InteractiveFly: GeneBrief

stromalin: Biological Overview | References

Gene name - Stromalin

Synonyms -

Cytological map position - 27C7-27C7

Function - subunit of the cohesin ring complex

Keywords - component of cohesin ring complex - regulates meiotic sister chromatid cohesion - important for chromosome condensation, DNA repair, and gene expression - negative regulator of synaptic vesicle pool size in dopamine neurons

Symbol - SA

FlyBase ID: FBgn0020616

Genetic map position - chr2L:6,950,421-6,954,586

NCBI classification - STAG domain

Cellular location - nuclear

NCBI links: EntrezGene, Nucleotide, Protein

Stromalin orthologs: Biolitmine

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).

Learning and memory are tightly regulated processes that require the activity of hundreds of genes to orchestrate the proper development of neural circuits and the underlying physiological changes necessary for cellular and synaptic plasticity. While many genes are known that define mechanisms required for the formation and consolidation of memory, far less is known about the genetic factors that constrain memory formation and their molecular and cellular mechanisms. Important conceptual insights about memory formation might come from elucidating the cellular mechanisms underlying this class of genetic element. Memory suppressor genes, named so by analogy to tumor suppressor genes, could, in principal, function by limiting memory acquisition, consolidation, or retrieval or by participating in active forgetting processes (Phan, 2018).

Several dozen and novel memory suppressor genes were recently identified in a large RNAi screen for effects on 3 hr aversive olfactory memory expression in Drosophila (Walkinshaw, 2015). They were classed as such because knockdown of these genes led to increased memory expression. A cohesin complex member, stromalin, was one such gene identified in the screen. The highly conserved cohesin complex is comprised of Stromalin (STAG1/2 in mammals) and three other subunits named structural maintenance of chromosomes 1 (SMC1), SMC3, and Rad21 (Phan, 2018).

Although the complex was first identified for its role in the proper segregation of chromosomes during cell division, evidence has emerged showing that the complex has other important biological functions. Cohesin complex mRNAs and proteins are present at moderate to high levels in both the Drosophila and mouse nervous systems, revealing potential roles beyond chromosome segregation. Elegant studies from two research groups have clearly shown that members of the complex have a post-mitotic role in the proper pruning of axons in Drosophila mushroom body neurons. Other studies have provided evidence for roles in gene expression, DNA repair, and cancer susceptibility. It is notable that absent from this list are clear and specific roles for the complex in learning and memory processes, other than the general cognitive disturbances observed in humans with cohesinopathies (Phan, 2018).

This study reports that RNAi knockdown of Stromalin in mushroom body and dopamine neurons leads to enhanced aversive olfactory memory in adult flies. stromalin functions during development as a negative regulator of both synaptic and dense core vesicle (DCV) number in the nervous system, limiting the strength of synaptic connections to suppress memory acquisition. Reducing Stromalin levels specifically increases the number of vesicles in neurons without detectably altering other features of the targeted neurons in adult flies, including synapse number, synapse volume, or neurite branching. These observations offer evidence that the size of the synaptic vesicle pool is regulated independently of other structural features of the neuron (Phan, 2018).

Memory suppressor genes offer a unique window for understanding the molecular and cellular mechanisms that constrain memory formation. In contrast to the many genes and gene products known to be required for acquisition and memory consolidation, there are but a handful of memory suppressor genes studied to the point of providing new conceptual insights into the processes of memory formation. Some function at the transcriptional level to control the formation of protein synthesis-dependent long-term memory (LTM). For instance, isoforms of the Creb transcription factor (Creb repressors) exist that inhibit the normal function of Creb activators to limit LTM. These are thought to function after initial memory acquisition through biochemical cascades that mobilize new protein synthesis required for LTM. Other memory suppressor genes actively repress communication between neurons. For instance, Drosophila SLC22A encodes a plasma membrane transporter that removes neurotransmitter from the synaptic cleft to terminate synaptic communication. Cyclin-dependent kinase 5 (Cdk5) promotes proteolysis of the NMDA receptor subunit NR2B, attenuating NMDA receptor signaling in mammalian neurons. It is notable that these and other previously described memory suppressor genes limit the memory capacity of adult organisms, while developmental negative regulation of adult memory is rare. Stromalin is unique, acting during a critical developmental window to constrain the strength of synaptic communication between neurons by limiting the size of the synaptic vesicle pool. This phenotype then persists into adult life (Phan, 2018).

The data argue that Stromalin regulates the SVs in DAn independent of other structural features of the neuron, such as cell number, the apparent ramification of DAn neuropil in the MB, and synapse number or size. These observations lead to the novel and important conclusion that the SV pool is under its own genetic regulation through Stromalin function. Prior to these results, SV pool size was thought to be a function of synapse or active zone size or some other aspect of neuronal morphology (Phan, 2018).

Surprisingly, Stromalin alters the number of both SVs and DCVs, suggesting that it has a shared role in the biosynthetic or degradative pathways for both types of vesicles that are distinct from the piccolo-bassoon transport vesicles that contain active zone proteins. Components of SVs and DCVs are generated in the endoplasmic reticulum (ER) and processed through the Golgi apparatus but are sorted separately into SV transport precursor vesicles containing SV proteins and into DCVs for anterograde transport toward the axon terminals. This study study provides the first evidence for a developmental genetic program that specifically controls the strength of synaptic connections by constraining the SV pool in neurons. It is hypothesized that Stromalin regulates SV and DCV number through its role in regulating gene expression (Phan, 2018).

Interestingly, the critical window for Stromalin's effects on the SV pool size occurs during the third-instar larval period. This developmental time point is well after the integration of DAn into the larval olfactory memory circuit and after the initial onset of DAn synaptogenesis onto MBn, since these synapses are already present at the first-instar larval stage. The γ MBn that are present in the larval brain prior to the mid-third-instar developmental stage undergo extensive axonal and dendritic restructuring during the pupal stage, such that the structural organization and connectivity of the larval γ MBn is distinct from that in the adult. It is during the mid-third-instar larval stage that the α'β' MBn develop, and these appear to persist into the adult fly relatively unchanged in structure. This developmental transition maps directly onto the critical window for Stromalin's effects on limiting synaptic vesicle pool size in DAn. Stromalin does not affect SV number at the earliest stages of neural circuit development and synaptogenesis but rather only upon emergence of the first set of MBn that persist and integrate into the adult neural circuitry. Stromalin may thus be specifically involved in a developmental program that adjusts the strength of DAn synaptic connectivity for adult-relevant neural circuitry and functions (Phan, 2018).

