InteractiveFly: GeneBrief

verthandi: Biological Overview | References

BIOLOGICAL OVERVIEW

Every time a cell divides, it is essential that both daughters receive the complete genetic information of their mother. In eukaryotes, the tight connection between the two copies of every chromosome generated by DNA replication (the sister chromatids) ensures the attachment of their kinetochores to spindle microtubules emanating from opposite poles so that the sister chromatids will be segregated into different daughter cells. Cohesin is a highly conserved multisubunit complex that holds sister chromatids together in mitotic cells (see Proposed functions for sister chromatid cohesion proteins in Dorsett, 2007; also see A Ring for Holding Sister Chromatids Together?; also see The cohesin complex and regulatory factors). At the metaphase to anaphase transition, proteolytic cleavage of the α kleisin subunit (Rad21) by separase causes cohesin's dissociation from chromosomes and triggers sister-chromatid disjunction. To investigate cohesin's function in postmitotic cells, where it is widely expressed, fruit flies were created whose Rad21 can be cleaved by TEV protease. Cleavage causes precocious separation of sister chromatids and massive chromosome missegregation in proliferating cells, but not disaggregation of polytene chromosomes in salivary glands. Crucially, cleavage in postmitotic neurons is lethal. In mushroom-body neurons, it causes defects in axon pruning, whereas in cholinergic neurons it causes highly abnormal larval locomotion. These data demonstrate essential roles for cohesin in nondividing cells and also introduce a powerful tool by which to investigate protein function in metazoa (Pauli, 2008).

The investigation of nonmitotic functions of proteins essential for cell proliferation poses a major technical challenge: namely, how to inactivate such proteins without compromising cell proliferation. A good example is the highly conserved multisubunit complex called cohesin, which holds the products of DNA replication (sister chromatids) together and thereby ensures their segregation to opposite poles of the cells during mitosis and meiosis (reviewed in Nasmyth, 2005; Hirano, 2006). Cohesin forms a large tripartite ring composed of a pair of Structural Maintenance of Chromosome (SMC) proteins, SMC1 and SMC3, and an α kleisin protein, Scc1/Rad21, whose cleavage by separase causes cohesin's dissociation from chromosomes and triggers sister-chromatid disjunction at the metaphase to anaphase transition. Sister-chromatid cohesion requires two other non-SMC subunits, namely, Pds5 and Scc3/SA, that bind to cohesin's α kleisin subunit. The establishment of cohesion depends on the cohesin loading complex Scc2/Scc4 and on the acetyl-transferase Eco1/Ctf7 (Pauli, 2008).

The fact that cohesin forms a ring whose opening releases it from chromatin has led to the suggestion that it holds sister DNAs together by using a topological mechanism (Gruber, 2003). Importantly, this type of function could also be of value in regulating aspects of chromosome organization that are independent of sister-chromatid cohesion and are not directly required for chromosome segregation. It is notable in this regard that the majority of cohesin is removed from chromosome arms during prophase/prometaphase in most eukaryotic cells by a separase-independent mechanism (Gandhi, 2006; Kueng, 2006). Only cohesin that subsequently persists on chromosomes is cleaved by separase at the onset of anaphase (Waizenegger, 2000). As a consequence, there exists a large pool of cohesin ready to reassociate with chromosomes as soon as cells exit from mitosis during telophase. Cohesin is therefore tightly associated with chromosomes for much of the cell-division cycle and could have important functions on unreplicated genomes (Pauli, 2008).

Much evidence has emerged recently that cohesin might have important roles in regulating gene expression (reviewed in Dorsett, 2007). Approximately half of the cases of a multisystem developmental disorder in humans called Cornelia de Lange syndrome (CdLS), which is characterized by mental retardation, upper limb abnormalities, growth delay, and facial dysmorphisms, are caused by mutations in genes encoding NIPBL/Delangin (the human Scc2 ortholog), SMC1A, or SMC3 (Deardorff, 2007; Krantz, 2004; Musio, 2006; Tonkin, 2004). Because even severe cases of CdLS appear not to be accompanied by defects in sister-chromatid cohesion, it has been suggested that CdLS is caused by misregulated gene expression during embryonic development. Consistent with this possibility, the Drosophila Scc2 ortholog, Nipped-B, facilitates long-range enhancer-promotor interactions, at least for certain genes whose regulatory sequences have been mutated (Dorsett, 2005; Rollins, 1999). Furthermore, mutations in mau-2, the Caenorhabditis elegans Scc4 ortholog, cause defects in axon guidance (Bernard, 2006; Takagi, 1997). Recently, two cohesin subunits, Scc1/Rad21 and SMC3 (Horsfield, 2007), have been implicated in expression of the hematopoietic transcription factors runx1 and runx3 in zebrafish (Pauli, 2008).

Despite these findings, it cannot be excluded that developmental 'cohesinopathies' are in fact caused by 'knock on' effects of compromising the establishment or maintenance of sister-chromatid cohesion. In the case of CdLS, for example, haploinsufficiency of NIPBL/Delangin might cause cell-type-specific sister-chromatid cohesion defects (Kaur, 2005) that would be overlooked by examining this process in only one type of cell. It is therefore vital to develop methods that permit observation of the effects on gene expression and development of eliminating cohesin's function completely without interfering with cell proliferation (Pauli, 2008).

