minor difference is due to the different methods (RNAi versus germline clone) or distinct functions of the two SMC proteins. Nevertheless, the results of these two experiments demonstrate that SMC1 and SMC3 are required for synapsis (Tanneti, 2011).

It is concluded that synapsis initiation during zygotene in Drosophila females occurs in three stages. In early zygotene, the centromeres are the first sites to accumulate the transverse filament protein C(3)G. Indeed, cohesion proteins SMC1, SMC3, and ORD are detected at the centromeres before meiotic prophase (prior to or during premeiotic S phase), which could explain why synapsis is first observed at the centromeres. Interestingly, the SC also forms first at the centromeres in budding yeast and depends on cohesion proteins. In mid-zygotene, synapsis initiates at a small number of euchromatic sites. These first two steps depend on the ORD protein. Finally, in late zygotene, synapsis initiates at a larger number of euchromatic sites. This stage requires C(2)M and appears to occur through a new set of initiation events rather than extending synapsis, or 'zipping up,' from the mid-zygotene initiation sites. Indeed, the synapsis initiation events in mid and late zygotene are independent and genetically separable, supporting a model where synapsis occurs through two independent waves of initiation events. In the absence of ORD, early and mid-zygotene synapsis events are skipped and the late zygotene initiation events occur with normal kinetics. This is not without consequence, however, because at the electron microscopy level, this synapsis is abnormal and tripartite SC is not visible. Both waves of synapsis initiation depend on the cohesin proteins SMC1 and SMC3, which may interact independently with C(2)M and ORD (Tanneti, 2011).

In addition to its role in centromere synapsis, ORD and the SMC proteins are required for the pairing and clustering of centromeres, whereas the SC components C(2)M or C(3)G are not. Thus, cohesion proteins may be able to function in a pairing role independent of DSBs, as Rec8 does in budding yeast for centromere coupling. It is suggested that the first euchromatic sites to initiate SC assembly in Drosophila are in regions where cohesion proteins are most abundant. This model is attractive because it provides a mechanism for SC initiation in the absence of DSBs. Interestingly, the number of euchromatic initiation sites in mid-zygotene or in c(2)M mutants approximates the number of crossovers in the genome. Not only do these mid-zygotene sites depend on ORD, but in ord mutants, crossing over is reduced to less than 10% of wild-type, even though DSBs occur normally. It is suggested that the reduction in crossing over in ord mutants is due to the absence of the synapsis initiation sites at mid-zygotene. Whether the synapsis initiation sites actually correspond to crossover sites awaits further study (Tanneti, 2011).

ORD may have a function similar to yeast Rec8 because it is required for synapsis at the centromeres and a subset of euchromatic sites. Interestingly, the findings with C(2)M, which is not an ortholog of Rec8, are also probably relevant to other species. Several recent studies have revealed Non-Rec8 Kleisin homologs in mouse and C. elegans (COH-3 and COH-). These parallels between the synapsis pathway in flies and that of organisms that depend on DSBs for synapsis could reflect the existence of a conserved underlying mechanism of synapsis. If synapsis initiation sites can be marked prior to DSB formation in a process involving cohesion proteins, and if proteins like Zip3 can be recruited in the absence of DSBs, as is true in C. elegans and likely in Drosophila, the timing of the DSB then becomes less of a determining factor in the process of synapsis (Tanneti, 2011).

Sisters Unbound is required for meiotic centromeric cohesion in Drosophila melanogaster

Regular meiotic chromosome segregation requires sister centromeres to mono-orient (orient to the same pole) during the first meiotic division (meiosis I) when homologous chromosomes segregate, and to bi-orient (orient to opposite poles) during the second meiotic division (meiosis II) when sister chromatids segregate. Both orientation patterns require cohesion between sister centromeres, which is established during meiotic DNA replication and persists until anaphase of meiosis II. Meiotic cohesion is mediated by a conserved four-protein complex called cohesin that includes two Structural Maintenance of Chromosomes (SMC) subunits (SMC1 and SMC3) and two non-SMC subunits. In Drosophila melanogaster, however, the meiotic cohesion apparatus has not been fully characterized and the non-SMC subunits have not been identified. This study identified a novel Drosophila gene called sisters unbound (sunn) (CG32088), which is required for stable sister chromatid cohesion throughout meiosis. sunn mutations disrupt centromere cohesion during prophase I and cause high frequencies of nondisjunction (NDJ) at both meiotic divisions in both sexes. SUNN co-localizes at centromeres with the cohesion proteins SMC1 and SOLO (Sisters on the loose/Vasa) in both sexes and is necessary for the recruitment of both proteins to centromeres. Although SUNN lacks sequence homology to cohesins, bioinformatic analysis indicates that SUNN may be a structural homolog of the non-SMC cohesin subunit Stromalin (SA), suggesting that SUNN may serve as a meiosis-specific cohesin subunit. In conclusion, these data show that SUNN is an essential meiosis-specific Drosophila cohesion protein (Krishnan, 2014).

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).

The Drosophila cohesin subunit Rad21 is a trithorax group (trxG) protein

The cohesin complex is a key player in regulating cell division. Cohesin proteins SMC1, SMC3, Rad21, and stromalin (SA), along with associated proteins Nipped-B, Pds5, and EcoI, maintain sister chromatid cohesion before segregation to daughter cells during anaphase. Recent chromatin immunoprecipitation (ChIP) data reveal extensive overlap of Nipped-B and cohesin components with RNA polymerase II binding at active genes in Drosophila. These and other data strongly suggest a role for cohesion in transcription; however, there is no clear evidence for any specific mechanisms by which cohesin and associated proteins regulate transcription. This study reports a link between cohesin components and trithorax group (trxG) function, thus implicating these proteins in transcription activation and/or elongation. The Drosophila Rad21 protein is encoded by verthandi (vtd), a member of the trxG gene family that is also involved in regulating the hedgehog (hh) gene. In addition, mutations in the associated protein Nipped-B show similar trxG activity i.e., like vtd, they act as dominant suppressors of Pc and hh^Mrt without impairing cell division. These results provide a framework to further investigate how cohesin and associated components might regulate transcription (Hallson, 2008).

In eukaryotic mitosis, accurate chromosome segregation requires paired sister chromatids to attach to opposite spindle poles. Sister chromatids are held together by the cohesin protein complex, which consists of four core subunits, Rad21/SCC1, stromalin (SA) and structural maintenance of chromosome (SMC) proteins SMC1 and SMC3. A widely accepted model postulates that cohesin forms a ring-like structure via interaction of the N- and C-termini of Rad21 with a SMC1/SMC3 heterodimer. With the participation of SCC2/Nipped-B, SCC4, EcoI/Ctf7, and Pds5 proteins, sister-chromatid cohesion is maintained until the onset of mitosis. Cleavage of Rad21 and the resulting removal of cohesin then allow separation of sister chromatids in anaphase. Mutation of genes encoding these subunits leads to errors in chromosome segregation and aneuploidy, which are hallmarks of cancer and a leading cause of birth defects in humans (Hallson, 2008).

Given the highly conserved role for cohesin in sister chromatid cohesion, it was unexpected to discover that cohesin and associated proteins might also play a distinct, independent role in regulating gene expression. Reduction in Nipped-B expression in Drosophila affects expression of the cut and Ultrabithorax genes, and mutations in the human orthologue, NIPBL, result in Cornelia de Lange Syndrome. In zebrafish, mutations in rad21 or Smc3 affect embryonic runx gene transcription in heterozygous mutant animals without compromising cell division, suggesting that these proteins may have functions in transcription that are distinct from a mitotic role. Recently, extensive overlap has been found of Nipped-B and cohesin components with RNA polymerase II binding at active genes and apparent exclusion from genes silenced by Polycomb group (PcG) genes. This intriguing chromatin immunoprecipitation (ChIP) result strongly suggests a role in transcription for cohesin and Nipped-B, although the mechanisms are unknown (Hallson, 2008).

Trithorax group (trxG) genes encode proteins implicated in transcriptional regulation. These genes were initially characterized as regulators of homeotic genes in Drosophila. The trxG genes are required to maintain activation of homeotic and other genes; many that have been molecularly characterized encode members of multimeric complexes with roles in transcriptional initiation and/or elongation. Typically, mutations in trxG genes suppress the phenotypes of mutations in PcG genes, whose function is to maintain the repressed state of homeotic genes and other developmentally important genes like hedgehog (hh), a gene required for cell signaling (Hallson, 2008).

As part of work toward a functional annotation of heterochromatin of Drosophila, the verthandi (vtd) locus, a member of the trxG gene family with Suppressor of Polycomb [Su(Pc)] activity, was characterized. The vtd locus also affects hh expression; vtd mutations are dominant suppressors of Moonrat (Mrt), a dominant gain of function allele of hh. However, because of its location deep within the centric heterochromatin of the left arm of chromosome, vtd has resisted characterization at the molecular level (Hallson, 2008).

This study reports that vtd mutations, isolated on the basis of their trxG phenotypes, map to the gene encoding the cohesin subunit Rad21 and exhibit corresponding defects in mitosis and sister chromatid cohesion. Mutations in Nipped-B also show trxG phenotypes, and as is the case for vtd, heterozygous mutant flies show trxG phenotypes without significantly affecting cell division. These results provide a link between sister chromatid cohesion proteins and trxG functions, thus suggesting that cohesion factors may act by facilitating transcription activation and/or elongation (Hallson, 2008).

Alleles of vtd have lesions in rad21, mutations or knockdowns of rad21 have vtd phenotypes, and vice versa, and a transgene containing rad21 rescues the lethality of vtd. It is also noteworthy that reductions in Rad21 or Nipped-B dosage alter gene expression without seriously affecting chromatid cohesion, suggesting that these may be separable functions for cohesin and associated proteins. Evidence has accumulated that cohesin and associated proteins have important roles in gene regulation, but the functional basis for this has been unclear. The simplest model that explains the existing data is that Rad21, like most other trxG proteins, facilitates transcription (Hallson, 2008).

In Drosophila, many trxG proteins are subunits of complexes with diverse roles in transcriptional activation. Trx and Ash1 encode SET domain proteins that methylate lysine 4 of histone H3 (H3K4), and Ash2 is a member of a complex that also methylates H3K4. Other trxG proteins (e.g., Brahma, Osa, Moira, Kismet) are members of ATP-dependent nucleosome remodeling complexes. However, despite concerted efforts from many laboratories, the precise mechanisms by which trxG proteins regulate transcription remain unclear. In addition to chromatin modification, trxG proteins appear to be directly involved in recruiting factors required for transcription elongation, and noncoding RNAs may also play a role in regulating some of the affected genes (Hallson, 2008).

The hypothesis that cohesin facilitates transcription is supported by the results of a recent genome-wide ChIP study, which shows preferential binding of Nipped-B and the cohesin subunits SMC1 and SA to transcribed regions, overlapping with RNA polymerase II (Pol II) binding sites. The colocalization of Nipped-B with cohesin on chromosomes, and physical association with SA and Rad21 in extracts further suggests that Nipped-B and cohesin act together (Hallson, 2008).

There are strong correlations between binding of cohesin components and active gene expression. The dosage sensitive suppression of the hh^Mrt gain of function allele by both vtd and Nipped-B mutations suggests that Nipped-B and cohesin both promote expression of hh. It is unknown, however, if this effect is direct. Cohesin or Nipped-B do not bind to the hh gene in any of the three cell lines examined, however, in at least two of these, PcG proteins actively silence hh. Genome-wide, PcG silencing and the resulting histone H3 lysine 27 trimethylation strongly anti-correlates with Nipped-B and cohesin binding. Thus, it would not be expected that cohesin binds hh in these cell lines even if it directly regulates hh. For example, although Nipped-B regulates Ubx expression in vivo, Nipped-B and cohesin are excluded from the silenced Ubx and Abd-A genes in Sg4 cells, but bind to the transcribed Abd-B gene. In cells in which Abd-B is silenced, cohesin does not bind to the Abd-B promoter region. Thus, it remains possible that Nipped-B and cohesin directly stimulate hh transcription in vivo (Hallson, 2008).

Identification of loss of function zebrafish rad21 alleles in a genetic screen for mutations that reduce expression of runx genes also suggests that cohesin promotes gene expression, but again, it is unknown if this effect is direct. Stronger evidence supporting the idea that cohesin directly stimulates transcription arises from a recent study on axon pruning in the Drosophila mushroom body. In this study, loss of function alleles of the Smc1 and SA genes were isolated in a screen for mutations that block pruning. The lack of pruning correlated with reduced expression of the ecdysone receptor (EcR) gene, and could be partially rescued by ectopic EcR expression. Nipped-B and cohesin bind to the transcribed portion of the EcR gene in all three cell lines examined, including the ML-DmBG3 line derived from third instar central nervous system, suggesting that they directly facilitate EcR expression (Hallson, 2008).