Insults that produce a loss of function of cohesin complex genes have been previously shown to cause developmental axonal and dendritic pruning defects in the γ subset of MBn. Membrane-GFP data, as well as the EM analysis on DAn neuropil volumes, failed to find similar differences in the DAn axonal ramifications of adult fly brains, arguing that DAn axons do not undergo the same Stromalin-dependent axonal pruning that occurs with γ MBn or that such pruning is transient and fails to persist into adulthood. stromalinRNAi was expressed in the γ MBn, and adult γ MBn morphology was examined using membrane-bound GFP staining but did not detect a pronounced morphological difference in these neurons. Presumably, a complete loss of function is required to detect the pronounced pruning defects observed previously (Pauli, 2008, Schuldiner, 2008). Moreover, impairing synaptic vesicle transport to axon terminals reversed the enhanced memory phenotype. Taken together, these data fail to support the hypothesis that developmental axonal pruning deficits of DAn lead to the enhancement in learning and memory scores in flies with Stromalin KD in these same neurons (Phan, 2018).

While this study focused on DAn, the data indicate that Stromalin's role in constraining synaptic vesicle pool size extends to other neurons of the Drosophila brain, since Syt:GFP increases were also detected with pan-neuronal KD and with KD in the cholinergic MB Kenyon cell neurons. The alteration of neurotransmitter release in a variety of neurons with Stromalin KD is likely to have a profound effect on a range of different behaviors, since mutations in genes affecting synaptic communication have been associated with many behavioral/cognitive, neurodevelopmental, neurodegenerative, and neuropsychiatric disorders. Similarly, it is predicted that broad expression of the unc104RNAi transgene would alter other behaviors and generally in ways opposite of stromalinRNAi with an appropriate level of expression. Thus, these transgenes offer valuable new tools for modulating SV content across neurons to probe effects on synaptic communication and behavioral processes. Interestingly, the stromalinRNAi effects were able to rescue the modest learning impairments caused by unc104RNAi expression in DAn, which suggests that increasing synaptic vesicle content may provide a potential symptomatic treatment for patients with KIF1A mutation (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).

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 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 Vtd in Drosophila (also known as Rad21) 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 (Kojic, 2018), 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 (Countryman 2018). 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).

A Cut/cohesin axis alters the chromatin landscape to facilitate neuroblast death

Precise control of cell death in the nervous system is essential for development. Spatial and temporal factors activate the death of Drosophila neural stem cells (neuroblasts) by controlling the transcription of multiple cell death genes through a shared enhancer. The activity of this enhancer is controlled by abdominalA and Notch, but additional inputs are needed for proper specificity. This study shows that the Cut DNA binding protein is required for neuroblast death, regulating reaper and grim downstream of the shared enhancer and of abdominalA expression. cut loss accelerates the temporal progression of neuroblasts from a state of low overall levels of H3K27me3 to a higher H3K27me3 state. This is reflected in an increase in H3K27me3 modifications in the cell death gene locus in the CNS on cut knockdown. This study also shows that cut regulates the expression of the cohesin subunit Stromalin. Stromalin and the cohesin regulatory subunit NippedB are required for neuroblast death, and knockdown of Stromalin increases H3K27me3 levels in neuroblasts. Thus, Cut and cohesin regulate apoptosis in the developing nervous system by altering the chromatin landscape (Arya, 2019).

This study reports that cut plays a previously unknown role in the regulation of NB death. cut is permissive for the expression of rpr and grim, acting downstream of the previously identified neuroblast regulatory region. cut loss increases the number of NBs with high levels of H3K27me3, indicating a role for cut in maintaining open chromatin in NBs. At the reaper- and grim- encoding RHG locus, this is reflected in higher levels of H3K27me3, associated with lower rpr and grim expression. Importantly, this study found that cut regulates the levels of the cohesin subunit SA in the CNS, and cohesin was shown to be required for NB death. This work demonstrates a novel connection between cut and cohesin in controlling the chromatin landscape and cell death in the developing CNS (Arya, 2019).

Previous work identified the Hox gene abdA as an important spatial signal for NB death in the embryo. A late pulse of AbdA in NBs is regulated by Notch activation that is dependent on Delta ligand expression in NB progeny. abdA is necessary and sufficient for NB death, and has been shown to bind to a shared enhancer, enh1, in the Neuroblast regulatory region. However, abdA is clearly expressed in many cells that do not die. Furthermore, mis-expression of abdA does not activate ectopic NB death prior to stage 13 of embryogenesis, suggesting that there are temporal and cell identity signals that regulate the competence of cells to respond to abdA (Arya, 2019).

This study identified cut as a novel regulator of NB death. Expression of cut in the embryonic CNS increases as NB death begins. However, most cells that normally express cut do not die, indicating that other factors coordinate with cut to regulate NB death. This study found that loss of cut inhibits rpr and grim transcription, but in contrast to abdA and N, cut does not act on enh1, as detected by enh1-GFP. In addition, cut knockdown blocks NB killing in response to abdA mis-expression, despite an expansion of enh1 expression. These data indicate that cut acts downstream of enh1, and suggests that cut acts in the nervous system as a permissive factor that regulates the competence of NBs to respond to other cell death signals (Arya, 2019).

This study found that cut functions in the CNS to restrict overall levels of repressive H3K27me3-marked chromatin. NBs have a significantly lower level of overall H3K27me3 than other tissues in the embryo, possibly associated with stem cell plasticity. As embryos age, the number of NBs with high overall levels of H3K27me3 increases. The cause and consequences of this transition are unknown, but could be related to a gradual restriction of NB fate (Arya, 2019).

Loss of cut promotes more NBs to acquire an H3K27me3 high state throughout later stages of embryogenesis. Interestingly, in both control and cut knockdown there is a temporal increase in the proportion of NBs with high H3K27me3. This suggests that additional temporal factors control this maturation of NBs to a more repressed state, but cut restrains the number of H3K27me3 high cells throughout this transition (Arya, 2019).

These data indicate that cut overexpression is sufficient to cause increased rpr and grim expression and apoptosis in NBs. This is not due to hyper-activation of enh1, as ectopic cut does not increase enh1-GFP expression and can cause NB death even in the absence of the neuroblast regulatory region. In addition, cut overexpression causes increased cell death in other cells that normally survive, as seen with heat shock-gal4. This suggests that ectopic cut could activate additional upstream apoptosis-inducing signals, directly activate rpr and grim expression, or could open the rpr region for activation by regulators that do not normally activate rpr and grim (Arya, 2019).