To analyze cohesin's function in a more sophisticated manner than hitherto possible in metazoa, the tobacco etch mosaic virus (TEV) protease was used to cleave cohesin's α kleisin subunit in Drosophila in a cell-type-specific and/or temporally controlled manner. This process opens the cohesin ring and presumably abolishes its topological embrace of chromatin fibers (Gruber, 2003). As expected, expression of TEV protease in proliferating cells of fly embryos whose sole form of Rad21 contains TEV-cleavage sites causes precocious separation of sister chromatids and has a devastating effect on chromosome segregation. More remarkably, TEV-induced Rad21 cleavage in postmitotic neurons is lethal. It causes defects in the developmental axon pruning of mushroom-body γ neurons within pupal brains and defects in cholinergic neurons that result in highly abnormal larval locomotion (Pauli, 2008).

To inactivate cohesin, cleavage of its α kleisin subunit (Rad21) was performed. Although this does not directly affect any known functional domain of Rad21, it severs and thereby opens cohesin's tripartite ring (see Schematic of the cohesin complex containing TEV-cleavable Rad21), leading to its rapid dissociation from chromosomes. To do this in Drosophila, it was necessary (1) to create a Rad21 mutant strain, (2) to complement the Rad21 mutation with a version of Rad21 that contains cleavage sites for a site-specific protease, and (3) to express a version of the protease that can accumulate within nuclei in a tissue-specific and/or time-dependent manner. TEV protease because it has been used successfully for this purpose in the budding yeast Saccharomyces cerevisiae (Pauli, 2008).

The Rad21 gene (CG17436) is located within the centric heterochromatin of chromosome 3L (Markov, 2003), but no mutants were available. To create Rad21 mutations, a P element inserted 4 kb upstream of the transcriptional start of Rad21 was remobilized by P element Transposase. Among the homozygous lethal stocks, four independent Rad21 deletion alleles were identified by using PCR (Rad21^ex3, Rad21^ex8, Rad21^ex15, Rad21^ex16). All four alleles lack exons 1 and 2, which encode the highly conserved N terminus of Rad21 that interacts with the ATPase head of SMC3 (Pauli, 2008).

Although it was known that TEV protease can inactivate protein function in budding yeast, it was unclear whether TEV could be used in a complex metazoan organism. This work shows that TEV can be expressed in a wide variety of Drosophila tissues without causing overt toxicity. More important, TEV expression induces quantitative cleavage of TEV-site-containing, but not wild-type, Rad21 protein; this is accompanied by penetrant phenotypes both in proliferating tissues and, more unexpectedly, in cells not engaged in mitosis, such as neurons and salivary gland cells (Pauli, 2008).

The system has many attractive features that should make it a powerful and versatile tool for studying protein function in vivo. (1) The method causes protein inactivation within a few hours and does not rely on a gradual depletion of the protein, as occurs in methods that interfere with the protein's synthetic capacity, such as recombinase-mediated gene deletion or RNA interference. (2) The system is reversible. By using Gal80ts, TEV protease can be turned both on and off. (3) It is possible to be certain that phenotypes are caused by cleavage of the target protein by comparing the effect of TEV expression in animals whose target protein either does or does not contain TEV sites. (4) By targeting the protease to particular locations inside or even (by using a secreted protease) outside cells, it should be possible to direct inactivation of the target protein to specific intra- or extracellular compartments. The restriction of protein inactivation to specific cellular compartments may be easier to devise by using TEV than degron systems relying on the much more complex process of ubiquitin-mediated proteolysis. Unlike the MARCM system, which uses FLP/FRT-induced mitotic recombination to generate homozygous mutant clones in proliferating tissues, TEV cleavage can be triggered in all cells of a given tissue and at any stage of development, features that will greatly facilitate phenotypic and biochemical analyses. Because many eukaryotic proteins contain multiple functional domains connected by unstructured polypeptide chains, protein inactivation through TEV cleavage should be applicable to a large variety of proteins. It could also be used to clip off protein domains and thereby alter protein activity (Pauli, 2008).

The first priority upon developing a system to cleave Rad21 was to use it to investigate the role of cohesin during mitosis. In yeast, cohesin has a vital role in holding sister chromatids together until all chromosomes have bioriented during mitosis, whereupon cleavage of Scc1/Rad21 by separase triggers sister-chromatid disjunction (reviewed in Nasmyth, 2005). The consequences of depleting Scc1/Rad21 from tissue-culture cells by using RNA interference are, on the whole, consistent with the above-mentioned notion (Coelho, 2003; Vass, 2003). However, results from depletion experiments have not been able to directly explain the effects of inactivating cohesin within a single cell cycle (Pxauli, 2008).

A situation was engineered in which efficient cleavage of Rad21 occurred precisely as embryonic cells embarked on cycle 14, causing a devastating effect on mitosis. Chromosomes enter mitosis with paired sister kinetochores; however, instead of stably biorienting on a metaphase plate, they disjoin precociously, usually segregating to opposite poles. Importantly, these highly abnormal movements all take place prior to the APC/C-dependent activation of separase. These observations imply that cohesin is essential for the sister-chromatid cohesion necessary to resist mitotic-spindle forces in metazoan organisms as well as in yeast (Pauli, 2008).

The finding that most sister chromatids (in cells with cleaved Rad21) disjoin to opposite spindle poles, albeit precociously, suggests that their chromosomes possess sufficient cohesion to establish a transient form of biorientation, though possibly with low accuracy. At this stage it is not possible to determine whether this cohesion is mediated by cohesin complexes that have survived Rad21 TEV cleavage or by an independent cohesive mechanism such as residual sister DNA catenation. It can nevertheless be concluded that the latter, if it exists, is incapable of resisting spindle forces and cannot therefore maintain sister-chromatid cohesion during a period in which the spindle-assembly checkpoint (SAC) has been activated and errors in chromosome biorientation are corrected. Thus, what really distinguishes cohesion mediated by cohesin from DNA catenation is its ability to be regulated by the SAC, and this may be the reason why eukaryotic cells appear to use cohesin for mitosis (Pauli, 2008).