The question remains as to whether the same cohesin complexes required for cohesion of sister chromatids also function in transcription regulation, or whether, analogous to trxG proteins, different cohesin subunits have different functions in transcription, presumably because they are members of different complexes. One might conclude the latter based upon the observation that reductions in Rad21, SA, or SMC1 all increase cut expression, whereas decreases in Nipped-B reduce cut expression. These effects are likely direct because cohesin and Nipped-B bind to a 180 kb region that encompasses the entire upstream regulatory and transcribed regions of cut in ML-DmBG3 cells. The expression of RNAi transgenes encoding for SA and Rad21 decreases the severity of the cut^K allele, whereas Nipped-B mutations enhance the cut^K phenotype, also suggesting that they have opposite effects at cut. Finally, in contrast to results with Nipped-B mutations, no consistent effects on cut expression were observed for vtd mutant heterozygotes; moreover, mutations in vtd and Nipped-B both suppress the phenotypes of Pc⁴ and Mrt, but mutations in Smc1 or pds5 did not. Similarly, it has been reported that null alleles of the cohesion factors sans and deco have no effect on the expression of cut when a functional chromosomal copy is present. Based on all of the above evidence, one might therefore conclude that different cohesin components may act differentially, possibly because, like trxG proteins, they are members of different regulatory complexes (Hallson, 2008).

However, it is also possible that the same cohesin complex involved in chromatid cohesion also regulates transcription, if binding at different loci results in different, gene specific consequences. Thus, in cut, which is activated by a remote wing margin enhancer located >80 kb upstream of the promoter, it has been proposed that cohesin could inhibit long range activation, and that Nipped-B facilitates activation by maintaining a dynamic cohesin binding equilibrium. In other genes, such as EcR or hh, cohesin might help maintain open chromatin to facilitate transcription by encircling a 10-nm fiber and preventing refolding to a higher order structure. As for the differences observed in the genetics of cohesin components, there are likewise other plausible explanations: differences in genetic background of mutant lines tested, differences in maternal expression/loading of required gene products in different heterozygous flies, or the possibility that the cut^K or Pc alleles are less sensitive to changes in rad21/vtd, SMC1, or pds5 gene dosage than they are to the gene dosage of Nipped-B. Consistent with this idea, effects of rad21 dosage on cut expression were observed when RNAi was used to deplete rad21 mRNA, presumably to levels lower than those available in vtd(⁺) heterozygotes. It was also reported that Nipped-B expression is not directly proportional to gene dosage. The data in this study also show that reductions in Nipped-B and rad21 dosage act in the same direction i.e., suppress Mrt and Pc, suggesting that both genes may contribute to gene activation. The fact that both the rad21 and Nipped-B genes are resident within a late-replicating, heterochromatic environment may also explain some differences in outcomes of genetics tests of cohesin subunit function (Hallson, 2008).

These results provide a link between cohesin binding and trxG gene function. It will be an interesting challenge for the future to determine how components involved in chromatid cohesion act at the molecular level to regulate transcription, particularly given other very recent evidence implicating cohesin in gene regulation. The discovery that vtd encodes the Rad21 cohesin subunit expands the known roles of cohesin and Nipped-B in Drosophila development to include regulation of hh, which like cut, Ubx, and EcR, has many developmental roles. Similar modulation of key developmental regulators in humans, each with multiple roles, could explain why Cornelia de Lange syndrome patients have multiple diverse developmental deficits (Hallson, 2008).

Regulation of meiotic cohesion and chromosome core morphogenesis during pachytene in Drosophila oocytes

During meiosis, cohesion between sister chromatids is required for normal levels of homologous recombination, maintenance of chiasmata and accurate chromosome segregation during both divisions. ORD activity is essential for the crucial decision each chromatid must make after the induction of DSBs (double strand breaks) - namely whether the broken chromatid will choose its sister or its homologue for repair. Strand invasion and crossovers are biased towards the homologue during meiosis, resulting in stable chiasmata that keep homologous chromosomes physically associated until anaphase I. In Drosophila, null mutations in the ord gene abolish meiotic cohesion, although how ORD protein promotes cohesion has remained elusive. This study shows that SMC (structural maintenance of chromosome) subunits of the cohesin complex (see A Ring for Holding Sister Chromatids Together?) colocalize with ORD at centromeres of ovarian germ-line cells. In addition, cohesin SMCs and ORD are visible along the length of meiotic chromosomes during pachytene and remain associated with chromosome cores following DNase I digestion. In flies lacking ORD activity, cohesin SMCs fail to accumulate at oocyte centromeres. Although SMC1 and SMC3 localization along chromosome cores appears normal during early pachytene in ord mutant oocytes, the cores disassemble as meiosis progresses. These data suggest that cohesin loading and/or accumulation at centromeres versus arms is under differential control during Drosophila meiosis. The experiments also reveal that the α-kleisin C(2)M is required for the assembly of chromosome cores during pachytene but is not involved in recruitment of cohesin SMCs to the centromeres. A model is presented for how chromosome cores are assembled during Drosophila meiosis and the role of ORD in meiotic cohesion, chromosome core maintenance and homologous recombination (Khetani, 2007).

Accurate segregation of chromosomes during meiosis relies on a number of dynamic changes in chromosome morphology that take place within the context of sister-chromatid cohesion. Meiotic cohesion is not only required for the correct segregation of sisters during the second meiotic division, but also ensures that recombinant homologous chromosomes remain physically associated until anaphase I. In addition, arm and centromeric cohesion must be regulated differently during meiosis. When the release of arm cohesion during meiosis I allows the segregation of homologues, centromeric cohesion must be protected and remain intact until anaphase II when sisters segregate to opposite poles (Khetani, 2007).

Cohesion between meiotic sister chromatids plays an essential role in assembly of the synaptonemal complex (SC), a tripartite proteinaceous structure that forms between homologous chromosomes during prophase I. During early prophase I, each pair of sister chromatids undergoes shortening along their longitudinal axes, resulting in the formation of 'chromosome cores' upon which the axial/lateral elements (AEs/LEs) of the SC assemble. During pachytene, SC central element proteins join each set of homologous AEs/LEs along their entire length resulting in synapsis of homologues. In many species (yeast, mice, Arabidopsis), meiotic double-strand breaks (DSBs) are essential for homologue synapsis; however, chromosome core formation (axial shortening) does not depend on DSBs. In addition, mutants that lack AE/LE components can still build chromosome cores (Khetani, 2007).

Crossovers between homologous chromosomes, in conjunction with sister chromatid cohesion, are essential for correct chromosome segregation during meiosis I. In most organisms, recombination between homologues takes place in the context of the SC. Although EM studies indicate that the ultrastructure of the SC is highly conserved, SC components in different organisms show surprisingly little sequence homology (Khetani, 2007).

During both mitosis and meiosis, sister-chromatid cohesion is mediated by an evolutionarily conserved protein complex called cohesin that contains two SMC and two non-SMC subunits (Lee, 2001; Petronczki, 2003). The α-kleisin subunit (Scc1/Mcd1/Rad21) bridges the two head domains of the SMC1-SMC3 dimer and thereby forms a ring that entraps DNA (Nasmyth, 2002; Shintomi, 2007). Several meiosis-specific cohesin subunits have been identified, including the α-kleisin Rec8, which has been shown to be crucial for meiotic cohesion and SC formation in all organisms examined (Khetani, 2007).

In Drosophila, four cohesin subunits have been uncovered through sequence analysis and the localization and function of mitotic cohesin has been examined in Drosophila embryos and tissue culture cells. However, little is known about the localization and dynamics of the cohesin complex during Drosophila meiosis (Khetani, 2007).

Drosophila oogenesis is an excellent system to study meiosis, as each Drosophila ovary is composed of approximately 10-30 ovarioles that contain a linear array of oocytes at progressive developmental stages from mitotic germ-line stem cells to metaphase-I-arrested oocytes. Meiosis initiates in the germarium, the most anterior structure of each ovariole. Germ-line stem cells in region 1 of the germarium undergo four rounds of synchronous mitotic divisions resulting in 16 interconnected cells that comprise a 'cyst'. As cysts mature, they move toward the posterior end of the ovariole. All germ cells within a 16-cell cyst undergo pre-meiotic S phase synchronously and prophase I of meiosis initiates in germarial region 2A where up to four cells per cyst initiate SC assembly. In addition, meiotic DSBs are induced in region 2A, but unlike several other organisms, synapsis in Drosophila does not depend on DSBs. As each cyst moves through the germarium, the SC breaks down in all but one nucleus so that, by region 3, full-length SC is restricted to the oocyte, which lies at the posterior end of the rounded cyst. As cysts continue to grow and mature, they leave the germarium and move into the 'vitellarium'. The oocyte remains in pachytene with full-length SC until vitellarial stage 6; however, the remaining 15 cells within each cyst adopt a nurse cell fate and enter an endo cell cycle, during which multiple rounds of S phase in the absence of intervening M phase results in polyploid cells (Khetani, 2007).

Meiotic cohesion in Drosophila depends on the novel protein, Orientation Disruptor (ORD). In mutants lacking ORD function, sister-chromatids segregate randomly through both meiotic divisions, consistent with complete absence of meiotic cohesion. In addition, homologous recombination is severely reduced in ord^null females and SC assembly and maintenance are disrupted. Immunolocalization studies have demonstrated that ORD is enriched at the centromeres of meiotic chromosomes in both males and females. In addition, ORD localization along the arms of female meiotic chromosomes coincides with that of the SC protein, C(3)G (Khetani, 2007 and references therein).

This study investigated the localization and dynamics of two cohesin subunits (dSMC1 and Cap/dSMC3) during early prophase I in Drosophila oogenesis. SMC1 and SMC3 localize along the arms and are enriched at the centromeres of all 16 cells within each germ-line cyst. In nuclei that build SC, cohesin subunits coalesce into chromosome cores that provide the scaffold for SC assembly. Formation of chromosome cores depends on the α-kleisin C(2)M, and the cohesion protein ORD is essential for cohesin loading at centromeres and for maintenance of chromosome cores. These data support the argument that during meiosis, the establishment of centromeric cohesion is regulated differently than on the arms. Moreover, these results provide insight into the interconnected roles of meiotic cohesion, chromosome cores and homologous recombination (Khetani, 2007).

Accumulation of cohesin SMCs on chromosomes of pre-meiotic cells: To analyze the behavior of cohesin subunits during meiotic progression in the Drosophila ovary, antibodies were generated against Drosophila SMC1 and SMC3 peptides. Following affinity-purification, SMC1 and SMC3 antibodies each recognize a single predominant band at the predicted molecular mass in embryo extracts and a doublet/triplet in ovary extracts. In addition, when germ-line clones are generated that are homozygous for the smc1 excision allele, smc1^exc46, no SMC1 signal above background is observed. Because affinity-purified SMC1 and SMC3 antibodies display very similar staining patterns in Drosophila ovaries, they were combined for most of the experiments described below to maximize signal intensity (here referred to as SMC1/3 or SMC) (Khetani, 2007).

Fixation and staining of intact ovarioles (whole-mount preparations) revealed several distinct cohesin SMC staining patterns within the germaria of wild-type females; multiple regions of each germarium contained bright foci, as well as diffuse staining and nuclei with a thread-like SMC signal. Although no cohesin SMC staining was detected in germarial stem cells and early cystoblasts in region 1, bright foci as well as diffuse SMC localization are visible in the nuclei of germ-line cysts within region 1. The bright SMC foci correspond to centromeres as confirmed by co-staining with CID, a centromere-specific histone H3 variant. The diffuse staining in pre-meiotic cells most probably corresponds to cohesin localization along chromosome arms. This same localization pattern has been observed (Webber, 2004) for the cohesion protein ORD in germ-line mitotic cysts (Khetani, 2007).

Thread-like cohesin SMC signal coincides with the SC in pachytene nuclei: As 16-cell cysts enter region 2A of the germarium, up to four nuclei in each cyst begin to assemble a SC and in these cells, SMC1/3 signal becomes visible as thread-like staining that coincides with the SC marker C(3)G. During the maturation of cysts and their progression through the germarium, thread-like SMC1/3 staining mimics that of the SC. As cysts move through the germarium, continuous linear SMC1/3 staining is visible in the two nuclei that contain full-length SC (pro-oocytes) but the SMC1/3 signal appears fragmented in the other C(3)G containing nuclei that will adopt a nurse cell fate (pro-nurse cells). Oocyte determination is complete by region 3 and, at this stage, the continuous thread-like SMC1/3 staining is restricted to the oocyte (Khetani, 2007).