The role of cut in activating NB death is in contrast to previous work suggesting that cut inhibits cell death in the developing posterior spiracle by directly inhibiting rpr expression. In the developing spiracle, cut is also required for normal differentiation. Several other tissues also require cut for normal differentiation, such as the bristle cells in the eye, and the developing trachea. In these tissues, cell death is also increased in the absence of cut. The role of cut in promoting cell survival in these tissues differs from its role in facilitating cell death in the CNS. This may reflect the diverse activities of cut as a transcriptional regulator, or could be due to cut's activity as a chromatin organizer, altering the landscape for binding by both activators and repressors of RHG gene transcription. Both pro-differentiation and pro-apoptotic roles of cut are consistent with its role as a potential tumor suppressor (Arya, 2019).

This study indicates that the rpr region is particularly sensitive to alterations in chromatin conformation, reflecting the need for rapid and robust transcription of the cell death genes in cells fated to die. Other factors that control histone modifications are involved in cell death. For example the dUTX H3K27me3 demethylase is required for Ecdysone Receptor-mediated activation of rpr expression in salivary gland death. This supports the finding that a more open chromatin conformation is particularly important for cell death gene activation. Expression of other components of the cell death pathway may also be controlled by changes in chromatin conformation. For example, treatment of Drosophila larvae with HDAC inhibitors, or HDAC1 knockdown, increases sensitivity to cell death activation through altered expression of caspases. Conversely, loss of Polycomb-mediated suppression is associated with loss of postembryonic NBs, although this may be due to ectopic abdA expression. There is also evidence for epigenetic regulation of genes important for cell death in the mammalian nervous system and in cancer. This study provides evidence that control of histone modifications in the rpr region is an important aspect of developmental cell death regulation (Arya, 2019).

Given the lack of evidence for a direct histone-modifying role of Cut in regulating cell death, alternative indirect mechanisms were investigated, and it was determined that cut promotes expression of the cohesin subunit SA. Similar to loss of cut, down-regulation of SA or Nipped-B results in ectopic NB survival. Cohesins are involved in sister chromatin cohesion, formation of topologically associated domains and in long-range enhancer promoter interactions. This latter function may be particularly important in Drosophila developmental cell death. Multiple cell death genes must be activated in different tissues in response to overlapping signals impinging on distinct regulatory enhancers. This suggests that three dimensional chromatin interactions, including those mediated by cohesin, are critical for facilitating precise gene activation in the RHG region (Arya, 2019).

Loss of one copy of the human Nipped-B homolog NIBPL, and of other cohesin components, is associated with Cornelia de Lange syndrome, a developmental disorder affecting growth, cognitive function and facial and limb morphology. This is likely due to the downregulation of developmentally important genes, as detected in NIBPL +/-MEFs. Nipped-B heterozygous flies also exhibit reduced growth, learning and memory deficits, abnormal brain morphology and reduced expression of many genes. Interestingly, Nipped-B heterozygotes are resistant to dMyc induced apoptosis, a phenotype also seen in the IRER mutants, suggesting that cohesin may also regulate cell death activated by the IRER enhancer. The current data suggest that control of cell death in the nervous system could also contribute to the Cornelia de Lange syndrome phenotype. Additional studies are needed to understand how cohesin activity is directed towards regulating the expression of specific genes (Arya, 2019).

Precise control of apoptotic gene expression is particularly important in the nervous system, the site of the majority of developmental cell death in flies, worms and mammals, and the tissue most affected by the absence of cell death. This work has led to a greater understanding of the temporal, spatial and tissue specific control of this death in flies through developmentally important transcription factors as well as regulation of chromatin accessibility and architecture. Given the conserved function of the pathways that were identified in this study, it is likely that these studies will provide insight into the regulation of cell death in human nervous system development and disease (Arya, 2019).

Dynamic and stable cohesins regulate synaptonemal complex assembly and chromosome segregation

Assembly of the synaptonemal complex (SC) in Drosophila depends on two independent pathways defined by the chromosome axis proteins C(2)M and ORD. Because C(2)M encodes a Kleisin-like protein and ORD is required for sister-chromatid cohesion, the hypothesis was tested that these two SC assembly pathways depend on two cohesin complexes. Through single- and double-mutant analysis to study the mitotic cohesion proteins Stromalin (SA) and Nipped-B (SCC2) in meiosis, evidence was provided that there are at least two meiosis-specific cohesin complexes. One complex depends on C(2)M, SA, and Nipped-B. Despite the presence of mitotic cohesins SA and Nipped-B, this pathway has only a minor role in meiotic sister-centromere cohesion and is primarily required for homolog interactions. C(2)M is continuously incorporated into pachytene chromosomes even though SC assembly is complete. In contrast, the second complex, which depends on meiosis-specific proteins SOLO, SUNN, and ORD is required for sister-chromatid cohesion, localizes to the centromeres and is not incorporated during prophase. Multiple cohesin complexes may provide the diversity of activities required by the meiotic cell (Gyuricza, 2016).

Based on similar mutant phenotypes and double-mutant analysis, it is proposed that SC assembly in Drosophila depends on two meiotic cohesin complexes. The first includes C(2)M, SA, and Nipped-B. The most important function of C(2)M/SA/Nipped-B is SC assembly, which is demonstrated by the more significant SC assembly defects observed in c(2)M mutants and SA or Nipped-B knockdowns compared to sunn, solo, or ord mutants. The cytological results suggest that, like C(2)M, SA has only a minor role in meiotic sister-centromere cohesion. Correlating with this difference is that C(2)M, SA, or Nipped-B are required for the accumulation of SMC proteins on the chromosome arms but not the centromeres. Furthermore, Nipped-B, like C(2)M, localizes to the chromosome arms but not the centromeres. These observations indicate a significant change in cohesin regulation. While SA and Nipped-B are required for sister-chromatid cohesion in mitotic cells, they have a new partner, C(2)M, for a non-cohesion function in meiosis. There are minor differences in the c(2)M and SA phenotypes, which has also been observed with solo and sunn, suggesting there could be additional minor complexes. SA and Nipped-B could be required to maintain sister-chromatid cohesion on the chromosome arms in late prophase, a function that C(2)M likely does not have (Gyuricza, 2016).

The second proposed meiotic cohesin complex includes SOLO, SUNN, and ORD, which are also highly diverged, making homology assignments difficult. Based on sequence features, SUNN may be a SA homolog, while SOLO has been shown to interact with SMC1 and to have sequence motifs similar to the SMC1 interaction domains of kleisins. The role of ORD in this context is unclear. It is possible that ORD is a positive regulator like Nipped-B. Genetic evidence shows that ord, sunn, and solo are required for sister-chromatid cohesion, which correlates with SMC1/3 and SC accumulation at the centromeres. In addition, there are elevated levels of sister-chromatid exchange and abnormal SC structure in ord and solo mutants (Gyuricza, 2016).