Mutations in Scc2's human ortholog as well as in SMC1 and SMC3 cause the developmental defects associated with CdLS (reviewed in Dorsett, 2007). It is unclear whether these defects are caused by mitotic errors during development or by defects in nonmitotic cohesin functions. The first clue that cohesin might indeed play key roles during development other than holding sister chromatids together was the finding that mutations in Drosophila Nipped-B (Rollins, 1999), the ortholog of Scc2, alters the expression of genes whose regulatory sequences have been mutated (Pauli, 2008).

If cohesin has nonmitotic functions during development, then these could occur in proliferating and nonproliferating (postmitotic) cells. To analyze cycling cells, it would be necessary to restrict analysis either to a short, specific cell-cycle stage (e.g., the G1 period) or to develop a means of differentially inactivating cohesin complexes engaged in nonmitotic functions, leaving intact those engaged in chromosome segregation. Analysis of postmitotic cells is easier. It is merely necessary to devise a protocol for inactivating cohesin only after cell proliferation has ceased (Pauli, 2008).

Cleavage of Rad21 induced by postmitotic pan-neuronal drivers causes lethality, suggesting that cohesin has key functions in neurons. To investigate these in greater detail, the effects of Rad21 cleavage in specific neuronal subtypes were analyzed. The finding that the proliferative defects caused by a SMC1 mutation in clones of mushroom-body neuroblasts are accompanied by defective pruning of axons (Schuldiner, 2008) led to an investigation of the effects of Rad21 cleavage in γ neurons. The results show that Rad21 cleavage abolishes the developmentally controlled pruning of both axons and dendrites in γ neurons. These defects cannot have been caused by failures in cell division because cleavage had no effect on the birth of γ neurons or on their initial axonal projections (Pauli, 2008).

Previous work on mau-2 (the C. elegans Scc4 ortholog) has already provided a link between cohesin and axon development (Benard, 2004). Whereas Mau-2 was reported to act as a guidance factor required for correct axon and cell migration, investigation of γ neurons in Drosophila suggests that cohesin mediates the elimination of axon projections and dendrites. However, the results do not rule out a function for cohesin in regulating axon guidance because Rad21 cleavage might not be complete when γ-neuron axons start growing out in the first place. Indeed, axon-projection defects were detected in developmentally arrested late pupae (Pauli, 2008).

It has not thus far been possible to show that γ-neuron pruning defects cause changes in animal behavior. Cleavage of cohesin in the entire population of cholinergic neurons, in contrast, has a dramatic effect, causing larvae to turn frequently, move their heads back and forth, and even crawl backward. Importantly, the neurons clearly survive without functional cohesin and must be at least partially active, because larvae are not paralyzed by cohesin cleavage, a phenotype seen when cholinergic transmission is switched off. The locomotion defects are not dissimilar to those caused by mutations in scribbler (sbb). sbb, also known as brakeless (bks) and master of thickveins (mtv), codes for a ubiquitously expressed corepressor of transcription (Haecker, 2007; references therein). Expression of a sbb transcript exclusively in cholinergic neurons is sufficient to rescue locomotion defects of sbb mutants (Suster, 2004). It therefore appears that the lack of sbb and cohesin in cholinergic neurons causes similar locomotion defects. Future work will have to show whether there is a link between sbb and cohesin. The finding that cohesin has roles in neurons that are essential for normal behavior is consistent with the notion that the mental retardation invariably found in patients with CdLS is also due to defective neuronal function, as opposed to defective cell proliferation during development (Pauli, 2008).

This study has shown that suppression of 201Y-Gal4-induced TEV expression, specifically in muscles, bypasses the early pupal arrest in Rad21^TEV-rescued flies and indicates that cohesin is essential in muscles as well as in neurons. In addition, although cohesin does not seem to be required for the maintenance of polytene-chromosome morphology, it is essential for normal progression through the endocycle in salivary glands. It is therefore conceivable that cohesin has key functions in most postmitotic cell types. What might these functions be? Cohesin is known to be required for efficient double-strand break repair as well as sister-chromatid cohesion (reviewed in Nasmyth, 2005), and it promotes repair by facilitating homologous recombination between sister chromatids. Its action in postmitotic neurons, however, must be on unreplicated chromatids. It is suggested therefore that cohesin's function in neurons and other postmitotic G0 cells is more likely to be in regulating gene expression. The finding that cohesin cleavage reduces the accumulation of EcR-B1 within γ neurons is consistent with this notion. Interestingly, recent data have shown that cohesin binds to the EcR gene in several fly cell lines (Misulovin, 2008). Future experiments should address whether cohesin acts as a general regulator of gene expression (Pauli, 2008).

In summary, definitive evidence is provided that the cohesin ring has essential functions in cells with unreplicated chromosomes. It will be important in the future to establish whether cohesin functions by trapping chromatin fibers, as it appears to do in cells that have replicated their genomes (Pauli, 2008).

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 (Kennison, 1988; Schulze, 2001). The vtd locus also affects hh expression; vtd mutations are dominant suppressors of Moonrat (Mrt), a dominant gain of function allele of hh (Schulze, 2001; Felsenfeld, 1995). 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 (Misulovin, 2008). 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 (Horsfield, 2007). Stronger evidence supporting the idea that cohesin directly stimulates transcription arises from a recent study on axon pruning in the Drosophila mushroom body (Schuldiner, 2008). 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, Dorsett (2005) has 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 (Dorsett, 2005). 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).