The oocyte nucleus will remain in pachytene for several hours as it progresses through the vitellarium. Electron microscopy has shown that that full-length tripartite SC is present as late as stage 6 of vitellarial development and these data have been supported by persistence of continuous threads of C(3)G immunostaining until the same stage (Page and Hawley, 2001). In vitellarial stages 2 to 6, thread-like SMC1/3 staining is observed in whole-mount preparations that is coincident with C(3)G signal. However, similar to C(3)G staining, the thread-like signal becomes weaker in these later stages and is accompanied by increased diffuse nuclear staining (Khetani, 2007).

Cohesin SMCs and ORD are present along chromosome cores during pachytene: Thread-like signals for cohesin SMCs as well as the cohesion protein ORD are restricted to germ-line cells that form SC (Webber, 2004). One possibility is that, together, these proteins contribute to the proteinaceous 'chromosome core' that has been proposed to serve as a scaffold for SC formation. If the cohesin complex and ORD are indeed part of the chromosome core, they should persist in the absence of DNA loops in SC-containing nuclei. To test this hypothesis, chromosome spreads of germarial cells were prepared to visualize proteins bound to meiotic chromosomes. In this procedure, soluble components are washed away, leaving only chromosomes and their associated proteins attached to the slide. DNase I treatment of chromosome spread slides resulted in loss of histone and DAPI staining, confirming that DNA loops had been digested. However, the thread-like SMC1/3 and ORD staining persisted in the absence of DNA loops, consistent with the model that ORD and the cohesin complex are components of the cores of meiotic chromosomes in SC-forming nuclei (Khetani, 2007).

Chromosome spread experiments also revealed that cohesin SMCs are associated with chromosome arms in all 16 nuclei of each germ-line cyst. However, in nuclei that do not build a SC, the SMC localization pattern is diffuse rather than thread-like. In addition, it was observed that during SC disassembly in non-oocyte nuclei, cohesin SMCs remain associated with chromosome arms and their staining pattern is indistinguishable from other pro-nurse cells. Association of ORD with chromosome arms in a pattern similar to SMC1/3 has been described previously (Webber, 2004). Soluble nuclear proteins are removed during the spread preparation. When transgenic flies expressing GFP-nls were used to generate spreads, diffuse SMC1 staining was visible in several nuclei but no corresponding GFP signal was detected. Therefore, it is concluded that the diffuse SMC1/3 staining observed in chromosome spreads represents cohesin SMCs stably associated with the chromatin. These data support the model that cohesin SMCs and ORD associate with the arms of all germ-line chromosomes and in cells that build SC, cohesion proteins coalesce into continuous threads that represent chromosome cores. Interestingly, diffuse SMC staining is often not visible in spread preparations of SC containing nuclei, suggesting that most or all of the cohesin complex in these cells is located along the cores, not the loops of meiotic chromosomes (Khetani, 2007).

Enrichment of cohesin SMCs at the centromeres of meiotic chromosomes: In addition to the thread-like staining pattern in pachytene cells, cohesin SMCs are enriched at the centromeres of wild-type meiotic chromosomes as confirmed by colocalization of the SMC1/3 foci with CID, the centromere specific histone H3 variant. The centromeres of Drosophila chromosomes are usually clustered together into a single chromocenter and each bright focus of SMC1/3 and ORD staining that is visible in the nuclei of whole-mount preparations corresponds to the chromocenter. In chromosome spreads, the chromocenter is frequently observed split into two or more regions. Interestingly, the increased resolution afforded by spread preparations indicates that the bright SMC1/3 signal at centromeres often extends beyond the area of CID staining. Cohesin SMCs exhibit the same extensive centromeric localization pattern as ORD (Webber, 2004), consistent with enrichment of these cohesion proteins within pericentric as well as centromeric heterochromatin. Robust SMC1/3 and ORD signals in the vicinity of the centromere are not restricted to nuclei that build SC. Instead, centromeric enrichment of these proteins is visible in all 16 cells of germarial cysts (Webber, 2004) whether they adopt a nurse cell or oocyte fate (Khetani, 2007).

As egg chambers progress into the vitellarium, the SMC1/3 signal associated with the chromocenter in nurse cells begins to assume a very distinctive pattern that resembles a cluster of finger-like projections. Interestingly, the onset of this staining pattern coincides with the beginning of the endo-reduplication cell cycle in nurse cells, during which DNA replication occurs repeatedly in the absence of cell division. These SMC1/3 finger-like projections are observed during early vitellarial stages when the polyploid nurse cell chromosomes exhibit polyteny, the precise alignment of multiple copies of sister chromatids. Unlike polytene chromosomes in the Drosophila salivary gland, nurse cell polytene chromosomes are short-lived. Around vitellarial stage 4, nurse cell chromosomes undergo a dramatic morphological change and no longer exhibit polyteny. Although the SMC1/3 signal remains enriched at the pericentric heterochromatin as nurse cell chromosomes transition out of polyteny, the pattern becomes more diffuse and less structured in these later stages (Khetani, 2007).

ORD is required for centromeric localization of cohesin SMCs during meiosis: ORD protein is necessary for both arm and centromeric cohesion during Drosophila meiosis. In mutant flies lacking ORD activity, chromosomes segregate randomly through both meiotic divisions, indicating that cohesion is completely absent. The localization pattern of ORD protein during early oogenesis closely mimics that of the cohesin SMC proteins. One possibility is that ORD controls the localization and/or function of the cohesin complex during meiosis. To study the localization dynamics of the cohesin complex in the absence of ORD, ovaries from ord⁵/Df (ord^null) females were examined. The ord⁵ mutation results in premature truncation of the ORD open reading frame and genetically behaves like a null-allele (Khetani, 2007 and references therein).

When whole-mount ord^null germaria are stained with SMC1 and SMC3 antibodies, bright centromeric foci are conspicuously absent throughout the germaria even though continuous thread-like staining is visible in region 2A. Within the germarium, SMC1/3 foci are undetectable in cells that form SC as well as the remaining cells of each cyst. ord^null oocytes also lack SMC1/3 centromeric foci after they exit the germarium. These data suggest that ORD is essential for normal accumulation of cohesin at oocyte centromeres and are consistent with the chromosome segregation defects observed in mutant flies. Absence of cohesin SMC localization at centromeres in the ord mutant was confirmed when chromosome spreads were immunostained for SMC1/3 and the centromere marker CID. Cohesin SMCs do not colocalize with CID foci in ord^null germaria; the CID signal corresponds to gaps in the thread-like SMC1/3 signal. These data suggest that ORD activity is required for loading and/or accumulation of centromeric cohesin during female meiosis (Khetani, 2007).

Interestingly, although centromeric SMC1/3 staining is never visible in oocytes of ord^null flies, a distinct centromeric staining pattern becomes detectable in nurse cells as cysts progress into the vitellarium. Even in the absence of ORD, finger-like projections of SMC1/3 staining were observed in the vicinity of nurse cell centromeres in ord^null mutant egg chambers by vitellarial stage 3, presumably when polytene chromosomes are present. Like wild type, this SMC1/3 staining becomes diffuse at later stages when polyteny is absent. These data argue that loading of cohesin subunits onto centromeres is controlled differently in oocytes and nurse cells, and once germ-line cells adopt a nurse cell fate, accumulation of cohesin at centromeres is no longer dependent on ORD function (Khetani, 2007).

ORD is necessary for maintenance of chromosome cores during early meiosis: Despite the centromeric defects that were observe in ord^null germaria, thread-like SMC1 and SMC3 staining along chromosome cores appear relatively normal during early pachytene even in the absence of ORD activity. In early region 2A (the anterior portion of region 2A), long continuous threads of SMC1 and SMC3 are visible in ord^null germaria, although SMC staining along cores is weaker than in wild type. However, both the intensity and integrity of cohesin thread-like staining deteriorates progressively as cysts mature and travel through the germarium. A gradual loss of thread-like C(3)G staining as cysts mature has also been observed (Webber, 2004) in ord^null germaria (Khetani, 2007).

To characterize the progressive deterioration of SMC1 and SMC3 thread-like staining in ord^null germaria, the integrity and intensity of the threads were scored in different regions of wild-type and mutant germaria. Careful analysis of the defects in several mutant germaria indicated that, by late region 2A (the posterior portion of region 2A), the intensity of the SMC1 and SMC3 thread-like staining was significantly reduced and fragmented threads were visible in a number of cells. For example, in late region 2A, a pronounced reduction in SMC1 signal intensity was observed in 43% of ord^null cysts but only 3% of wild-type cysts. Similarly, fragmented SMC1 threads were observed in 15% of late region 2A mutant cysts, but no fragmentation was visible at this stage in wild type. In older mutant cysts, loss of the thread-like SMC1 and SMC3 staining became more prominent. By region 3, no mutant oocyte nucleus exhibited robust continuous thread-like SMC1 or SMC3 staining. At this stage, 45% of ord^null oocyte nuclei contained no visible SMC1 staining and 53% contained severely fragmented threads. SMC3 signal was undetectable in approximately 52% of region 3 ord^null oocyte nuclei and about 30% had short dim fragments. Interestingly, the anti-SMC1 and anti-SMC3 antibodies appear to have different affinities for their respective antigens in wild-type nuclei; SMC1 signal along chromosome cores was consistently more robust than that for SMC3. This difference may reflect variation in epitope accessibility for the two proteins and is most likely the cause for quantitative differences in the defects observed for SMC1 and SMC3 in mutant germaria. However, deterioration of the thread-like signal followed the same trend for both proteins and reinforces the conclusion that ORD activity is required to maintain chromosome cores during early pachytene. Notably, these defects first become manifest after the onset of homologous recombination, namely the induction of DSBs in region 2A (Khetani, 2007).

ORD is not required for stable association of cohesin SMCs with chromosome arms during pachytene: The loss of thread-like staining in whole-mount preparations of ord^null germaria initially suggested that cohesin dissociates from chromosome arms during pachytene in the absence of ORD activity, consistent with the essential role of ORD in arm cohesion. However, it was reasoned that it was also possible that cohesin SMCs might remain associated with chromatid arms in the absence of ORD, but loss of thread-like staining might occur because the longitudinal compaction of meiotic chromosome cores depends on ORD function. If cohesin SMCs remain associated with chromosome arms during pachytene in ord^null females but chromosome cores are unstable, the thread-like SMC1/3 signal would disappear. However, it is difficult to detect diffuse localization of cohesin SMCs along chromosome arms in whole-mount preparations. Therefore, chromosome spreads were prepared from ord^null germaria and immunostained for SMC1/3 and C(3)G proteins. Because in spread preparations, the temporal arrangement of individual cysts within each germaria is not maintained, semi-intact cysts were sought that contained a maximum of one or two nuclei with C(3)G staining, reasoning that these most probably represent region 2B cysts. At this stage in ord^null germaria, C(3)G thread-like signal has begun to fragment. Diffuse SMC1/3 staining is readily evident in nuclei that also contain fragmented C(3)G and SMC1/3 threads. Moreover, the intensity of diffuse SMC1/3 signal in these nuclei is very similar to that of adjacent pro-nurse cell nuclei. Because soluble nuclear protein is removed during the spread preparation, the diffuse SMC1/3 signal that was observed represents cohesin subunits that are associated with the chromatin but not organized into chromosome cores. These data argue that, in the absence of ORD activity, chromosome cores disassemble but cohesin SMCs remain associated with chromosome arms (Khetani, 2007).

Temporal relationship between SMC1/3 and C(3)G defects in ord^null oocytes: The progressive deterioration of chromosome cores in ord^null germaria is reminiscent of the fragmentation and loss of thread-like staining observed by Webber (Webber, 2004) for the SC central element component, C(3)G. However, a careful comparison of the quantitative analyses of cohesin SMC and C(3)G localization defects (Webber, 2004) indicates that the onset of SMC1/3 localization defects appear to precede those for C(3)G. This is not completely unexpected given that chromosome cores have been proposed to serve as the scaffold upon which SC axial/lateral and central element components can assemble. If disruption of chromosome cores in ord^null oocytes causes a subsequent loss of the central element between homologues, defects in the C(3)G staining pattern should closely follow disintegration of SMC1/3 threads. To test this hypothesis, SMC1/3 and C(3)G defects were examined simultaneously in individual nuclei at different stages in whole-mount preparations of intact ord^null germaria (Khetani, 2007).