Surprisingly, this study found an important role for the meiotic cohesins SUNN and ORD in mitotic germline cells, which is consistent with prior observations that ORD localizes to centromeric foci in premeiotic cells and ord mutants have defects in mitotically dividing germline cells. Since Rec8 in C. elegans is also observed in premeiotic cells, it may be a conserved feature of meiotic cohesins required for sister-chromatid cohesion that they accumulate and function in premeiotic mitotic germline cells (Gyuricza, 2016).

A striking difference between C(2)M/SA and SOLO/SUNN is that C(2)M incorporation is continuous during pachytene even after the SC is fully assembled. This may result in a dynamic SC, which has been observed in budding yeast. Paradoxically, this study also found that C(2)M must load during a narrow window of early prophase in order to support crossover formation. Cohesins have been shown to be loaded during prophase in a number of systems including Drosophila. In contrast, centromeric SOLO/SUNN can only be loaded prior to meiotic prophase. Sister-chromatid cohesion in mitotic cells is established during S-phase and in mammals, Rec8 cohesin cannot be replenished and dissociates with age. Whereas in mitotic cells the dynamic and stable cohesin complexes involve the same four core subunits, in meiosis, there may be separate cohesin complexes that differ in their regulation and capacity to be replaced or replenished. These observations complement Weng (2014) who showed there was cohesin replenishment during meiotic prophase, although after pachytene and possibly not at the centromeres. Interestingly, mouse Nipbl (Nipped-B) and the meiosis-specific SMC1β show pronounced accumulation starting at leptotene, indicating that, as in Drosophila, some mouse cohesins are loaded during pachytene while other cohesins are stable (Gyuricza, 2016).

Meiosis-specific cohesin complexes appear to be a highly conserved feature of meiosis. While there is some variation in the constituents of each cohesin complex, the results suggesting two major pathways contributing to SC assembly help explain the results with coh-3/coh-4 and rec-8 in C. elegans or rad21l and rec8 in mouse. Only the double mutants in each case eliminate all SC. The role of the respective kleisins could also be conserved. Like C(2)M, Rad21L has been proposed to be primarily responsible for inter-homolog chromosome interactions. Multiple cohesin complexes may be required because some cohesins need to be loaded at a specific time (S-phase for cohesion) while others need to be exchanged during pachytene. A dynamic cohesin complex may be important to provide plasticity to the meiotic chromosomes and allow them to respond to double-stranded breaks (DSBs) and regulate crossover formation, crossover interference, and chromosome segregation. Alternatively, different cohesin complexes may accumulate at different locations. If meiotic cohesins, directly or indirectly, interact with SC proteins, they may have a strong influence on the pattern of SC assembly and influence the frequency and distribution of DSBs and crossovers (Gyuricza, 2016).

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 has identified a novel Drosophila gene called sisters unbound (sunn), which is required for stable sister chromatid cohesion throughout meiosis. sunn mutations disrupt centromere cohesion during prophase I and cause high frequencies of non-disjunction (NDJ) at both meiotic divisions in both sexes. SUNN co-localizes at centromeres with the cohesion proteins SMC1 and SOLO 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).

Histone chaperone NAP1 mediates sister chromatid resolution by counteracting protein phosphatase 2A

Chromosome duplication and transmission into daughter cells requires the precisely orchestrated binding and release of cohesin. This study found that the Drosophila histone chaperone NAP1 is required for cohesin release and sister chromatid resolution during mitosis. Genome-wide surveys revealed that NAP1 and cohesin co-localize at multiple genomic loci. Proteomic and biochemical analysis established that NAP1 associates with the full cohesin complex, but it also forms a separate complex with the cohesin subunit

  • stromalin (SA). NAP1 binding to cohesin is cell-cycle regulated and increases during G2/M phase. This causes the dissociation of protein phosphatase 2A (PP2A) from cohesin, increased phosphorylation of SA and cohesin removal in early mitosis. PP2A depletion led to a loss of centromeric cohesion. The distinct mitotic phenotypes caused by the loss of either PP2A or NAP1, were both rescued by their concomitant depletion. In is concluded that the balanced antagonism between NAP1 and PP2A controls cohesin dissociation during mitosis (Moshkin, 2013).

    As reflected by their name, a major activity of histone chaperones is to prevent illicit liaisons and guide newly synthesized histones to sites of chromatin assembly. This study describes a mitotic function for the canonical histone chaperone NAP1 that is unrelated to nucleosome assembly. NAP1 was found to bind cohesin and block dephosphorylation of SA by PP2A, thereby promoting cohesin dissociation from the chromosome arms. Consequently, chromosomal binding of cohesin during mitosis is controlled by the balance between the opposing activities of NAP1 and PP2A (Moshkin, 2013).

    NAP1 is part of a large assemblage including the full cohesin complex and PP2A. In addition, NAP1 and SA form a subcomplex, which lacks the other cohesin subunits and PP2A. An attractive scenario is that the NAP1-SA module or NAP1 alone competes with PP2A-bound SA within the full cohesion complex. PP2A displacement by NAP1 allows stable phosphorylation of cohesin and its dissociation during early mitosis. NAP1 might also act as a direct inhibitor of PP2A catalytic activity, because a mammalian NAP1 homolog, SET, has been identified as a potent PP2A inhibitor, which promotes sister chromatid segregation during mouse oocyte miosis (Qi, 2013; Chambon, 2012). In addition, NAP1 might help cohesin phosphorylation by tethering Polo kinase to cohesin. In fact, a potential association between NAP1 and Polo kinase was detected. However, the dramatic chromosome condensation defects after Polo kinase depletion precluded a functional evaluation of a possible role of NAP1 in its function. Nevertheless, although additional NAP1 activities cannot be excluded, functional experiments established that blockage of PP2A suffices to explain the crucial role of NAP1 during sister chromatid resolution (Moshkin, 2013).

    NAP1 not only regulates the chromosomal distribution of cohesin and PP2A, but also that of MeiS332, a fly homolog of Sgo. The function of MeiS332 and PP2A appears to be largely conserved from mammals to flies because they bind each other and depletion of either factor causes a loss of centromeric cohesion. Either knockdown of NAP1 or over-expression of PP2A caused spreading of MeiS332 onto the arms of mitotic chromosomes, accompanying the loss of sister chromatid resolution. Thus, the balanced antagonism between NAP1 and PP2A controls chromosomal association of both cohesin and MeiS332 during mitosis (Moshkin, 2013).