A direct role for cohesin in gene regulation and ecdysone response in Drosophila salivary glands

Developmental abnormalities observed in Cornelia de Lange syndrome have been genetically linked to mutations in the cohesin machinery. These and other recent experimental findings have led to the suggestion that cohesin, in addition to its canonical function of mediating sister chromatid cohesion, might also be involved in regulating gene expression. This study report that cleavage of cohesin's kleisin subunit in postmitotic Drosophila salivary glands induces major changes in the transcript levels of many genes. Kinetic analyses of changes in transcript levels upon cohesin cleavage reveal that a subset of genes responds to cohesin cleavage within a few hours. In addition, cohesin binds to most of these loci, suggesting that cohesin is directly regulating their expression. Among these genes are several that are regulated by the steroid hormone ecdysone. Cytological visualization of transcription at selected ecdysone-responsive genes reveals that puffing at Eip74EF ceases within an hour or two of cohesin cleavage, long before any decline in ecdysone receptor could be detected at this locus. It is concluded that cohesin regulates expression of a distinct set of genes, including those mediating the ecdysone response (Pauli, 2010).

The regulation of gene expression essential for normal animal development is largely mediated by sequence-specific transcription factors. One of the more mysterious aspects of developmentally regulated transcription concerns how transcription factors bound to remote regulatory sequences modulate transcription of genes many kilobases away while having no effect on neighboring genes. These distant factors must either slide long distances along chromatin fibers or else interact directly with those factors bound close to the start of transcription, with intervening chromatin forming a loop. Because of their proposed roles in chromatin looping, it is suspected that factors that regulate chromatin topology might have key roles in modulating transcription. One such factor is cohesin, a multisubunit complex essential for sister chromatid cohesion and necessary for mitotic chromosome segregation. Cohesin's Smc1, Smc3, and Rad21/Scc1 subunits form a three-membered ring, within which sister chromatin fibers are entrapped in a process that requires a separate cohesin loading factor composed of the Scc2 and Scc4 proteins. By entrapping unreplicated DNAs, cohesin could, in principle, hold distant sequences of the same chromatid together (in cis) using the same topological principle by which sister DNAs are held together in trans (Pauli, 2010).

Cohesin clearly functions in processes besides sister chromatid cohesion because it is associated with chromatin in most, if not all, quiescent cells and is essential for the pruning of postmitotic neurons, at least partly by regulating levels of ecdysone receptor. Whether or not cohesin regulates transcription has hitherto been investigated mainly by analyzing the effects of its depletion using RNA interference (RNAi). Depletion of its Rad21/Scc1 subunit causes 2-fold changes in expression of the H19 and IGF2 genes in HeLa cells and little or no effect on inducibility of the gene encoding Interferon-γ in T cells, despite destroying a putative loop between its enhancer and promoter sequences. In Drosophila BG3 tissue culture cells, up to 10- to 50-fold changes in the level of transcripts from the enhancer of split and invected-engrailed loci were detected 6 days after RNAi treatment. Intriguingly, substantial changes in mRNA levels for these transcripts were only observed 3 days following RNAi treatment. Though insightful, these experiments have a number of limitations. The effects on transcription are either modest or they are only seen long after cohesin depletion and might therefore be secondary effects due to chromosome missegregation, defective DNA repair, or some other hitherto-uncharacterized state of stress induced by a loss of cohesin activity (Pauli, 2010).

Another line of evidence hinting at a role for cohesin in transcriptional control is the finding that inactivation of one allele of Nipped-B, the Drosophila ortholog of Scc2, alters long-range enhancer-promoter interactions at the homeotic loci cut and Ultrabithorax (Ubx), at least when compromised by a gypsy retrotransposon. Moreover, mutating Rad21 in zebrafish reduces expression of the hematopoietic transcription factors RUNX1 and RUNX3 during development, whereas mutations in mau-2, the Caenorhabditis elegans Scc4 ortholog, cause defects in axon guidance. Particularly striking is the finding that Cornelia de Lange syndrome (CdLS), a multisystem developmental disorder, is caused (in more than 50% of cases) by haplodeficiency of NIPBL/Delangin, the human Scc2/Nipped-B ortholog. Because tissue culture cells derived from CdLS patients have apparently normal sister chromatid cohesion, dysregulated gene expression during embryonic development has been suggested as a potential cause. There are indeed minor changes in the expression of certain genes in NIPBL± mice (up to 2.5-fold) and CdLS patient-derived cell lines (up to 4-fold), but these so far do little to explain the developmental defects associated with CdLS, which could, in principle, be due to defective DNA repair at crucial stages of development (Pauli, 2010).

Ideally, an investigation of cohesin's role in transcription should aim to observe the immediate consequences of the complex's inactivation in cells that are neither undergoing mitosis nor replicating their DNA. Sister chromatid cohesion is normally destroyed at the onset of anaphase by separase-mediated cleavage of cohesin's Rad21/Scc1 α-kleisin subunit, which destroys its topological entrapment of chromatin fibers by opening the cohesin ring (Uhlmann, 2000; Gruber, 2003). This process can be reproduced in an inducible manner using tobacco etch virus protease (TEV) in strains of Drosophila melanogaster whose α-kleisin Rad21 contains TEV cleavage sites. This study describes the effect on gene expression of TEV-induced Rad21 cleavage in a nonproliferating tissue, which constitutes conclusive evidence that cohesin has a direct role in regulating transcription (Pauli, 2010).