Continuous thread-like staining is evident for both C(3)G and SMC1/3 in early region 2A nuclei of ord^null germaria with extensive overlap between the two signals. However, by late region 2A, the SMC1/3 signal appears more fragmented than the C(3)G signal, with fewer SMC1/3 threads and more punctate staining. This difference is most obvious in region 3, where intact threads of C(3)G staining are still evident but the SMC1/3 pattern consists primarily of puncta. These data support the hypothesis that premature breakdown of chromosome cores induces the defects in C(3)G staining that were observed in ord germaria. However, the residual C(3)G threads that remain when SMC1/3 thread-like staining disappears raises the intriguing possibility that aligned C(3)G proteins might form polymers that remain transiently stable, even if they are no longer associated with chromosome cores (Khetani, 2007).

It was also asked whether C(3)G is required to maintain chromosome core integrity; thread-like SMC1/3 staining is still visible in c(3)G⁶⁸/Df mutant germaria in which the SC fails to form. These data indicate that intact SC is not necessary for chromosome core formation or maintenance. Interestingly, SMC1/3 threads appear more numerous in c(3)G mutant nuclei than in wild type, consistent with the inability of homologues to synapse in the absence of C(3)G. In the absence of synapsis, the homologous cores would not be intimately associated and cohesin staining would be visible along individual chromosome cores. A similar staining pattern has been reported for the putative lateral element component C(2)M in c(3)G mutants (Manheim, 2003) providing further support for this model (Khetani, 2007).

C(2)M is required for chromosome core formation during pachytene: To explore the mechanistic interplay between cohesion proteins (ORD, cohesin SMCs), and an α-kleisin involved in SC assembly (C(2)M), the localization of SMC1/3 and GFP-ORD were examined in females homozygous for the c(2)M-null allele, c(2)M^EP[2115] (Manheim, 2003). To observe ORD localization, a c(2)M^EP[2115] stock homozygous for a functional GFP-ORD transgene (Balicky, 2002) was generated and it was confirmed that X-chromosome meiotic nondisjunction in c(2)M^EP[2115];P{GFP-ORD} females (24.44%, n=753) was similar to that previously reported for c(2)M^EP[2115] homozygotes (29.3%) (Manheim, 2003; Khetani, 2007).

At first glance, the staining pattern for SMC1/3 and GFP-ORD in c(2)M^[EP2115] germaria appeared very similar to that previously observed for C(3)G in this mutant (Manheim, 2003). Thread-like SMC1/3 staining is completely absent in the germaria of whole-mount ovaries. Instead, SMC1/3 staining is restricted to patches and foci (Khetani, 2007).

In c(2)M^[EP2115] females, the chromosome segregation defects are severe in meiosis I, but negligible during meiosis II (Manheim, 2003). This suggests that centromeric cohesion in these mutants is intact. To test whether the patches of SMC1/3 and GFP-ORD staining correspond to centromeres, c(2)M^[EP2115] ovaries were co-immunostained with anti-CID antibodies. In these whole-mount preparations, CID foci largely coincide with ORD and SMC1/3 patches in pro-oocyte and oocyte nuclei in all regions of c(2)M-mutant germaria. These data argue that neither loading nor maintenance of cohesin SMCs at centromeres depends on the activity of C(2)M protein, consistent with low levels of meiosis II segregation defects in c(2)M-mutant females (Manheim, 2003; Khetani, 2007).

Absence of thread-like SMC1/3 signal in whole-mount preparations of c(2)M-mutant germaria raises the possibility that C(2)M activity is required for loading cohesin subunits onto chromosome arms. Alternatively, cohesin subunits may localize normally to the arms, but fail to coalesce into chromosome cores in the absence of C(2)M. In this case, diffuse chromatin-bound cohesin signal would probably go undetected in whole-mount ovary preparations. To address this possibility, chromosome spreads prepared from c(2)M^EP[2115];P{GFP-ORD} germaria were examined. In the c(2)M mutant, each nucleus contained one to four centromeric foci in which SMC1/3 and GFP-ORD always colocalized. These foci also coincided with C(3)G foci in the subset of cells that contained C(3)G signal. This staining pattern is consistent with that observed in whole-mount preparations of c(2)M germaria. However, in the chromosome spreads, diffuse chromosomal ORD and SMC1/3 staining also was observed in all mutant c(2)M pro-oocytes and pro-nurse cells. Moreover, the intensity of diffuse SMC1/3 and ORD signal is comparable in nuclei with and without C(3)G patches/foci (pro-oocytes and pro-nurse cells, respectively) (Khetani, 2007).

The data for chromosome spread localization indicate that in the absence of C(2)M, ORD and cohesin SMCs are loaded and maintained on both the arms and centromeres of meiotic chromosomes. However, absence of thread-like ORD and SMC1/3 staining argues that assembly of chromosome cores requires C(2)M activity. Because stable chromosome cores and lateral elements are a prerequisite for SC formation, their absence most probably explains the lack of thread-like C(3)G signal in c(2)M mutant germaria (Khetani, 2007).

This study has describe temporal and spatial changes in cohesin localization during early prophase in wild-type Drosophila ovaries as well as in mutants with compromised cohesion and/or homologous recombination. Drosophila oogenesis provides a unique opportunity to examine important changes in chromosome morphology that occur during meiotic prophase. Because not all germ-line cells adopt an oocyte fate, the 16-cell cyst allows direct comparison of nuclei that assemble meiotic chromosome cores (and SC) with those that do not. Importantly, this dynamic transformation in chromosome structure depends upon and must occur within the context of functional sister-chromatid cohesion. In addition, these events are crucial for homologous recombination and, therefore, accurate segregation of meiotic chromosomes (Khetani, 2007).

Analysis of chromosome spread preparations from wild-type ovaries indicates that the cohesin subunits SMC1 and SMC3 localize along the arms of chromosomes in all 16 cells of each cyst. However, thread-like cohesin SMC staining is only observed in the nuclei that build a SC. The simplest model to explain the differences observed in pro-nurse cells and pro-oocytes is that multiple cohesin complexes come together to form long continuous threads of cohesin staining in nuclei that build SC. It is proposed that chromosomes in pro-nurse cells maintain an extended interphase-like organization in which cohesin complexes localize along the arms but fail to assemble into this higher order structure. By contrast, the formation of chromosome cores in a subset of nuclei occurs when multiple cohesin complexes along the arms coalesce into threads and, thereby, bring about the shortening of the longitudinal axes of meiotic chromosomes (Khetani, 2007).

Formation of chromosome cores represents the first step in the organized assembly of the SC. Although DSBs are required for synapsis of homologues in a number of species, lateral elements are still visible in mutants that fail to make DSBs and therefore lack tripartite SC. Moreover, chromosome cores have been proposed to serve as the scaffold upon which SC components organize and assemble. Genetic and cytological analyses in a number of organisms have confirmed that the cohesin complex plays an integral role in SC assembly. This work argues that cohesin SMCs as well as the cohesion protein ORD are stable components of meiotic chromosome cores, which remain intact when DNase I treatment removes chromatin loops. Interestingly, in chromosome spread preparations, localization of cohesin subunits and ORD appears to be restricted to chromosome cores; diffuse staining is not detectable in the areas between threads. These data are consistent with the model that, in Drosophila meiotic cells, cohesion proteins localize predominantly along the chromatid axes and are not found decorating the loops. Similar arguments have been made for cohesin localization in S. cerevisiae and mouse meiotic cells (Khetani, 2007).

Evidence is provided that the meiosis-specific protein, C(2)M, is required for chromosome core formation in Drosophila oocytes. Chromosome spread preparations indicate that in the absence of C(2)M activity, SMC1 and SMC3 diffuse staining is visible throughout the nuclei of all 16 cells within each cyst, indicative of the association of cohesin subunits with chromosome arms. However, no thread-like SMC1/3 or ORD staining is observed in c(2)M-mutant germaria. These data indicate that C(2)M protein controls an early step in the formation of chromosome cores. It is proposed that, by virtue of its ability to interact with cohesin SMC proteins (Heidmann, 2004), C(2)M drives the association of cohesin complexes to form meiotic chromosome cores. Failure in this process would prohibit subsequent assembly of the SC in c(2)M mutant germaria as evidenced by lack of thread-like immunostaining for the transverse filament protein C(3)G (Manheim, 2003). These results also are consistent with EM localization of C(2)M protein along the lateral elements of the wild-type Drosophila SC (Anderson, 2005; Khetani, 2007).

Surprisingly, it was found that the cohesion protein ORD is required for the maintenance of chromosome cores during early pachytene in Drosophila. In the absence of ORD activity, thread-like SMC1/3 staining is visible in region 2A of the germarium; however, the intensity and integrity of SMC1/3 threads deteriorate as pachytene progresses within the germarium. Analysis of chromosome spread preparations indicates that, although chromosome cores disassemble, the cohesin subunits SMC1 and SMC3 remain associated with the chromosome arms in ord^null germaria (Khetani, 2007).

Quantitative analyses of temporal progression of SMC1/3 and C(3)G defects in ord mutant germaria (Webber, 2004), as well as co-immunostaining experiments that simultaneously monitored cohesin subunits and C(3)G in individual ord^null nuclei, argue that the onset of chromosome core dissolution precedes fragmentation of thread-like epifluorescent signal for the SC marker, C(3)G. These data demonstrate that initial assembly of cores is not sufficient for stable SC; instead, maintenance of chromosome cores is an ongoing requirement to preserve SC integrity (Khetani, 2007).

Why do cores disassemble in the absence of ORD activity? By immunofluorescence, continuous thread-like C(2)M and C(3)G staining is transiently present during early pachytene in ord^null germaria; however, at the same stage, normal tripartite SC is not detectable by EM (Webber, 2004). Therefore, although the highly organized SC ultrastructure is absent, some aspects of SC assembly still occur in the absence of ORD function [namely recruitment of C(2)M and C(3)G]. These data suggest that ORD is required to recruit additional proteins along the chromosome cores and/or lateral/axial elements that are required for core integrity. Alternatively, ORD itself might be required to maintain C(2)M-mediated organization of the cores into stable structures (Khetani, 2007).

Programmed cycles of stress and relaxation along meiotic chromosomes have been proposed to govern several critical events during prophase. One possibility is that in the absence of ORD function - although chromosome cores assemble - they are unable to withstand normal changes in compression and/or relaxation, and subsequently buckle. Interestingly, it has been reported that, during wild-type pachytene, the SC shortens significantly as cysts move through the Drosophila germarium. If chromosome cores that assemble in the absence of ORD are inherently unstable, programmed shortening of the SC could cause additional stress that results in fragmentation of the cores. Curiously, it has been observed that in later stages (stages 3-6 of the vitellarium), continuous thread-like SMC1/3 staining often reappears in ord^null oocytes. These stages loosely correspond to the time after which the SC reaches its shortest length and starts to expand in wild type. Chromosome cores might be able to reassemble at these later stages in ord^null ovarioles if decompaction of the chromosome axes reduces stress (Khetani, 2007).

Breakdown of chromosome cores in ord^null germaria is also temporally linked to the onset of DSBs. Both the timing (early 2A) and the number of DSBs are normal in ord^null germaria (Webber, 2004). However, fragmentation of SMC1/3 thread-like staining is detected in late region 2A, after the onset of DSBs. Therefore, induction of DSBs might contribute to destabilization of chromosome cores that are compromised due to lack of ORD protein. A similar model has been proposed to explain the phenotypes associated with disruption of the Pds5 orthologue (Spo76) in Sordaria (Storlazzi, 2003). Although AEs are continuous during early prophase I in spo76-1 mutants, they fragment prematurely in a DSB-dependent fashion (Khetani, 2007).

The disassembly of chromosome cores in ord^null germaria is most probably responsible for the severe reduction in crossovers between homologues in mutant females. ORD activity is essential for the crucial decision each chromatid must make after the induction of DSBs - namely whether the broken chromatid will choose its sister or its homologue for repair. Strand invasion and crossovers are biased towards the homologue during meiosis, resulting in stable chiasmata that keep homologous chromosomes physically associated until anaphase I. Previous experiments have shown that ORD activity is required for homologue bias during meiotic recombination in Drosophila. In ord^null females, the frequency of crossovers between homologues is decreased, while that between sister chromatids is significantly increased (Webber, 2004). These data combined with current analyses suggest that chromosome cores are necessary for homologue bias during meiosis, and partner choice takes place in late region 2A or region 2B of the Drosophila germarium (Khetani, 2007).