    One level of regulation involves changes in NAP1's subcellular localization and chromatin binding through the cell cycle. At prophase there is a strong increase in the level of nuclear NAP1, but by metaphase, NAP1 and cohesin have dissociated from the chromosomes. Thus, the dynamic behavior of NAP1 correlates well with its function in promoting cohesin release at early mitosis. Regulation of NAP1 localization may involve cyclin B-cdc2/cdk1 kinase complexes. Previously it was found that yeast and vertebrate NAP1 are phosphorylated by cyclin B-cdc2 and that yeast cyclin B requires NAP1 for its full range of mitotic functions (Moshkin, 2013).

    It is suggested that histone chaperones are at the hubs of specialized protein networks that perform a wide variety of tasks in chromosome biology. Through association with distinct partners, NAP1 is able to perform different functions. By acting as a histone chaperone, NAP1 mediates chromatin assembly. Through recruitment of the histone H3 deacetylase and H3K4 demethylase complex RLAF, NAP1 controls gene-selective silencing at developmental loci. Finally, by binding cohesin and blocking SA dephosphorylation by PP2A, NAP1 mediates sister chromatid resolution during mitosis. These results emphasize the surprisingly diverse- and specific regulatory functions of histone chaperones in chromosome biology (Moshkin, 2013).

  • Drosophila Nipped-B protein supports sister chromatid cohesion and opposes the Stromalin/Scc3 cohesion factor to facilitate long-range activation of the cut gene

    The Drosophila melanogaster Nipped-B protein facilitates transcriptional activation of the cut and Ultrabithorax genes by remote enhancers. Sequence homologues of Nipped-B, Scc2 of Saccharomyces cerevisiae, and Mis4 of Schizosaccharomyces pombe are required for sister chromatid cohesion during mitosis. The evolutionarily conserved Cohesin protein complex mediates sister chromatid cohesion, and Scc2 and Mis4 are needed for Cohesin to associate with chromosomes. This study shows that Nipped-B is also required for sister chromatid cohesion but that, opposite the effect of Nipped-B, the stromalin/Scc3 component of Cohesin inhibits long-range activation of cut. To explain these findings, a model is proposed based on the chromatin domain boundary activities of Cohesin in which Nipped-B facilitates cut activation by alleviating Cohesin-mediated blocking of enhancer-promoter communication (Rollins, 2004).

    These experiments addressed two questions: (1) does Nipped-B, in addition to facilitating remote activation of cut and Ultrabithorax, participate in mitotic sister chromatid cohesion, and (2) does Cohesin participate in long-range activation of cut? The first was motivated by the sequence similarity of Nipped-B to yeast adherins required for sister chromatid cohesion, and the second was motivated by the published observations that the yeast adherins are required for the Cohesin complex to associate with chromosomes. The results indicate that the cooperation between adherins and Cohesin that occurs in yeast is conserved in Drosophila. The findings also indicate, however, that the SA/Scc3 subunit of Cohesin opposes Nipped-B in long-range activation of the cut gene (Rollins, 2004).

    The high rate of precocious sister chromatid separation (PSCS) in homozygous and heteroallelic Nipped-B mutants and the increased lethality of Rad21/Scc1 RNAi in flies heterozygous for a Nipped-B mutation observed in this study are consistent with the findings that the yeast Scc2 and Mis4 homologues of Nipped-B are required for Cohesin to associate with chromosomes. Although PSCS was detected in third-instar neuroblasts in RNAi experiments, it is unlikely that the synthetic lethality of a heterozygous Nipped-B mutation and Rad21/Scc1 RNAi is caused by changes in long range gene activation, because as discussed below, Nipped-B and Cohesin appear to have opposing roles in gene activation. In the RNAi experiments, it is possible that the pupal lethality is caused by PSCS in a subpopulation of critical cells, but the possibility cannot be ruled out that Nipped-B and Cohesin cooperate with each other in other essential functions (Rollins, 2004).

    No physical association between the yeast adherins and Cohesin has been detected, nor do they colocalize on chromosomes. No chromosomal population of Nipped-B could be detected, but if Nipped-B acts primarily as a chaperone for loading Cohesin onto chromosomes, only a small fraction of Nipped-B may be transiently interacting with chromosomes at any time. The mechanisms by which yeast adherins facilitate Cohesin chromosome binding are unclear, but the synthetic lethality between a heterozygous Nipped-B mutation and Rad21/Scc1 RNAi observed in this study indicates that this functional connection is conserved in metazoans. In budding yeast, Cohesin begins to associate with chromosomes in late G1, while in fission yeast, Caenorhabditis elegans, Drosophila, and mammalian cells it begins to associate in telophase. Nipped-B is detected in the nucleus at all stages that have a nuclear membrane, indicating that it could be involved in Cohesin chromosomal association beginning in telophase and thus could influence all potential interphase functions of Cohesin, in addition to sister chromatid cohesion (Rollins, 2004).

    The finding that SA/Scc3 RNAi reduces the severity of the ctK wing-nicking phenotype indicates that the SA/Scc3 component of Cohesin inhibits cut expression. This is the opposite to the role of Nipped-B at cut. Multiple Nipped-B mutations were recovered in a screen for mutations that increase the severity of a wing-nicking phenotype displayed by a cut allele with a weak gypsy insulator insertion. Reduced mRNA levels indicated that some of these Nipped-B mutations are loss-of-function alleles, and viability of homozygous Nipped-B mutants was rescued by a transgene expressing a Nipped-B cDNA from a Chip gene promoter. Thus, Nipped-B protein facilitates activation of cut by the wing margin enhancer (Rollins, 2004).

    The effect of Nipped-B on cut expression is likely direct. Nipped-B does not regulate cut by altering the activities of known cut regulators because it is most limiting for cut expression when there is a gypsy insertion at cut while the other known regulators are more limiting with other types of cut mutations. Moreover, heterozygous Nipped-B loss-of-function alleles reduce cut expression, and partial reduction of Nipped-B is unlikely to cause an equal or greater change in the expression of another cut regulator. Although the effects of Nipped-B on gene expression were most apparent with gypsy insertion alleles of cut, a measurable effect was observed in heterozygous females with a wild-type cut allele and an allele in which the wing margin enhancer is deleted. Thus, Nipped-B also facilitates the activation of wild-type cut (Rollins, 2004).