Development of a method to cleave Rad21 with TEV protease in a time- and tissue-specific manner has enabled assessment of the immediate and long-term consequences of cohesin inactivation on transcription in third-instar salivary glands from Drosophila. This postmitotic tissue was chosen to ensure that any effects of cohesin inactivation on the transcriptional apparatus could not be attributed to indirect or knock on effects of chromosome missegregation or defective DNA repair due to the absence of cohesin's canonical function, namely sister chromatid cohesion. Despite this precaution, cohesin cleavage causes, from 24 hr onward, major changes in cellular physiology, some of which most likely reflect a general stress-related response. It cannot at this stage be ascertained whether these highly pleiotropic events are triggered by changes in gene expression that precede them or by the loss of a novel currently unknown cohesin function. In either case, the observations demonstrate that it is very difficult to attribute functions to cohesin in regulating gene expression merely by observing the long-term consequences of its inactivation. Changes in gene expression that occur only 24 hr or more after cohesin's removal from chromosomes could be secondary or tertiary events triggered by fundamental changes in cell physiology (Pauli, 2010).

The current observations reveal an additional complication in interpreting gene expression changes. Several of the genes whose expression is affected by cohesin cleavage are genes regulated by the ecdysone receptor, whose abundance declines after 8 hr, presumably because of an almost immediate, cohesin cleavage-dependent decline in its mRNA. Thus, the precipitous decline in Sgs1 mRNAs that takes place between 8 and 16 hr could be caused by the lack of ecdysone receptor and not by the lack of cohesin per se. Such phenomena could explain many late responses to cohesin inactivation (Pauli, 2010).

Given these considerations, it is clear that in order to attribute a role for cohesin in regulating a gene on the basis of changes in its expression upon cohesin inactivation, it is necessary to demonstrate a change in transcription as soon as cohesin has been removed from chromosomes and, crucially, long before any major change in cell physiology or in the concentration of other transcription regulators. Two genes stand out in this regard, namely EcR encoding the ecdysone receptor and Eip74EF encoding an ecdysone-dependent transcription factor. Eip74EF is a particularly good candidate, because heavy transcription of this gene in third-instar larvae gives rise to a cytologically visible puff. Cohesin is associated with this puff, and its removal by Rad21 cleavage is accompanied by an immediate cessation of puffing. Crucially, contraction of band 74 caused by Rad21's removal takes place several hours before any decline in ecdysone receptor associated with it. It is therefore suggested that cohesin present at Eip74EF has a direct role in maintaining transcription of the gene. There is no reason to believe that the same is not also true for EcR, though this has not observed it at a cytological level. Because transcription of most genes is unaffected by cohesin cleavage, it is striking that transcription of EcR, as well as of a direct target gene, Eip74EF, appears to be directly regulated by cohesin. Ecdysone-responsive genes in general are enriched in cohesin domains and preferentially misregulated following cohesin cleavage; this suggests a common aspect of the transcription process at these loci that renders them particularly dependent on cohesin. It is conceivable that the interplay between the core set of gene regulatory mechanisms (transcription factors, enhancers, promoters, etc.) was insufficient to achieve the precise control that was required to orchestrate the dramatic ecdysone-induced changes that occur during the larval-to-pupal metamorphosis. It is also conceivable that cohesin, because of its ability to encircle chromatin strands, was particularly suited to fulfill this role, either by facilitating interactions between distant DNA elements in cis or by its ability to slide along DNA (Pauli, 2010).

Although Eip74EF may be the best example of a gene directly regulated by cohesin, it is by no means the only candidate. Reduced puffing at its twin, the adjacent Eip75B, also occurs before any obvious decline in ecdysone receptor at this locus. Although the drop in Eip75B mRNAs that occurs 8 hr after induction of Rad21 cleavage may be due to a decline in ecdysone receptor, the more modest decrease that occurs earlier may be due to a direct effect of cohesin's dissociation from the locus. There are other genes, for example comm2, whose mRNAs decline rapidly upon cohesin cleavage, and these may also be directly regulated by cohesin. Interestingly, transcripts from at least two genes, namely ush and Mst87F, rise rapidly after cohesin cleavage, suggesting that although cohesin promotes transcription at certain genes, it exerts repression at others (Pauli, 2010).

Cohesin's canonical function is to mediate sister chromatid cohesion. It is currently thought to perform this by entrapping sister DNAs inside a tripartite ring formed by its Smc1, Smc3, and Rad21/Scc1 subunits. This raises the important question of whether cohesin regulates gene expression using a similar topological principle. With this in mind, it has been repeatedly proposed that cohesin might regulate gene expression by facilitating the formation or maintenance of loops between remote regulatory elements and promoter regions. Such loops have not been visualized directly but have instead been inferred from coprecipitation of remote DNA sequences following formaldehyde fixation. According to this somewhat indirect assay, long-term cohesin depletion reduces interaction between an enhancer at the 3' end of the H19 gene with a remote CCCTC-binding factor (CTCF) binding site that controls imprinting of the IGF2-H19 locus. Loss of the putative loop between the CTCF binding site and the H19 enhancer is thought to enable the enhancer to activate the neighboring IGF2 gene. Cohesin depletion also disrupts a similar type of long-range interaction between distant (cohesin-associated) CTCF sites at the INFG locus, though in this case, cohesin depletion has little effect on inducibility of the locus by cytokine. The observation that cohesin in Drosophila, unlike its enrichment at CTCF binding sites in human cells, is associated with large domains raises the possibility that it can also regulate transcription by means other than the formation of loops between remote regulatory elements. By entrapping DNAs inside rings capable of sliding along chromatin, cohesin complexes may provide a potentially mobile platform for the stable association of other factors necessary for regulating (positively or negatively) the movement of polymerases through transcription units. Cohesin's intriguing potential to modulate chromatin, together with its binding to regions covering several transcription units, is seemingly at odds with the finding that differentially expressed genes are not clustered in the genome. Whatever the activity is that cohesin brings along, the data suggest that its absence affects only a subset of genes that are normally exposed to it. The identification of ecdysone-responsive genes as a class of cohesin-dependent genes highlights that there might exist still-unknown common determinants or gene-specific regulators that render a gene susceptible to changes in cohesin binding (Pauli, 2010).