In ord^null germaria, SMC1 and SMC3 fail to accumulate at centromeres, but appear to localize normally along chromosome arms within all 16 cells of each germ-line cyst. These results suggest that distinct pathways mediate cohesin loading on the arms and centromeres during Drosophila meiosis. It cannot be differentiated whether centromeric loading of SMC1 and SMC3 is completely ablated or whether cohesin SMCs are able to load at centromeres but are quickly removed when ORD activity is absent. Regardless, accumulation of cohesin subunits at the centromeres of meiotic chromosomes appears to depend on ORD function. By contrast, ORD activity is not required for stable association of SMC1 and SMC3 along chromosome arms. Curiously, after germ-line cells adopt a nurse cell fate, ORD is no longer necessary for centromeric accumulation of cohesin; the clustered finger-like projections of SMC1/3 staining at nurse cell centromeres in ord^null ovarioles (stages 3-4) are indistinguishable from that in wild type. However, even at these later stages when cohesin subunits are visible at nurse cell centromeres, SMC1 or SMC3 are never detected at the centromeres of oocytes in ord^null ovarioles. Therefore, the data implicate ORD in a meiosis-specific pathway for cohesin loading and/or accumulation at centromeres (Khetani, 2007).

Co-immunostaining experiments with CID and SMC1/3 antibodies indicate that absence of cohesin subunits appears to be restricted to the centromeres in ord^null germaria; within the resolution limits of the chromosome spread images, the area lacking SMC1/3 signal is approximately the same size as the CID staining. These data suggest that cohesin loading and/or accumulation at pericentromeric heterochromatin occurs in the absence of ORD function. However, the striking enrichment of SMC1/3 in pericentromeric heterochromatin prominent in wild type is not observed. Therefore, the analysis of defects of cohesin localization in ord^null germaria suggest that normal loading and/or accumulation of cohesin is regulated differently even within the different domains of heterochromatin in and around centromeres. In addition, the chromocenter appears to be less stable in ord^null oocytes, raising the possibility that changes in heterochromatin structure in the absence of ORD activity diminishes the ability of centromeres to associate (Khetani, 2007).

Chromosome spread experiments clearly indicate that SMC1 and SMC3 are stably associated with the chromosome arms of both pro-nurse cells and pro-oocytes within ord^null germaria. However, whether the localization of cohesin subunits represents functional cohesin is not clear. From genetic and cytological studies, it is known that meiotic cohesion is completely absent in ord^null oocytes by the time that meiotic chromosomes make microtubule attachments (Bickel, 2002; Bickel, 1997). Separated sister chromatids have not been detected during early pachytene in ord^null germaria by FISH (Webber, 2004). However, catenation might hold sisters together at this time, thereby masking defects arising from the absence of cohesin-mediated cohesion (Khetani, 2007).

Stepwise loading of cohesin subunits during meiotic prophase has been described for a number of organisms. In worms and grasshoppers, stable association of cohesin SMCs in the absence of non-SMC subunits has been reported for meiotic chromosomes. One possibility is that, in the absence of ORD, cohesin SMCs load without their non-SMC partners. Another possibility is that the entire cohesin complex loads in ord^null ovaries but cohesin-mediated cohesion is not established. At least two reports have indicated that, in S. cerevisiae, the binding of cohesin to specific genomic locations is insufficient for cohesin-mediated cohesion. In addition, recent work has been elegantly demonstrated that different populations of chromatin-bound mitotic cohesin exist within Hela cells. This work suggests that, during replication and the establishment of cohesion, a subset of chromatin-bound cohesin complexes is converted from 'dynamically associated' to 'irreversibly bound'. ORD might be necessary for the establishment of meiotic sister-chromatid cohesion but not for association of cohesin subunits with the chromosomes. Alternatively, cohesion might be established in the absence of ORD activity but not maintained (Khetani, 2007).

In most species, a meiosis-specific α-kleisin subunit (Rec8) promotes meiotic cohesion by interacting with the heads of the SMC subunits and closing the cohesin ring. Surprisingly, an obvious Rec8 orthologue has not been identified in the Drosophila genome. Although its limited sequence homology to Rec8 in other organisms has led to the proposal that C(2)M functions as the meiosis-specific α-kleisin subunit of the cohesin complex in Drosophila (Schleiffer, 2003), phenotypic analysis of c(2)M mutant flies is inconsistent with this hypothesis (Manheim, 2003). In contrast to other Drosophila mutations that disrupt meiotic cohesion, defects of meiosis II segregation are negligible in c(2)M females and accurate chromosome segregation during male meiosis also does not depend on C(2)M function (Manheim, 2003). Moreover, female germ-line expression of a mutated C(2)M transgene in which putative separase cleavage sites were disrupted did not result in meiotic segregation defects. Finally, localization of C(2)M protein in whole-mount preparations (Manheim, 2003) indicates that, like C(3)G protein, C(2)M is restricted to the subset of cells within each germ-line cyst that build SC. Therefore, it is proposed that the kleisin domain in C(2)M allows it to interact with cohesin SMC subunits and that C(2)M plays an essential role in building meiotic chromosome cores, but does not promote meiotic cohesion (Khetani, 2007).

In several respects, the behavior of ORD protein is consistent with it performing the role of Rec8 during Drosophila meiosis. Null mutations in ord eliminate meiotic cohesion in both sexes and ORD colocalizes extensively with cohesin SMC subunits during early pachytene in females. During male meiosis, ORD remains associated with spermatoctye centromeres throughout prophase I (E. M. Balicky, Regulation of chromosome segregation by ORD and dRING during Drosophila meiosis, PhD thesis, Dartmouth College, Hanover, NH, 2004) and is not lost until anaphase II when centromeric cohesion is released (Balicky, 2002). Moreover, retention of ORD at centromeres until anaphase II depends on the activity of the Drosophila Shugoshin ortholog, Mei-S332 (E. M. Balicky, PhD thesis, Dartmouth College, Hanover, NH, 2004). It has not been possible to detect ORD (or cohesin SMCs) on meiotic chromosomes during late oogenesis; however, given that ORD is required for cohesion in both sexes and localizes to spermatocyte centromeres until anaphase II, it seems likely that lack of signal in mature oocytes is due to antibody accessibility issues and/or detection limitations, not the absence of the protein (Khetani, 2007).

Although several pieces of data are consistent with the model that ORD protein provides Rec8 activity in Drosophila, the size of ORD protein (479 amino acids) may be too small to bridge the heads of the Drosophila SMC1/3 dimer. In addition, ORD does not share obvious sequence homology with Rec8, Scc3/SA or regulators of cohesin (Pds5, Scc4, Scc2). One possibility is that during Drosophila meiosis, two proteins collaborate to provide Rec8 function. Such is the case for Drosophila separase, which is composed of two subunits encoded by separate genes. ORD may cooperate with Rad21 or another unidentified protein to provide Rec8 function during meiosis. Why flies would use an altered mechanism to accomplish such a highly conserved activity is an enigma. However, further analysis of the regulation of meiotic cohesion in Drosophila should provide important evolutionary insights into fundamental aspects of recombination and chromosome segregation during meiosis (Khetani, 2007).

Effects of sister chromatid cohesion proteins on cut gene expression during wing development in Drosophila

The cohesin protein complex is a conserved structural component of chromosomes. Cohesin binds numerous sites along interphase chromosomes and is essential for sister chromatid cohesion and DNA repair. This study tests the idea that cohesin also regulates gene expression. This idea arose from the finding that the Drosophila Nipped-B protein, a functional homolog of the yeast Scc2 factor that loads cohesin onto chromosomes, facilitates the transcriptional activation of certain genes by enhancers located many kilobases away from their promoters. Cohesin binds between a remote wing margin enhancer and the promoter at the cut locus in cultured cells, and reducing the dosage of the Smc1 cohesin subunit increases cut expression in the developing wing margin. cut expression is increased by a unique pds5 gene mutation (see CG17509) that reduces the binding of cohesin to chromosomes. On the basis of these results, it is posited that cohesin inhibits long-range activation of the Drosophila cut gene, and that Nipped-B facilitates activation by regulating cohesin-chromosome binding. Such effects of cohesin on gene expression could be responsible for many of the developmental deficits that occur in Cornelia de Lange syndrome, which is caused by mutations in the human homolog of Nipped-B (Dorsett, 2005).

To identify general facilitators of enhancer-promoter communication, genetic screens were conducted to isolate factors that support activation of the cut gene by a wing margin-specific enhancer located 85 kbp upstream of the promoter. The region between this enhancer and the promoter contains many enhancers that activate cut in specific tissues during embryogenesis and larval development. In addition to tissue-specific activators that bind to the wing margin enhancer, these screens identified two proteins, Chip and Nipped-B, that are expressed in virtually all cells, and facilitate the expression of diverse genes. Chip interacts with many DNA-binding proteins, and likely supports the cooperative binding of proteins to enhancers and to sites between enhancers and promoters (Dorsett, 2005).

Nipped-B functions by a different mechanism. Unlike other cut regulators, Nipped-B is more limiting for cut expression when enhancer-promoter communication is partially compromised by a weak gypsy insulator than it is when the enhancer is partially inactivated by a small deletion, leading to the idea that Nipped-B specifically facilitates enhancer-promoter communication. Nipped-B homologs in Saccharomyces cerevisiae, S. pombe and Xenopus (Scc2, Mis4 and Xscc2), known collectively as adherins, load the cohesin protein complex onto chromosomes, Nipped-B is required for sister chromatid cohesion, and thus is a functional adherin. The fact that Nipped-B is an adherin raises the critical question, addressed here, of whether or not cohesin plays a role in enhancer-promoter communication. In all metazoans examined, cohesin loading starts in late anaphase, and it is not removed from the chromosome arms until prophase. Cohesin, therefore, is a structural component of chromosomes during interphase, when gene expression occurs (Dorsett, 2005).

Cohesin consists of two Smc proteins, Smc1 and Smc3, and two accessory subunits, Rad21 (Mcd1/Scc1) and Stromalin (Scc3/SA). Cohesin forms a ring-like structure. One idea is that adherins, such as Nipped-B, temporarily open the ring and allow it to encircle the chromosome. It is proposed that cohesin encircles both sister chromatids after DNA replication to establish cohesion. Cohesin binds every 10 kbp or so along the chromosome arms in yeast. If it binds at a similar density in metazoans, it could potentially affect the expression of many genes (Dorsett, 2005).

Determining if the effects of Nipped-B on gene expression are mediated through cohesin is pertinent to Cornelia de Lange syndrome (CdLS, OMIM #122470), which is caused by heterozygous loss-of-function mutations in the human homolog of Nipped-B, Nipped-B-Like (NIPBL, GenBank Accession Number NM_133433. CdLS results in numerous birth defects, including slow physical and mental growth, upper limb deformities, gastroesophageal and cardiac abnormalities. These developmental deficits likely reflect changes in gene expression similar to those caused by heterozygous Nipped-B mutations (Dorsett, 2005).

This study examines binding of cohesin to the cut gene, and the effects that the Pds5 sister chromatid cohesion factor has on cut expression and cohesin binding to chromosomes. The results are consistent with the idea that cohesin inhibits the activation of cut by the wing margin enhancer (Dorsett, 2005).

RNAi experiments have shown that slightly reducing the Stromalin (Scc3) and Rad21 (Mcd1/Scc1) subunits of cohesin increased expression of the cut gene in the developing wing margin. To determine if the Smc1 cohesin subunit plays a similar role, a null allele of the smc1 gene was generated by excision of a viable P transposon insertion near the transcription start site. The smc1^exc46 allele is recessive lethal and chromosome squashes show precocious sister chromatid separation (Dorsett, 2005).

The effects of smc1^exc46 on the mutant phenotype displayed by the ct^K gypsy transposon insertion allele of cut were used to determine changes in cut expression. The ct^K gypsy insulator partially blocks activation of cut by the wing margin enhancer, causing a scalloped wing phenotype sensitive to the dosage of factors that regulate cut. A decrease in cut expression increases nicks in the wing margin, and an increase in expression leads to fewer nicks. The wing-nicking assay is a highly specific and sensitive measure of the activation of cut by the wing margin enhancer, in the developing margin cells of the wing discs during the 24-hour period centered around pupariation (Dorsett, 2005).