    All three SA/Scc3 RNAi insertions and one of three Rad21/Scc1 insertions reduced the number of nicks displayed by the ctK gypsy insertion. It is thought likely that the Cohesin complex, and not just one or two of its subunits, is responsible for reducing cut expression. Scc1 and Scc3 operate together as a unit in both Drosophila and C. elegans. Thus, it is unlikely that they work independently of each other in regulating gene expression. Indeed, Rad21/Scc1 RNAi in cultured Drosophila cells reduces both Rad21/Scc1 and SA/Scc3 proteins, and data presented in this study indicate that Rad21/Scc1 and SA/Scc3 may regulate each other's transcript levels. However, the possibility cannot be ruled out that Rad21/Scc1 and SA/Scc3 work independently of the Smc1 and Cap/Smc3 Cohesin subunits, which form another stable subcomplex. Initial attempts to reduce expression of the SMC subunits by RNAi were unsuccessful (Rollins, 2004).

    It is unlikely that the effects of SA/Scc3 on cut expression occur by reducing the expression of a cut activator. The small reductions in SA/Scc3 expression in these experiments are unlikely to cause equal or larger changes in the activities of other cut regulators. Also effects are not seen of Cohesin RNAi on ct53d, which has a small deletion in the enhancer and is affected by all known cut regulators except Nipped-B. It is most likely, therefore, that SA/Scc3 acts directly at cut or by reducing the ability of Nipped-B to facilitate activation (Rollins, 2004).

    The possibility that the negative effect of SA/Scc3 on cut expression may be specific to gypsy insertion alleles cannot be ruled out, it is thought improbable. The negative effect is likely to be related to the positive effect of Nipped-B, and Nipped-B facilitates the expression of wild-type cut. If SA/Scc3 does specifically affect gypsy insertion alleles, however, it may interact with the gypsy insulator and contribute to enhancer blocking. This is consistent with evidence that Cohesin functions at chromosomal boundaries in yeast. Certain Smc1 and Smc3 mutations reduce the ability of a boundary that flanks the HMR silent mating-type locus to block the spread of gene-silencing Sir protein complexes, and Scc1 associates with this boundary. It has also been proposed that Cohesin binding sites are boundaries that control the extent of chromosome loop formation by Condensin. This proposal is based in part on the observation that Cohesin is needed to reestablish chromosome condensation upon returning temperature-sensitive Condensin mutants to the permissive temperature. In Drosophila, the gypsy insulator partially blocks the negative effects of heterochromatin on the expression of a euchromatic gene, suggesting that it has boundary activity, and in yeast, the Su(Hw) protein that binds the gypsy insulator also blocks the spread of gene-silencing complexes. If SA/Scc3 or Cohesin increases insulation by gypsy, Nipped-B could facilitate activation by reducing their association with the insulator (Rollins, 2004).

    A more general version of the 'Cohesin insulator' model is preferred, in which native Cohesin binding sites in the 85-kb region separating the wing margin enhancer from the cut promoter act as insulators and impede the formation of structures needed to bring the wing margin enhancer close to the promoter. In yeast, Cohesin binds every 10 kb or so along the chromosomes. The spacing of Cohesin in Drosophila has not been investigated, but multiple complexes could bind in the 85-kb interval between the wing margin enhancer and the cut promoter. Assuming that Nipped-B, perhaps by opening the Cohesin ring, facilitates both the loading and the removal of Cohesin from chromosomes, could explain how Nipped-B facilitates the activation of wild-type cut. By opening the Cohesin ring, Nipped-B would help achieve equilibrium between the bound and unbound states by providing opportunities to load or remove Cohesin from chromosomes. This mechanism would be distinct from proteolytic removal of Cohesin by separase at the metaphase-to-anaphase transition but could be involved in the removal of Cohesin from chromosome arms in prophase. In heterozygous Nipped-B mutants, which retain substantial Nipped-B activity, the equilibrium endpoint would not be altered, but it might take longer to achieve equilibrium. Thus, reduced Cohesin binding to chromosomes would not be expected, but the lower Nipped-B levels would reduce the windows of opportunity for removal of Cohesin needed to allow long-range activation. This model also predicts that Nipped-B does not have to stably associate with chromosomes, which could explain why no chromosomally bound Nipped-B was detected by immunostaining (Rollins, 2004).

    Finally, in a simple indirect model it could be supposed that, similar to its role in loading Cohesin, Nipped-B could also facilitate chromosomal binding of another protein complex that assists long-range enhancer-promoter interactions. In this case, there would be competition between Cohesin and the long-range activation complex for Nipped-B, and reduction of Cohesin would make Nipped-B more available to facilitate long-range activation. This and the insulator model described above are not mutually exclusive, but both explain how Nipped-B cooperates with Cohesin in sister chromatid cohesion but opposes the effect of Cohesin proteins on cut expression (Rollins, 2004).

    Depletion of Drad21/Scc1 in Drosophila cells leads to instability of the cohesin complex and disruption of mitotic progression

    The coordination of cell cycle events is necessary to ensure the proper duplication and dissemination of the genome. The consequences of depleting Rad21 and SA (also known as Scc3; Losada, 1998), two non-SMC subunits of the cohesin complex, by dsRNA-mediated interference in Drosophila cultured cells, were examined. A bona fide cohesin complex exists in Drosophila embryos. Strikingly, the Drad21/Scc1 and SA/Scc3 (Stromalin) non-SMC subunits associate more intimately with one another than they do with the SMCs. Defects were observed in mitotic progression in cells from which Drad21 has been depleted: cells delay in prometaphase with normally condensed, but prematurely separated, sister chromatids and with abnormal spindle morphology. Much milder defects are observed when SA is depleted from cells. The dynamics of the chromosome passenger protein, INCENP, are affected after Drad21 depletion. The surprising observation was made that SA is unstable in the absence of Drad21; however, the converse is not true. Interference with Drad21 in living Drosophila embryos also has deleterious effects on mitotic progression. It is concluded that Drad21, as a member of a cohesin complex, is required in Drosophila cultured cells and embryos for proper mitotic progression. The protein is required in cultured cells for chromosome cohesion, spindle morphology, dynamics of a chromosome passenger protein, and stability of the cohesin complex, but apparently not for normal chromosome condensation. The observation of SA instability in the absence of Drad21 implies that the expression of cohesin subunits and assembly of the cohesin complex are be tightly regulated (Vass, 2003; Online text).