The Drosophila MI-2 chromatin-remodeling factor regulates higher-order chromatin structure and cohesin dynamics in vivo

dMi-2 is a highly conserved ATP-dependent chromatin-remodeling factor that regulates transcription and cell fates by altering the structure or positioning of nucleosomes. dMi-2 plays an unanticipated role in the regulation of higher-order chromatin structure in Drosophila. Loss of dMi-2 function causes salivary gland polytene chromosomes to lose their characteristic banding pattern and appear more condensed than normal. Conversely, increased expression of dMi-2 triggers decondensation of polytene chromosomes accompanied by a significant increase in nuclear volume; this effect is relatively rapid and is dependent on the ATPase activity of dMi-2. Live analysis revealed that dMi-2 disrupts interactions between the aligned chromatids of salivary gland polytene chromosomes. dMi-2 and the cohesin complex are enriched at sites of active transcription; fluorescence-recovery after photobleaching (FRAP) assays showed that dMi-2 decreases stable association of cohesin with polytene chromosomes. These findings demonstrate that dMi-2 is an important regulator of both chromosome condensation and cohesin binding in interphase cells (Fasulo, 2012).

Cohesin has been the topic of intensive study due to its critical role in sister chromatid cohesion during mitosis, and its roles in gene regulation and DNA repair. The complex forms a ring-like structure that encircles chromosomes beginning in telophase, and mediates sister chromatid cohesion upon DNA replication. Cohesin binding is dynamic, but unusually stable compared to most DNA-binding proteins. Interphase cohesin is continuously loaded by the kollerin complex containing Nipped-B and released from chromosomes by the releasin complex containing Pds5 and Wap. The current studies revealed an intriguing connection between dMi-2 and the cohesin complex, and argue that dMi-2 facilitates removal of cohesin from chromosomes during interphase. This activity is not restricted to situations in which dMi-2 is expressed at unusually high levels, since a twofold reduction in dMi-2 dosage counteracts the developmental consequences of reduced dosage of Nipped-B. These findings add dMi-2 to the list of factors that regulate cohesin binding (Fasulo, 2012).

Cohesin regulates transcription by multiple mechanisms, including long-range interactions between insulators, enhancers and promoters via the formation of DNA loops, repression in collaboration with Polycomb proteins, and controlling transition of paused polymerase to elongation. The observed suppression of a dominant Nipped-B mutant phenotype by reduced dMi-2 gene dosage suggests that regulation of cohesin chromosome binding may be one mechanism by which dMi-2 controls gene expression (Fasulo, 2012).

The live analysis of a LacO array tagged with GFP in living cells is consistent with a potential role for dMi-2 in chromosome cohesion. The array is organized in a compact disc due to cohesion between precisely aligned chromatids. The over-expression of dMi-2 caused the LacO array to disperse into hundreds of discrete foci, presumably due to the disruption of interactions between sister chromatids. The over-expression of dMi-2 also disrupted the organization of mitotic chromosomes along their longitudinal axes, possibly by interfering with chromosomal interactions in cis that contribute to the organization of chromosome shape (Fasulo, 2012).

The findings of this study show that dMi-2 plays unanticipated roles in both the regulation of higher-order chromosome structure and cohesin dynamics. Is there a causal relationship between the two activities? The sudden removal of cohesin in late larval development by targeted proteolysis does not dramatically alter polytene structure and thus cohesin may not be critical for maintenance of polytene structure once fully established. However, genetic studies of pds5 have revealed a role for both cohesin binding and sister chromatid cohesion in forming the normal structure of polytene chromosomes. A pds5 null allele and an allele encoding an N-terminally truncated protein alter polytene chromosome structure in distinctive ways, but in both cases the size and normal banding pattern are disrupted. Taken together, the above considerations prevent the conclusion that dMi-2 promotes chromosome decondensation by destabilizing cohesin binding. However, because dMi-2 over-expression causes a large reduction in both the amount of stable cohesin and its chromosomal residence time, it can be concluded that cohesin binding has been reduced, and that it is also likely that cohesion is affected (Fasulo, 2012).

The internal diameter of cohesin is ~35 by 50 nm; it can therefore encircle only one 30 nm or two 10 nm chromatin fibers. Interactions between cohesin complexes are thought to contribute to chromatid cohesion and presumably anchor chromatin loops to form 'hubs' of high transcriptional activity . The destabilization of cohesin binding therefore may be a secondary consequence of changes in chromatin structure catalyzed by dMi-2. Further work will be necessary to test this possibility and clarify the causal relationship, if any, between changes in chromosome structure and cohesin binding catalyzed by dMi-2 (Fasulo, 2012).

It is intriguing that dMi-2, an antagonist of cohesin binding and well-characterized transcriptional repressor, co-localizes with cohesin at sites of active transcription. Although cohesin subunits and Nipped-B were not identified as stable subunits of dMi-2 containing complexes in cultured cells, the extensive overlap between their chromosomal distributions suggests that chromatin structure and gene activity may be dependent on a fine balance of opposing dMi-2 and cohesin activities. Cohesin selectively binds and regulates active genes that have paused RNA polymerase, and can both positively and negatively regulate these genes by multiple mechanisms, including controlling the transition of paused polymerase to elongation. It is possible that dMi-2 may also influence this transition by regulating cohesin binding and the chromatin structure at the pause sites. Intriguingly, mouse Mi-2ß and the NuRD complex bind active and poised gene promoters in thymocytes, and have both negative and positive effects on expression of these genes. The Mi-2/NuRD complex regulates the expression of genes involved in lymphocyte differentiation [and is also involved in stem cell renewal and determination. As in Drosophila, mammalian cohesin also regulates many genes critical for growth and development. These findings raise the interesting possibility that Mi-2 may regulate cellular differentiation in vertebrates by modulating chromosome condensation and cohesin activity (Fasulo, 2012).