In repeated experiments, the heterozygous smc1^exc46 mutation reduced the number of ct^K wing margin nicks relative to the number observed with the heterozygous parental chromosome. The difference was significant. It is concluded that, similar to the effects of reducing the Stromalin (Scc3) and Rad21 (Mcd1/Scc1) cohesin subunits, reducing the levels of the Smc1 subunit increases cut expression. Because all three cohesin subunits have a similar effect, it is concluded that the cohesin complex inhibits cut expression (Dorsett, 2005).

Mutations in genes that modulate cohesin activity for effects on cut expression were tested. The separation anxiety (san) and deco (eco, as listed in FlyBase) genes encode putative acetyltransferase proteins that are required for sister chromatid cohesion and the association of cohesin with centromeric regions. Separase (Sse) encodes a protease that cleaves cohesin to permit sister chromatid separation. Mutations in these genes did not have significant effects on the ct^K mutant phenotype. It is possible that heterozygosity for these mutations do not sufficiently alter cohesin activity to change cut expression. Alternatively, these proteins may affect cohesin only at the centromere (Dorsett, 2005).

If cohesin directly regulates cut, it would be expected to bind to the cut locus. Salivary gland polytene chromosomes were immunostained with anti-Smc1 and anti-Stromalin (Scc3) to determine whether cohesin binds to cut. In wild type, Stromalin and Smc1 co-localize on polytene chromosomes as expected. Distinct regions of cohesin staining were seen in both bands and interbands. It is concluded that cohesin binds many sites along all chromosome arms. Staining was observed in some chromosomal puffs, suggesting that cohesin associates with transcribed loci. To test this, it was determined whether cohesin binds to heat-shock puffs. After 20 minutes of heat shock at 37°C, cohesin staining was observed in the 93D puff, but not in the others. Thus, cohesin localization does not correlate with transcription (Dorsett, 2005).

The examination of several nuclei showed that the chromosome band containing the cut locus (7B3-4) consistently displayed cohesin staining. This band contains 150 kbp of DNA, and four genes other than cut, in the regulatory region between the wing margin enhancer and the cut promoter. At least three of these genes are testis specific. Salivary glands do not express cut, but because cohesin is a constitutive chromosomal component, and probably binds to cut in most cells, these data are consistent with the view that the effects of cohesin on cut expression in the wing margin are direct (Dorsett, 2005).

Further support is provided by chromatin immunoprecipitation experiments, which show that cohesin binds to the regulatory region of cut in Drosophila cultured Kc cells of embryonic origin. An 85-kbp region was examined encompassing the wing margin enhancer and the promoter; four cohesin-binding sites were detected. Two binding sites were centered 0.5- and 4-kbp upstream of the promoter, one was centered about 30.5-kbp upstream of the promoter, and another small broad peak was 68-kbp upstream of the promoter. The same sites were seen with both antisera, and neither pre-immune serum showed enrichment of any sequences. Thus, in addition to the non-dividing polytene salivary cells, cohesin also binds cut in predominantly diploid dividing cells of embryonic origin. Based on the assumption that cohesin is a constitutive chromosomal component, and the finding that it binds to the cut locus in two very different cell types, it is posited that cohesin also binds cut in the developing wing margin cells and that the effects of cohesin on cut expression in the developing wing are direct (Dorsett, 2005).

The possibility was considered other factors recruited by cohesin could inhibit cut activation. In fungi, the Pds5 (Spo76) protein is required for sister chromatid cohesion. Pds5 associates with cohesin sites on chromosomes, and requires cohesin for association (Dorsett, 2005 and references therein).

By sequence analysis, the CG17509 gene was identified as the likely pds5 homolog. The P{EPgy2}CG17509^EY06473 transposon insertion in the first exon is homozygous viable. It was mobilized to generate two recessive lethal mutations, pds5^e3 and pds5^e6, that fail to complement each other, and a deletion of the region [Df(2R)BSC39]. Both homozygous mutants and the heteroallelic combination are lethal in late third instar to early pupal stages of development. Late third instar larvae of both mutants display small or missing imaginal discs, and the larval brains are approximately half the volume of wild type, consistent with a mitotic defect (Dorsett, 2005).

Neuroblast metaphase nuclei from mutant third instar larvae were examined for cohesion defects. Despite examining more than 30 metaphases from each, no normal metaphases were found in the pds5 mutants. Nearly all displayed aneuploidy, and most displayed precocious sister chromatid separation. By contrast, 15.4% of the pds5^e3/+ heterozygote metaphases showed aneuploidy and 12.8% showed sister chromatid separation, similar to the frequencies observed with wild-type neuroblasts. It is concluded that the pds5^e3 and pds5^e6 mutations affect chromosome segregation and sister chromatid cohesion, and that CG17509 encodes a functional Pds5 homolog (Dorsett, 2005).

The effect of the pds5 mutations on the ct^K phenotype was tested relative to the viable P element insertion used to generate them. Unexpectedly, the two mutations had different effects. The pds5^e3 mutation slightly increased the number of wing margin nicks, indicating that it decreased cut expression, whereas pds5^e6 increased cut expression. Although a small increase in wing margin nicks was consistently observed with pds5^e3, the wing nicking was not significantly different from that seen with the parental chromosome. The decreased nicking associated with the pds5^e6 allele, however, was significantly different from that of the parental chromosome. It is concluded that the pds5^e6 mutation dominantly increases cut expression, and that the pds5^e3 mutation may cause a small decrease (Dorsett, 2005).

pds5 expression was examined in the two pds5 mutants to determine why they have different effects on cut expression. Northern blots revealed a pds5 transcript of the expected size (4.6 kb) in embryos prior to zygotic gene expression, indicating that it is maternal. The transcript was present at 10- to 25-fold lower levels in larvae. To avoid detecting maternal pds5 mRNA, the transcripts produced in pds5^e3 and pds5^e6 second instar larvae were examined. No pds5 transcripts were detected in homozygous pds5^e3 mutants, but a shorter transcript (3.65 kb) was seen at levels similar to those of wild type in both heterozygous and homozygous pds5^e6 mutants. A wild-type sized transcript was present in heterozygous pds5^e6 mutants, but was undetectable in homozygotes. The viable parental P insertion line used to generate both lethal pds5 alleles produced wild-type levels of a wild-type sized transcript (Dorsett, 2005).

The Northern blots indicate that pds5^e3 is a null allele, and PCR analysis of mutant genomic DNA revealed that sequences starting at the P insertion site and extending upstream of the transcription start site are missing. Thus, a reduction in Pds5 dosage slightly decreases cut expression. It is concluded, therefore, that wild-type Pds5 does not contribute to the inhibition of cut expression by cohesin, but may slightly decrease the inhibitory effect (Dorsett, 2005).

The presence of a new transcript in the pds5^e6 mutant suggested that it could produce a mutant protein lacking an activity crucial for sister chromatid cohesion that somehow interferes with the inhibition of cut by cohesin. PCR analysis of pds5^e6 genomic DNA revealed that the region from the P insertion site through exon 5 is missing. 5' RACE analysis of the pds5^e6 transcript shows that it starts 67 nucleotides upstream of the wild-type start site predicted by EST analysis. The pds5^e6 transcript extends from the start site to the P insertion site. The next 17 nucleotides are from the end of the P insertion, followed by 12 nucleotides of internal P sequence fused to the pds5 sequence 11 nucleotides downstream of the exon 6 5' splice site. The exon 6 sequences present in the mutant transcript contain six in-frame AUG codons, two of which match a consensus (RNVATGR) for Drosophila translation initiation sites. Thus the pds5^e6 mutant transcript encodes a protein lacking the N terminus (Dorsett, 2005).

The effects of the two pds5 mutations on cut expression correlate with a difference in cohesin binding to chromosomes. Although the salivary glands of the homozygous pds5 mutants are substantially reduced, polytene chromosomes were obtained from both. Morphology was altered enough to make it difficult to identify specific loci. Nevertheless, individual chromosome tips could be identified, and developmental puffs, including the puff at 2B, were present in both mutants, indicating that the chromosomes are transcribed. In size-matched third instars, the pds5^e3 mutant chromosomes were thicker than wild type, and the pds5^e6 mutant chromosomes were thinner. The pds5^e3 null allele did not reduce staining for Smc1 or Stromalin, although the pattern appeared less discrete. By contrast, pds5^e6 mutant chromosomes showed strongly reduced staining for Smc1 and Stromalin. Loss of cohesin staining was observed in multiple nuclei from multiple pds5^e6 salivary glands. These results indicate that the pds5^e6 mutant either blocks loading of cohesin onto chromosomes, or facilitates removal. The reduction of cohesin binding caused by pds5^e6, which dominantly increases cut expression, is consistent with the hypothesis that cohesin inhibits cut expression (Dorsett, 2005).

The demonstration that cohesin binds to the cut locus in polytene chromosomes, and to multiple sites between the remote wing margin enhancer and the promoter in cultured cells, supports the hypothesis that the effects of cohesin on cut expression in the wing margin are direct. The wing margin enhancer does not activate cut in salivary glands or Kc cells, but it is not technically feasible to examine the association of cohesin with cut in the developing margin cells in which the wing margin enhancer functions. Based on the association of cohesin with cut in two diverse cell types, it is posited that cohesin also binds cut in the developing wing margin cells, and inhibits activation by the wing margin enhancer. Such a direct effect of cohesin could explain why small reductions (<20%) in cohesin subunits induced by RNAi have detectable effects on cut expression (Dorsett, 2005).

Both pds5 mutations tested cause similar sister chromatid cohesion defects, but only pds5^e6 reduces the binding of cohesin to chromosomes and increases cut expression. This provides additional evidence that binding of cohesin to chromosomes is required for it to inhibit cut activation, and also shows that Pds5 itself is not required to inhibit gene expression (Dorsett, 2005).

The negative effects of cohesin on cut expression raise the possibility that cohesin contributes to the silencing of euchromatic genes placed in heterochromatin. Cohesin binds more densely in centromeric heterochromatin in yeasts and metazoans, and, in S. pombe, heterochromatin proteins recruit cohesin. Moreover, RNAi-mediated silencing of a non-centromeric gene in S. pombe causes the recruitment of heterochromatin proteins and cohesin to the silenced gene (Dorsett, 2005).

The idea is favored that cohesin inhibits enhancer-promoter communication in cut. This idea originates from the allele-specific effects of Nipped-B mutations on cut expression. The known cut regulators required for activation by the wing margin enhancer, including scalloped, vestigial, mastermind, Chip, Nipped-A and l(2)41Af, all display different cut allele specificities than Nipped-B. In contrast to these factors, Nipped-B is most limiting when enhancer-promoter communication is partially compromised by a weak gypsy insulator, suggesting that Nipped-B facilitates long-range communication (Dorsett, 2005).

Binding of cohesin to multiple sites between the wing margin enhancer and the cut promoter is consistent with the hypothesis that Nipped-B facilitates enhancer-promoter communication by regulating the binding of cohesin to chromosomes. To explain how Nipped-B aids activation, it is theorized that Nipped-B can remove cohesin from chromosomes. In a simple model, Nipped-B facilitates the cohesin-binding equilibrium. When Nipped-B is partially reduced but not abolished, it takes longer to achieve equilibrium, but the extent of cohesin binding is not altered and there is little effect on sister chromatid cohesion. The reduced cohesin on-off rates, however, would diminish the opportunities for gene activation that require cohesin removal or repositioning (Dorsett, 2005).

It is not known how cohesin inhibits long-range activation, but mechanisms can be envisioned for various long-range activation models. For example, cohesin could inhibit the folding or looping of the chromosome that is required to bring the enhancer into contact with the promoter. Alternatively, cohesin could block a Chip-mediated spread of protein binding between the enhancer and promoter in linking models for long-range activation, or block the transfer or tracking of RNA polymerase from the enhancer to the promoter, as appears to occur in the chicken beta-globin gene locus (Dorsett, 2005).

This study found that Drosophila Pds5 is required for sister chromatid cohesion, consistent with studies on fungal Pds5. To explain the effects of the pds5^e6 mutation on cohesin chromosome binding and cut expression, it is proposed that it produces a mutant protein that blocks cohesin binding, or causes cohesin to be released from chromosomes. This agrees with observations on vertebrate and S. pombe Pds5 suggesting that Pds5 has both positive and negative effects on cohesion, possibly by regulating the association of cohesin with chromosomes (Dorsett, 2005).