    This study demonstrates that Drad21 is in a cohesin complex with SMC1, SMC3, and SA in Drosophila embryos. Drad21 depletion results in SA instability; intriguingly, however, the converse is not true. This result suggests that SA must interact with Drad21 in order to be stable (perhaps SA is synthesized only after Drad21 accumulates in the cell). This may help to ensure a 1:1 ratio between these subunits (as observed in cohesin complexes in S. cerevisiae; Haering, 2002). Upon Drad21 depletion, dramatic effects are seen on mitotic progression; cells are delayed in prometaphase with prematurely separated sister chromatids and abnormal spindle morphology. In contrast, no premature separation of sister chromatids was seen or significant effects on the cell cycle when SA is depleted, suggesting that the Drad21 phenotype is likely specific to the interference with Drad21 only (Vass, 2003).

    Chromosome condensation in either the Drad21- or the SA-depleted cells appeared normal, as judged by overall size and shape of chromosomes, localization of the Barren non-SMC condensin subunit, and the centromeric domain occupied by the CID centromeric protein. Since chromosomes also exhibited normal condensation in Scc1-knockout DT40 cells, human cells expressing a dominant-negative N-terminal truncation of Scc1, and after immunodepletion of cohesin from Xenopus egg extracts, it appears likely that cohesins act independently of condensation machinery in metazoan chromosome structure (Vass, 2003).

    The dispersed single chromatids observed in Drad21-depleted Drosophila cells were in contrast to the separate, albeit proximal, chromatids after the knockout of Scc1 from DT40 chicken cells. Perhaps for that reason, INCENP appeared along chromosome arms in metaphase Scc1-knockout DT40 cells (INCENP distribution in later mitotic stages was not reported). In contrast, in early mitotic S2 cells depleted of Drad21, INCENP appeared diffusely localized, possibly because chromatids were no longer close to one another. Later, mitotic cells with aberrant INCENP localization fell into three groups: cells that displayed INCENP staining on single chromatids, cells that showed INCENP transferring onto the spindle even though chromatids had failed to congress to a metaphase plate, and cells in which INCENP associated with microtubules, but was not restricted to the central spindle. Drad21-depleted cells that progressed into the final mitotic stages indicated that INCENP could, however, still localize to the central region of the cell, even though chromosome segregation had not occurred. These results suggest that the correct localization of INCENP to the centromeric domain and its subsequent translocation to the central spindle at the metaphase-to-anaphase transition is dependent on the presence of cohesion between sister centromeres. However, even in the absence of chromatid cohesion, INCENP was able to achieve microtubule localization, albeit in an abnormal temporal and spatial manner (Vass, 2003).

    It is unlikely that the separate chromatids observed after Drad21 depletion would form bipolar spindle attachments and align at a metaphase plate. The metaphase checkpoint should be activated, resulting in prometaphase delay. A potential role for sister chromatid cohesion and kinetochore attachment in the metaphase checkpoint has been suggested, with the correct alignment of all sister kinetochores clearly required to establish bipolarity and loss of Mad2 metaphase checkpoint signaling. Why cells are delayed and not arrested by the metaphase checkpoint may be a reflection of compromised checkpoints in Drosophila cultured cells (derived from embryos), since these cells are extremely difficult to synchronize in response to numerous cell cycle inhibitors (Vass, 2003).

    Molecular cloning and expression of stromalin protein from Drosophila melanogaster: homologous to mammalian stromalin family of nuclear proteins

    This study reports the cloning of a new cDNA from Drosophila melanogaster that encodes an open reading frame of 1116 amino acid residues. It is the insect homolog of the previously reported stromalin (SA) family of nuclear proteins in mammals. Taking into account the identical domain present in all the SA family members characterized to date, polymerase chain reaction (PCR) was carried out using degenerate oligonucleotides from the 5' and 3' ends of one of those regions of the molecule and cDNA from D. melanogaster embryos. The homologous domain was isolated of the putative Drosophila SA molecule (DSA). This cDNA fragment was used as a radiolabeled probe for screening a cDNA library from Drosophila embryos, and a full-length cDNA for the SA homolog was cloned from an insect. The protein shows a good degree of identity with the mammalian stromalins SA-1 and SA-2, with the N and C ends being the most divergent regions of the molecule. The mRNA coding for this protein shows a molecular size of about 3.7 kb by Northern blot analysis and is essentially expressed in embryonic stage. The in situ hybridization experiments indicate that the DSA messenger is expressed mainly in neurogenic territories in the embryonic development of Drosophila. The DSA protein has been cloned and expressed in a baculovirus system, and polyclonal antibodies were generated against the recombinant molecule. Western blot analysis using these antibodies detected a main band corresponding to about 120 kDa, principally in embryos (Valdeolmillos, 1998).

    Functions of Stromalin orthologs in other species

    Cohesin SA2 is a sequence-independent DNA-binding protein that recognizes DNA replication and repair intermediates

    Proper chromosome alignment and segregation during mitosis depend on cohesion between sister chromatids, mediated by the cohesin protein complex, which also plays crucial roles in diverse genome maintenance pathways. Current models attribute DNA binding by cohesin to entrapment of dsDNA by the cohesin ring subunits (SMC1, SMC3, and RAD21 in humans). However, the biophysical properties and activities of the fourth core cohesin subunit SA2 (STAG2) are largely unknown. Using single-molecule atomic force and fluorescence microscopy imaging as well as fluorescence anisotropy measurements, this study has established that SA2 binds to both dsDNA and ssDNA, albeit with a higher binding affinity for ssDNA. SA2 can switch between the 1D diffusing (search) mode on dsDNA and stable binding (recognition) mode at ssDNA gaps. Although SA2 does not specifically bind to centromeric or telomeric sequences, it does recognize DNA structures often associated with DNA replication and double-strand break repair, such as a double-stranded end, single-stranded overhang, flap, fork, and ssDNA gap. SA2 loss leads to a defect in homologous recombination-mediated DNA double-strand break repair. These results suggest that SA2 functions at intermediate DNA structures during DNA transactions in genome maintenance pathways. These findings have important implications for understanding the function of cohesin in these pathways (Countryman, 2018).

    Distinct roles of cohesin-SA1 and cohesin-SA2 in 3D chromosome organization

    Two variant cohesin complexes containing SMC1, SMC3, RAD21 and either SA1 (also known as STAG1) or SA2 (also known as STAG2) are present in all cell types. This paper reports their genomic distribution and specific contributions to genome organization in human cells. Although both variants are found at CCCTC-binding factor (CTCF) sites, a distinct population of the SA2-containing cohesin complexes (hereafter referred to as cohesin-SA2) localize to enhancers lacking CTCF, are linked to tissue-specific transcription and cannot be replaced by the SA1-containing cohesin complex (cohesin-SA1) when SA2 is absent, a condition that has been observed in several tumors. Downregulation of each of these variants has different consequences for gene expression and genome architecture. The results suggest that cohesin-SA1 preferentially contributes to the stabilization of topologically associating domain boundaries together with CTCF, whereas cohesin-SA2 promotes cell-type-specific contacts between enhancers and promoters independently of CTCF. Loss of cohesin-SA2 rewires local chromatin contacts and alters gene expression. These findings provide insights into how cohesin mediates chromosome folding and establish a novel framework to address the consequences of mutations in cohesin genes in cancer (Kojic, 2018).