Enhancer of Split Gene Complex: architecture and coordinate regulation by Notch, Cohesin and Polycomb group proteins

The cohesin protein complex functionally interacts with Polycomb group (PcG) silencing proteins to control expression of several key developmental genes, such as the Drosophila Enhancer of split gene complex [E(spl)-C]. The E(spl)-C contains twelve genes that inhibit neural development. In a cell line derived from central nervous system, cohesin and the PRC1 PcG protein complex bind and repress E(spl)-C transcription, but the repression mechanisms are unknown. The genes in the E(spl)-C are directly activated by the Notch receptor. This study shows that depletion of cohesin or PRC1 increases binding of the Notch intracellular fragment (NICD) to genes in the E(spl)-C, correlating with increased transcription. The increased transcription likely reflects both direct effects of cohesin and PRC1 on RNA polymerase activity at the E(spl)-C, and increased expression of Notch ligands. By chromosome conformation capture this study found that the E(spl) C is organized into a self-interactive architectural domain that is co-extensive with the region that binds cohesin and PcG complexes. The self-interactive architecture is formed independently of cohesin or PcG proteins. It is posited that the E(spl)-C architecture dictates where cohesin and PcG complexes bind and act when they are recruited by as yet unidentified factors, thereby controlling the E(spl)-C as a coordinated domain (Schaaf, 2013b).

These studies investigated the regulation of the E(spl)-C complex by cohesin, PRC1, and the Chromator/Putzig (Chro-Z4/Pzg) protein complex in CNS derived BG3 cells, in which the E(spl)-C has a rare restrained state with a cohesin-H3K27me3 overlap. The E(spl)-C has a highly self-interactive structure that is unexpectedly independent of these protein complexes and the level of gene expression. Depletion of any of these three protein complexes, however, significantly increases E(spl)-C transcription. The effects of these three protein complexes on E(spl)-C expression likely reflect changes in expression of Notch ligands, and in the cases of cohesin and PRC1, potentially direct effects on activator and Pol II activity at the E(spl)-C genes (Schaaf, 2013b).

Chromosome conformation capture (3C) analysis revealed that the E(spl)-C has a structure in which all positions within the complex interact with each other at a high frequency, but not with flanking regions. Surprisingly, this study found that this architecture is independent of cohesin, the PcG complexes, the Chro-Pzg/Z4 complex, transcription, and stage of the cell cycle. Thus it is not known which factors establish this striking architecture, which defines the E(spl)-C as a structurally independent domain. It is also not yet known which factors control recruitment of cohesin and PcG complexes to the locus. It is speculated, however, that this architecture coordinates transcriptional control of the entire E(spl)-C, based on the finding that in BG3 cells, cohesin, PRC1, and the ubiquityl-Histone H2 (H2Aub) and H3K27me3 histone modifications made by the PRC1 and PRC2 complexes are co-extensive within this architectural domain. Although no known insulators or boundary elements flank the E(spl)-C, and depletion of the CP190 protein required for activity of all known Drosophila insulators does not alter E(spl)-C expression, it is likely that the unknown factors that form this structure limit the spread of these protein complexes and histone marks. The E(spl)-C architectural domain may be evolutionarily significant, because Notch-regulated Enhancer of split complexes with similar structures are conserved in insects and crustaceans over 420 million years (Schaaf, 2013a).

Possible clues to the identities of the factors that control the E(spl)-C architecture and/or the recruitment of cohesin and PcG complexes may arise in genetic screens for factors that alter E(spl)-C sensitive phenotypes, such as the Nspl-1 rough eye and bristle phenotypes. These phenotypes are sensitive to mutations in the E(spl)-C and cohesin genes in a highly dosage-sensitive manner, and modest changes in the E(spl)-C architecture or recruitment of cohesin or PcG proteins may have similar effects (Schaaf, 2013a).

There is coordinate regulation of gene complexes by cohesin in mammalian cells. The Protocadherin beta (Pchdb) gene complex is downregulated in the embryonic fibroblasts and brains of mice heterozygous mutant for the Nipbl cohesin loading factor, and brains of mice that are homozygous mutant for the SA1 cohesin subunit, and cohesin is involved in enhancer-promoter looping in the Protocadherin alpha (Pchda) complex, helping determine which genes in the complex are active. While this is a positive role for cohesin, as opposed to the repressive role that occurs in the E(spl)-C, it is possible that the protocadherin gene clusters also have a higher order architecture that dictates how cohesin functions within the gene complex. Recent genome-wide analysis also indicates that there are constitutive higher order looping architectures that may organize cell-type specific interactions on a shorter scale, and that cohesin contributes to both types of structures (Schaaf, 2013a).

Prior studies showed that depletion of cohesin or PRC1 increases expression of the Serrate Notch ligand gene. This likely explains part of the increase in E(spl)-C transcription upon cohesin and PRC1 depletion, because the E(spl)-C genes are directly activated by Notch. Consistent with this idea, this study detected increases in NICD association with the HLHmβ and HLHm3 genes upon cohesin or PRC1 depletion. EDTA treatment confirms that increasing Notch activation increases NICD binding to the E(spl)-C genes (Schaaf, 2013a).