Vertebrates contain two Pds5 isoforms that associate with chromosomal cohesin. Reduction of Pds5 partially decreases sister chromatid cohesion. Consistent with the finding that the pds5^e3 null mutation does not reduce the binding of cohesin to polytene chromosomes, and with previous work on S. cerevisiae and S. pombe Pds5, Xenopus Pds5 is not required for the binding of cohesin to chromosomes. One report suggested that S. cerevisiae Pds5 is required for the association of cohesin with chromosomes, but it is possible that this discrepancy might be caused by differences in the mutant alleles, similar to the differences found between pds5^e3 and pds5^e6 (Dorsett, 2005 and references therein).

Depletion of Pds5 from Xenopus extracts increases the amount of cohesin associated with chromatin. A similar increase in cohesin binding could explain the slight decrease in cut expression caused by the pds5^e3 null allele. Consistent with the idea that wild-type Pds5 partially reduces cohesin binding, deletion of S. pombe pds5 partially suppresses a temperature-sensitive mutation in mis4, which encodes the homolog of the Nipped-B and Scc2 cohesin-loading factors (Dorsett, 2005 and references therein).

Because wild-type Pds5 appears to partially reduce the binding of cohesin to chromosomes, it is speculated that the pds5^e6 mutation increases this activity, which may be related to the cohesin-loading function of Nipped-B/Scc2. Scc2 interacts with cohesin, and is thought to open the cohesin ring. In synchronized yeast cells, cohesin loads at Scc2-binding sites and translocates away. Like Nipped-B/Scc2, Pds5 contains several HEAT repeats, and thus might also open the cohesin ring during DNA replication to allow it to encompass both sister chromatids. It could play a similar role in the snap model, in which cohesin complexes bound to the two sisters interlock to hold the sisters together. If the pds5^e6 mutant protein interacts with cohesin non-productively, it could block access to Nipped-B and prevent loading. Alternatively, when the mutant Pds5 attempts to establish cohesion, it might fail, releasing cohesin from the chromosome. Wild-type Pds5 might partially reduce cohesin binding by competing with Nipped-B for cohesin, or by occasionally failing to establish cohesion (Dorsett, 2005).

The effects of cohesin on cut expression are likely pertinent to the etiology of Cornelia de Lange syndrome (CdLS). CdLS is caused by mutations in the Nipped-B-Like (NIPBL) human homolog of Nipped-B. Most missense mutations that cause CdLS affect residues conserved in Nipped-B. CdLS is characterized by several physical and mental deficits, including slow growth, mental retardation, and upper limb, gastroesophageal and cardiac deformities. Heterozygous loss-of-function NIPBL mutations cause CdLS, and thus the developmental changes likely reflect gene expression effects similar to those caused by heterozygous Nipped-B mutations. At least some birth defects in CdLS, such as limb truncations and cardiac abnormalities, could be caused by changes in expression of the known homeotic genes. The observations presented here indicate that cohesin likely plays a role in CdLS by inhibiting the long-range gene control of homeotic genes (Dorsett, 2005).

The possibility that some developmental changes in CdLS reflect reduced sister chromatid cohesion cannot be ruled out. A recent study found evidence for cohesion deficits in 41% of CdLS patients compared with 9% of controls. Also, the autosomal recessive Roberts syndrome has some similarities to CdLS, and is caused by mutations in a human homolog of the Eco1/Eso1/Deco cohesion factor. Cells from Roberts patients display defects in sister chromatid cohesion. Homozygous Drosophila deco¹ mutants appear to affect cohesin binding only at centromeric regions, and, as described above, dominant effects of deco mutations on cut gene expression are not seen, leading to the the idea that most CdLS developmental deficits reflect changes in gene expression instead of in sister chromatid cohesion (Dorsett, 2005).

Chromosomal cohesin forms a ring

The cohesin complex is essential for sister chromatid cohesion during mitosis. Its Smc1 and Smc3 subunits are rod-shaped molecules with globular ABC-like ATPases at one end and dimerization domains at the other connected by long coiled coils. Smc1 and Smc3 associate to form V-shaped heterodimers. Their ATPase heads are thought to be bridged by a third subunit, Scc1, creating a huge triangular ring that could trap sister DNA molecules. This study addressed whether cohesin forms such rings in vivo. Proteolytic cleavage of Scc1 by separase at the onset of anaphase triggers its dissociation from chromosomes. N- and C-terminal Scc1 cleavage fragments remain connected due to their association with different heads of a single Smc1/Smc3 heterodimer. Cleavage of the Smc3 coiled coil is sufficient to trigger cohesin release from chromosomes and loss of sister cohesion, consistent with a topological association with chromatin (Gruber, 2003; full text of article).

The preferential association of Scc1's N-terminal fragment with Smc3's head and its C-terminal one with that of Smc1 is conserved in its meiotic counterpart Rec8. Scc1 and Rec8 share very little sequence homology except within their first and last 100 amino acids, which are generally conserved amongst Scc1 and Rec8 homologs from a wide variety of eukaryotes. These conserved terminal sequences must contain the SMC head interaction domains because fragments containing the first 115 amino or the last 115 amino acids of Scc1 formed complexes specifically with Smc3 or Smc1, respectively. There is no apparent similarity between Scc1's Smc3 binding N-terminal 115 amino acids with its Smc1 binding 115 C-terminal ones. This asymmetry presumably corresponds to an asymmetry of the Smc1/3 heterodimer's two heads, to which these sequences bind. The finding that these N- and C-terminal domains of Scc1 and Rec8 are homologous to those of ScpA proteins in bacteria and barren subunits of condensin implies that the heads of all SMC-like proteins may be connected in a similar manner. It is for this reason that that members of this family are called kleisins (from the Greek word for closure: kleisimo). Cohesin's asymmetry is presumably shared by condensin, which contains an Smc2/Smc4 heterodimer but contrasts with the symmetry of bacterial SMC proteins. If the latter bind the ScpA, as is currently suspected, then the molecular symmetry of the SMC dimer would predict that they bind at least two molecules of ScpA in a symmetric fashion (Gruber, 2003).

This image of Scc1-SMC interactions, inferred merely from co-expression of different subunits and their fragments in insect cells, is in full agreement with analysis of cohesin released from yeast chromosomes after cleavage by separase in vivo. Thus, both Scc1's N-terminal cleavage fragment and a fragment containing Smc3's head domain are released from the rest of separase-cleaved cohesin by severance of Smc3's coiled coil, while the C-terminal Scc1 fragment remains associated with the central domain severed from its heads, presumably due to the latter's association with Smc1. Soluble yeast cohesin appears to possess the same fundamental geometry; namely Scc1's N-terminal half is connected with Smc3's head while Scc1's C-terminal half is connected to Smc3's central domain via its interaction with Smc1 (Gruber, 2003).

The ring hypothesis postulates that Scc1, Smc1 and Smc3 form a triangle and predicts that all three subunits and fragments therefore should remain associated with each other when any single side of the triangle has been broken irrespective of where this break is. The finding that Smc3 holds together N- and C-terminal cleavage fragments of Scc1 while Scc1 holds together the two halves of Smc3 when its arm has been severed by double cleavage with the TEV protease clearly confirms this prediction. It is remarkable that breakage of any one the triangle's sides destroys its closure and hence its potential for trapping DNA without affecting its three corners, i.e., subunit interactions (Gruber, 2003).

Though these findings together with the electron micrographs of soluble cohesin are consistent with cohesin being an asymmetric monomeric complex, they do not by themselves rule out the possibility that it forms dimers when bound to chromatin, especially after DNA replication when cohesin complexes on different sister chromatids could in principle interact to generate cohesion. Available evidence is, however, inconsistent with this notion. For example, differently tagged versions of the same cohesin subunit cannot be coimmunoprecipitated from cohesin released from chromatin by micrococcal nuclease digestion. One specific dimer model would have it that the two Scc1 molecules within a cohesin dimer connect Smc1 and Smc3 heads not from the same but from different heterodimers. If this were so, then one might predict that cleavage of just one of the Scc1 molecules might be sufficient to release cohesin dimers from chromatin. This study found that TEV protease treatment of chromatin from a heterozygous diploid strain carrying wild-type and TEV cleavable Scc1 released Scc1 fragments but not intact Scc1. Therefore the notion is favored that both soluble and chromatin bound cohesin form an asymmetric monomeric complex (Gruber, 2003).

An important but yet unresolved issue is the mechanism by which the cohesin and other SMC protein-containing complexes bind to chromosomes. One model predicts that each of the two SMC head domains binds to one sister chromatid. It has been suggested on the basis of electron spectroscopic images of condensin complexes bound to DNA that the double helix might be wrapped around the SMC head domains. It is, however, unclear from the existing crystal structure of these domains how DNA could conceivably be wrapped round a domain whose dimensions are much smaller than those of a nucleosome (Gruber, 2003).

The finding that chromosomal cohesin forms a closed ring raises the possibility that cohesin's interaction with DNA is topological and not chemical in nature. By passing through cohesin's ring, chromosomal DNA could be topologically trapped by cohesin. This model makes no prediction as to the actual path of DNA as it passes through cohesin's ring and does not exclude the possibility of DNA being wrapped around the ring as opposed to simply passing once through it. A topological interaction of this nature would explain the resistance to high salt concentrations of cohesin's association with chromosomes as well as its resistance to DNA intercalating agents such as ethidium bromide. It would also explain why the ring's severance, be it within Scc1 or Smc3, is sufficient to release cohesin from chromosomes either in vitro or in vivo without apparently affecting any of cohesin's subunit interactions. If cohesin embraces chromosomal DNA in this manner, then sister chromatid cohesion could arise from the passage of sister DNA molecules through the same cohesin ring. Sister DNA molecules could also conceivably pass through different but interacting cohesin rings; though this would predict hitherto undetected interactions between cohesin complexes. Further experiments will be required to establish whether DNA really passes inside cohesin's ring (Gruber, 2003).

Though the experiments are consistent with DNA's passage through cohesin's ring, they shed little or no insight into the mechanism by which transient opening of the ring permits entry of the double helix. Such a process would be analogous to the entry of a climbing rope into a carabiner, which is a ring with a gate. In this model, Scc1 could be considered cohesin's gate and the binding of ATP to the SMC head domains and/or its hydrolysis might regulate the opening and shutting of cohesin's gate. A key question is whether Scc1 connects the head domains of Smc1 and Smc3 when they are bound to ATP and have thereby themselves 'dimerized' or whether it only connects the two heads after ATP has been hydrolyzed. In the latter case, cohesin could switch between two types of ring: one in which the heads are bound to each other but the Scc1 gate is open and another in which Scc1 alone connects the heads. Such a system would permit DNA strands to enter cohesin's ring without pre-existing strands exiting. Even if this hypothesis is correct, it remains a mystery how some gates can apparently be reopened without destroying Scc1, as presumably occurs during prophase in metazoan cells, while others can only be opened by cleavage of Scc1 at anaphase onset (Gruber, 2003).

Given the similar structure of Scc1, barren, ScpA, and the other members of the kleisin superfamily, it is hard not to believe that all complexes composed of SMC proteins operate using a similar topological principle to that proposed for cohesin, namely passage of DNA inside two arms held together by Scc1-like bridges (kleisins), which can be opened and shut. Such devices are presumably indispensable for regulating the packing of DNA within cells because unlike nucleosomes, they clearly existed in the common ancestor of all life forms on this planet and have been retained in almost all organisms ever since. Cohesin may be unique in holding sister DNAs within a single ring whereas condensin may have the ability to pass the same DNA molecule more than once through its ring, thereby acting as a DNA coil securing device (Gruber, 2003).

The structure of the cohesin ATPase elucidates the mechanism of SMC-kleisin ring opening

Genome regulation requires control of chromosome organization by SMC-kleisin complexes. The cohesin complex contains the Smc1 and Smc3 subunits that associate with the kleisin Scc1 to form a ring-shaped complex that can topologically engage chromatin to regulate chromatin structure. Release from chromatin involves opening of the ring at the Smc3-Scc1 interface in a reaction that is controlled by acetylation and engagement of the Smc ATPase head domains. To understand the underlying molecular mechanisms, this study has determined the 3.2-Å resolution cryo-electron microscopy structure of the ATPgammaS-bound, heterotrimeric cohesin ATPase head module and the 2.1-Å resolution crystal structure of a nucleotide-free Smc1-Scc1 subcomplex from Saccharomyces cerevisiae and Chaetomium thermophilium. ATP-binding and Smc1-Smc3 heterodimerization were found to promote conformational changes within the ATPase that are transmitted to the Smc coiled-coil domains. Remodeling of the coiled-coil domain of Smc3 abrogates the binding surface for Scc1, thus leading to ring opening at the Smc3-Scc1 interface (Muir, 2020).