    A conserved domain in the Scc3 subunit of cohesin mediates the interaction with both mcd1 and the cohesin loader complex

    The Structural Maintenance of Chromosome (SMC) complex, termed cohesin, is essential for sister chromatid cohesion. Cohesin is also important for chromosome condensation, DNA repair, and gene expression. Cohesin is comprised of Scc3, Mcd1, Smc1, and Smc3. Scc3 also binds Pds5 and Wpl1, cohesin-associated proteins that regulate cohesin function, and to the Scc2/4 cohesin loader. SCC3 was mutagenized to elucidate its role in cohesin function. A 5 amino acid insertion after Scc3 residue I358, or a missense mutation of residue D373 in the adjacent stromalin conservative domain (SCD) induce inviability and defects in both cohesion and cohesin binding to chromosomes. The I358 and D373 mutants abrogate Scc3 binding to Mcd1. These results define an Scc3 region extending from I358 through the SCD required for binding Mcd1, cohesin localization to chromosomes and cohesion. Scc3 binding to the cohesin loader, Pds5 and Wpl1 are unaffected in I358 mutant and the loader still binds the cohesin core trimer (Mcd1, Smc1 and Smc3). Thus, Scc3 plays a critical role in cohesin binding to chromosomes and cohesion at a step distinct from loader binding to the cohesin trimer. Residues Y371 and K372 within the SCD are critical for viability and chromosome condensation but dispensable for cohesion. However, scc3 Y371A and scc3 K372A bind normally to Mcd1. These alleles also provide evidence that Scc3 has distinct mechanisms of cohesin loading to different loci. The cohesion-competence, condensation-incompetence of Y371 and K372 mutants suggests that cohesin has at least one activity required specifically for condensation (Orgil, 2015).


    Search PubMed for articles about Drosophila Stromalin

    Arya, R., Gyonjyan, S., Harding, K., Sarkissian, T., Li, Y., Zhou, L. and White, K. (2019). A Cut/cohesin axis alters the chromatin landscape to facilitate neuroblast death. Development. PubMed ID: 30952666

    Countryman, P., Fan, Y., Gorthi, A., Pan, H., Strickland, J., Kaur, P., Wang, X., Lin, J., Lei, X., White, C., You, C., Wirth, N., Tessmer, I., Piehler, J., Riehn, R., Bishop, A. J. R., Tao, Y. J. and Wang, H. (2018). Cohesin SA2 is a sequence-independent DNA-binding protein that recognizes DNA replication and repair intermediates. J Biol Chem 293(3): 1054-1069. PubMed ID: 29175904

    Gyuricza, M. R., Manheimer, K. B., Apte, V., Krishnan, B., Joyce, E. F., McKee, B. D. and McKim, K. S. (2016). Dynamic and stable cohesins regulate synaptonemal complex assembly and chromosome segregation. Curr Biol 26(13):1688-1698. PubMed ID: 27291057

    Kojic, A., Cuadrado, A., De Koninck, M., Gimenez-Llorente, D., Rodriguez-Corsino, M., Gomez-Lopez, G., Le Dily, F., Marti-Renom, M. A. and Losada, A. (2018). Distinct roles of cohesin-SA1 and cohesin-SA2 in 3D chromosome organization. Nat Struct Mol Biol 25(6): 496-504. PubMed ID: 29867216

    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): 947-965. PubMed ID: 25194162

    Moshkin, Y. M., Doyen, C. M., Kan, T. W., Chalkley, G. E., Sap, K., Bezstarosti, K., Demmers, J. A., Ozgur, Z., van Ijcken, W. F. and Verrijzer, C. P. (2013). Histone chaperone NAP1 mediates sister chromatid resolution by counteracting protein phosphatase 2A. PLoS Genet 9: e1003719. PubMed ID: 24086141

    Orgil, O., Matityahu, A., Eng, T., Guacci, V., Koshland, D. and Onn, I. (2015). A conserved domain in the scc3 subunit of cohesin mediates the interaction with both mcd1 and the cohesin loader complex. PLoS Genet 11(3): e1005036. PubMed ID: 25748820

    Pauli, A., Althoff, F., Oliveira, R. A., Heidmann, S., Schuldiner, O., Lehner, C. F., Dickson, B. J. and Nasmyth, K. (2008). Cell-type-specific TEV protease cleavage reveals cohesin functions in Drosophila neurons. Dev Cell 14(2): 239-251. PubMed ID: 18267092

    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

    Rollins, R. A., Korom, M., Aulner, N., Martens, A. and Dorsett, D. (2004). Drosophila Nipped-B protein supports sister chromatid cohesion and opposes the Stromalin/Scc3 cohesion factor to facilitate long-range activation of the cut gene. Mol. Cell. Biol. 24: 3100-3111. PubMed ID: 15060134

    Schuldiner, O., Berdnik, D., Levy, J. M., Wu, J. S., Luginbuhl, D., Gontang, A. C. and Luo, L. (2008). piggyBac-based mosaic screen identifies a postmitotic function for cohesin in regulating developmental axon pruning. Dev Cell 14(2): 227-238. PubMed ID: 18267091

    Valdeolmillos, A., Villares, R., Buesa, J. M., Gonzalez-Crespo, S., Martinez, C. and Barbero, J. L. (1998). Molecular cloning and expression of stromalin protein from Drosophila melanogaster: homologous to mammalian stromalin family of nuclear proteins. DNA Cell Biol 17(8): 699-706. PubMed ID: 9726252

    Vass, S. et al. (2003). Depletion of Drad21/Scc1 in Drosophila cells leads to instability of the cohesin complex and disruption of mitotic progression. Curr. Biol. 13: 208-218. PubMed ID: PubMed ID; Online text

    Walkinshaw, E., Gai, Y., Farkas, C., Richter, D., Nicholas, E., Keleman, K. and Davis, R. L. (2015). Identification of genes that promote or inhibit olfactory memory formation in Drosophila. Genetics 199(4): 1173-1182. PubMed ID: 25644700

    date revised: 12 May, 2019

    Home page: The Interactive Fly © 2011 Thomas Brody, Ph.D.