Jmjd2c/Kdm4c facilitates the assembly of essential enhancer-protein complexes at the onset of embryonic stem cell differentiation

Jmjd2/Kdm4 H3K9-demethylases (see Drosophila Kdm4A) cooperate in promoting mouse embryonic stem cell (ESC) identity. However, little is known about their importance at the exit of ESC pluripotency. This study uncovered that Jmjd2c facilitates this process by stabilizing the assembly of Mediator-Cohesin complexes at lineage-specific enhancers. Functionally, Jmjd2c is required in ESCs to initiate appropriate gene expression programs upon somatic multi-lineage differentiation. In the absence of Jmjd2c, differentiation is stalled at an early post-implantation epiblast-like stage, while Jmjd2c-knockout ESCs remain capable of forming extra-embryonic endoderm derivatives. Dissection of the underlying molecular basis revealed that Jmjd2c is re-distributed to lineage-specific enhancers during ESC priming for differentiation. Interestingly, Jmjd2c-bound enhancers are co-occupied by the H3K9-methyltransferase G9a/Ehmt2, independently of its H3K9-modifying activity. Loss of Jmjd2c abrogates G9a recruitment and furthermore destabilizes loading of the Mediator and Cohesin components Med1 and Smc1a at newly activated and poised enhancers in ESC-derived epiblast-like cells. These findings unveil Jmjd2c-G9a as novel enhancer-associated factors, and implicate Jmjd2c as a molecular scaffold for the assembly of essential enhancer-protein complexes with impact on timely gene activation (Tomaz, 2017).

X-linked Cornelia de Lange syndrome owing to SMC1L1 mutations

Cornelia de Lange syndrome is a multisystem developmental disorder characterized by facial dysmorphisms, upper limb abnormalities, growth delay and cognitive retardation. Mutations in the NIPBL gene, a component of the cohesin complex, account for approximately half of the affected individuals. This study reports that mutations in SMC1L1 (also known as SMC1), which encodes a different subunit of the cohesin complex, are responsible for CdLS in three male members of an affected family and in one sporadic case (Musio, 2006).

Rad50 depletion impacts upon ATR-dependent DNA damage responses

The Mre11/Rad50/NBS1 (MRN) complex is mutated in inherited genomic instability syndromes featuring cancer predisposition, mental retardation and immunodeficiency. It functions both in DNA double-strand break repair and in controlling the ataxia telangiectasia mutated (ATM) kinase during the response to these lesions. Patients inheriting homozygosity for an NBS1 hypomorphic allele (see Drosophila Nbs) display reduced phosphorylation of signaling factors such as Chk1, but not of chromatin-associated factor H2AX, after stresses that activate the ATM-related kinase, ATR. Therefore, whether MRN has a global controlling role over the ATR kinase was tested through the study of MRN deficiencies generated via RNA interference. MRN is shown to be required for ATR-dependent phosphorylation of structural maintenance of chromosomes 1 (Smc1), which acts within chromatin to ensure sister chromatid cohesion and to effect several DNA damage responses. Novel phenotypes were uncovered, caused by MRN deficiency, that support a functional link between this complex, ATR and Smc1, including hypersensitivity to UV exposure, a defective UV responsive intra-S phase checkpoint and a specific pattern of genomic instability. In addition, certain ATR-dependent responses do not require MRN. These studies demonstrate that there is indeed a controlling role for MRN over the ATR kinase and have established that the downstream events under this control are broad, including both chromatin-associated and diffuse signaling factors, but may not be universal. These studies contribute to an understanding of the central role that MRN plays in damage detection and signaling, which serve to maintain genomic stability and resist neoplastic transformation (Zhong, 2005).

Phosphorylation of SMC1 is a critical downstream event in the ATM-NBS1-BRCA1 pathway

Structural maintenance of chromosomes (SMC) proteins (SMC1, SMC3) are evolutionarily conserved chromosomal proteins that are components of the cohesin complex, necessary for sister chromatid cohesion. These proteins may also function in DNA repair. SMC1 is a component of the DNA damage response network that functions as an effector in the ATM/NBS1-dependent S-phase checkpoint pathway. SMC1 associates with BRCA1 and is phosphorylated in response to IR in an ATM- and NBS1-dependent manner. Using mass spectrometry, it has been established that ATM phosphorylates S957 and S966 of SMC1 in vivo. Phosphorylation of S957 and/or S966 of SMC1 is required for activation of the S-phase checkpoint in response to IR. The phosphorylation of NBS1 (Nijmegen breakage syndrome gene product is a part of the hMre11 complex, a central player associated with double-strand break repair) by ATM is required for the phosphorylation of SMC1, establishing the role of NBS1 as an adaptor in the ATM/NBS1/SMC1 pathway. The ATM/CHK2/CDC25A pathway is also involved in the S-phase checkpoint activation, but this pathway is intact in NBS cells. These results indicate that the ATM/NBS1/SMC1 pathway is a separate branch of the S-phase checkpoint pathway, distinct from the ATM/CHK2/CDC25A branch. Therefore, this work establishes the ATM/NBS1/SMC1 branch, and provides a molecular basis for the S-phase checkpoint defect in NBS cells (Yazdi, 2002).

Involvement of the cohesin protein, Smc1, in Atm-dependent and independent responses to DNA damage

Structural maintenance of chromosomes (SMC) proteins play important roles in sister chromatid cohesion, chromosome condensation, sex-chromosome dosage compensation, and DNA recombination and repair. Protein complexes containing heterodimers of the Smc1 and Smc3 proteins have been implicated specifically in both sister chromatid cohesion and DNA recombination. The protein kinase Atm phosphorylates Smc1 protein after ionizing irradiation. Atm phosphorylates Smc1 on serines 957 and 966 in vitro and in vivo, and expression of an Smc1 protein mutated at these phosphorylation sites abrogates the ionizing irradiation-induced S phase cell cycle checkpoint. Optimal phosphorylation of these sites in Smc1 after ionizing irradiation also requires the presence of the Atm substrates Nbs1 and Brca1. These same sites in Smc1 are phosphorylated after treatment with UV irradiation or hydroxyurea in an Atm-independent manner, thus demonstrating that another kinase must be involved in responses to these cellular stresses. Yeast containing hypomorphic mutations in SMC1 and human cells overexpressing Smc1 mutated at both of these phosphorylation sites exhibit decreased survival following ionizing irradiation. These results demonstrate that Smc1 participates in cellular responses to DNA damage and link Smc1 to the Atm signal transduction pathway (Kim, 2002).

SMC1: an essential yeast gene encoding a putative head-rod-tail protein is required for nuclear division and defines a new ubiquitous protein family

The smc1-1 mutant was identified initially as a mutant of Saccharomyces cerevisiae that had an elevated rate of minichromosome nondisjunction. The wild-type SMC1 gene has been cloned. The sequence of the SMC1 gene predicts that its product (Smc1p) is a 141-kD protein, and antibodies against Smc1 protein detect a protein with mobility of 165 kD. Analysis of the primary and putative secondary structure of Smc1p suggests that it contains two central coiled-coil regions flanked by an amino-terminal nucleoside triphosphate (NTP)-binding head and a conserved carboxy-terminal tail. These analyses also indicate that Smc1p is an evolutionary conserved protein and is a member of a new family of proteins ubiquitous among prokaryotes and eukaryotes. The SMC1 gene is essential for viability. Several phenotypic characteristics of the mutant alleles of smc1 gene indicate that its product is involved in some aspects of nuclear metabolism, most likely in chromosome segregation. The smc1-1 and smc1-2 mutants have a dramatic increase in mitotic loss of a chromosome fragment and chromosome III, respectively, but have no increase in mitotic recombination. Depletion of SMC1 function in the ts mutant, smc1-2, causes a dramatic mitosis-related lethality. Smc1p-depleted cells have a defect in nuclear division as evidenced by the absence of anaphase cells. This phenotype of the smc1-2 mutant is not RAD9 dependent. Based upon the facts that Smc1p is a member of a ubiquitous family, and it is essential for yeast nuclear division, it is proposed that Smc1p and Smc1p-like proteins function in a fundamental aspect of prokaryotic and eukaryotic cell division (Strunnikov, 1993).

Search PubMed for articles about Drosophila Smc1

Dorsett, D., Eissenberg, J. C., Misulovin, Z., Martens, A., Redding, B. and McKim, K. (2005). Effects of sister chromatid cohesion proteins on cut gene expression during wing development in Drosophila. Development 132(21): 4743-4753. PubMed ID: 16207752

Gruber S. Haering, C. H. and Nasmyth, K. K. (2003). Chromosomal cohesin forms a ring. Cell 112: 765-777. PubMed ID: 12654244

Hallson, G., et al. (2008). The Drosophila cohesin subunit Rad21 is a trithorax group (trxG) protein. Proc. Natl. Acad. Sci. 105(34): 12405-10. PubMed ID: 18713858

Hatkevich, T., Boudreau, V., Rubin, T., Maddox, P. S., Huynh, J. R. and Sekelsky, J. (2019). Centromeric SMC1 promotes centromer clustering and stabilizes meiotic homolog pairing. PLoS Genet 15(10): e1008412. PubMed ID: 31609962

Kim, S. T., Xu, B. and Kastan, M. B. (2002). Involvement of the cohesin protein, Smc1, in Atm-dependent and independent responses to DNA damage. Genes Dev 16(5): 560-570. PubMed ID: 11877376

Khetani, R. S. and Bickel, S. E. (2007). Regulation of meiotic cohesion and chromosome core morphogenesis during pachytene in Drosophila oocytes. J Cell Sci 120(Pt 17): 3123-3137. PubMed ID: 17698920

Krishnan, B., Thomas, S. E., Yan, R., Yamada, H., Zhulin, I. B. and McKee, B. D. (2014). Sisters Unbound is required for meiotic centromeric cohesion in Drosophila melanogaster. Genetics 198(3): 947-65. PubMed ID: 25194162

Muir, K. W., Li, Y., Weis, F. and Panne, D. (2020). The structure of the cohesin ATPase elucidates the mechanism of SMC-kleisin ring opening. Nat Struct Mol Biol 27(3): 233-239. PubMed ID: 32066964

Musio, A., et al. (2006). X-linked Cornelia de Lange syndrome owing to SMC1L1 mutations. Nat. Genet. 38: 528-530. PubMed ID: 16604071

Perkins, A. T., Das, T. M., Panzera, L. C. and Bickel, S. E. (2016). Oxidative stress in oocytes during midprophase induces premature loss of cohesion and chromosome segregation errors. Proc Natl Acad Sci U S A 113: E6823-E6830. PubMed ID: 27791141

Phan, A., Thomas, C. I., Chakraborty, M., Berry, J. A., Kamasawa, N. and Davis, R. L. (2018). Stromalin constrains memory acquisition by developmentally limiting synaptic vesicle pool size Neuron. PubMed ID: 30503644

Pherson, M., Misulovin, Z., Gause, M. and Dorsett, D. (2019). Cohesin occupancy and composition at enhancers and promoters are linked to DNA replication origin proximity in Drosophila. Genome Res. PubMed ID: 30796039

Strunnikov, A. V., Larionov, V. L. and Koshland, D. (1993). SMC1: an essential yeast gene encoding a putative head-rod-tail protein is required for nuclear division and defines a new ubiquitous protein family. J Cell Biol 123(6 Pt 2): 1635-1648. PubMed ID: 8276886

Tanneti, N. S., Landy, K., Joyce, E. F. and McKim, K. S. (2011). A pathway for synapsis initiation during zygotene in Drosophila oocytes. Curr Biol 21(21): 1852-1857. PubMed ID: 22036181

Tomaz, R. A., et al. (2017). Jmjd2c/Kdm4c facilitates the assembly of essential enhancer-protein complexes at the onset of embryonic stem cell differentiation. Development [Epub ahead of print]. PubMed ID: 28087629

Webber, H. A., Howard, L. and Bickel, S. E. (2004). The cohesion protein ORD is required for homologue bias during meiotic recombination. J Cell Biol 164(6): 819-829. PubMed ID: 15007062

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

Yazdi, P. T., Wang, Y., Zhao, S., Patel, N., Lee, E. Y. and Qin, J. (2002). SMC1 is a downstream effector in the ATM/NBS1 branch of the human S-phase checkpoint. Genes Dev. 16(5): 571-82. 11877377

Zhong, H., et al. (2005). Rad50 depletion impacts upon ATR-dependent DNA damage responses. Hum. Mol. Genet. 14: 2685-2693. 16087684

date revised: 10 June 2024

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