Because cohesin and PRC1, unlike the Chro-Pzg/Z4 complex, bind directly to the E(spl)-C, it is also possible that they also directly control association of NICD with the Su(H) protein bound upstream of the active genes. For example, they could potentially interact with NICD or the Su(H) complex, and interfere with NICD association, or somehow facilitate ubiquitination and degradation of NICD. The lack of an effect of cohesin or PRC1 depletion on NICD association with E(spl)-C genes after EDTA treatment does not rule out this possibility, because under these conditions, the amount of NICD is no longer limiting (Schaaf, 2013a).

It remains to be determined if the multiple effects of cohesin on Notch function seen in Drosophila, including regulation of Notch ligand and target genes, also occur in mammals. If so, this could underlie many of the development deficits seen in Cornelia de Lange syndrome, caused by mutations in NIPBL and cohesin subunit genes. Mutations in Notch receptor and ligand genes cause Alagille and other syndromes that affect many of the same tissues as CdLS (Schaaf, 2013a).

The possibility cannot be ruled out that cohesin and PRC1 directly repress E(spl)-C transcription independently of any effects on Notch ligand expression or NICD association with the E(spl)-C genes. This is because both bind throughout the complex, and the PRC1-generated H2Aub repressive histone mark is co-extensive with the E(spl)-C architectural domain. Importantly, all genes in BG3 cells that show rare overlap of cohesin and the PRC2-generated H3K27me3 modification, such as the invected and engrailed gene complex, show substantial increases in transcription upon cohesin or PRC1 depletion, even though they are not Notch activated. It is highly unlikely that cohesin or PRC1 depletion increases the expression of all the diverse activators that control these genes, and more likely that cohesin and PRC1 directly repress their transcription (Schaaf, 2013a).

At all genes examined that are strongly repressed by cohesin, cohesin restricts the transition of paused RNA Pol II into elongation, irrespective of whether or not they have the H3K27me3 mark (Fay, 2011). PRC1 restricts entry of paused Pol II into elongation at active genes that bind cohesin and PRC1, but lack PRC2 and the H3K27me3 modification (Schaaf, 2013b). It is thus posited that cohesin and PRC1 together restrict transition of the paused Pol II present at the active E(spl)-C genes into elongation. Because co-depletion of cohesin and PRC1 does not synergistically increase transcription, it is thought likely that they function together at the same step. Cohesin and PRC1 directly interact with each other, and cohesin facilitates binding of PRC1 to active genes that lack the H3K27me3 mark. Cohesin depletion, however, does not significantly alter PRC1 association with the E(spl)-C, likely because PRC1 binding is stabilized by the known interaction of PRC1 with H2K27me3. PRC1 is thus not sufficient to repress E(spl)-C transcription in the absence of cohesin, indicating that cohesin has roles that extend beyond its interaction with PRC1 (Schaaf, 2013a).

The cohesin subunit Rad21 is required for synaptonemal complex maintenance, but not sister chromatid cohesion, during Drosophila female meiosis

Replicated sister chromatids are held in close association from the time of their synthesis until their separation during the next mitosis. This association is mediated by the ring-shaped cohesin complex that appears to embrace the sister chromatids. Upon proteolytic cleavage of the alpha-kleisin cohesin subunit at the metaphase-to-anaphase transition by separase, sister chromatids are separated and segregated onto the daughter nuclei. The more complex segregation of chromosomes during meiosis is thought to depend on the replacement of the mitotic alpha-kleisin cohesin subunit Rad21/Scc1/Mcd1 by the meiotic paralog Rec8. In Drosophila, however, no clear Rec8 homolog has been identified so far. Therefore, this study has analyzed the role of the mitotic Drosophila alpha-kleisin Rad21 during female meiosis. Inactivation of an engineered Rad21 variant by premature, ectopic cleavage during oogenesis results not only in loss of cohesin from meiotic chromatin, but also in precocious disassembly of the synaptonemal complex (SC). The lateral SC component C(2)M can interact directly with Rad21, potentially explaining why Rad21 is required for SC maintenance. Intriguingly, the experimentally induced premature Rad21 elimination, as well as the expression of a Rad21 variant with destroyed separase consensus cleavage sites, do not interfere with chromosome segregation during meiosis, while successful mitotic divisions are completely prevented. Thus, chromatid cohesion during female meiosis does not depend on Rad21-containing cohesin (Urban, 2014 -- PubMed ID: 25101996).

Cohesin organizes chromatin loops at DNA replication factories

Genomic DNA is packed in chromatin fibers organized in higher-order structures within the interphase nucleus. One level of organization involves the formation of chromatin loops that may provide a favorable environment to processes such as DNA replication, transcription, and repair. However, little is known about the mechanistic basis of this structuration. This study demonstrates that cohesin participates in the spatial organization of DNA replication factories in human cells. Cohesin is enriched at replication origins and interacts with prereplication complex proteins. Down-regulation of cohesin slows down S-phase progression by limiting the number of active origins and increasing the length of chromatin loops that correspond with replicon units. These results give a new dimension to the role of cohesin in the architectural organization of interphase chromatin, by showing its participation in DNA replication (Guillou, 2010).

The first part of this study describes a physical interaction between cohesin and the MCM complex in human cells that is consistent with a previous report of an interaction between Smc1 and Mcm7. Whether the association of cohesin with chromatin depends on the previous formation of pre-RCs at origins has been a matter of discussion. This study shows that cohesin associates normally with chromatin after the down-regulation of ORC or MCM, arguing that cohesin loading is independent of pre-RC formation in human cells, as it happens in yeast or Drosophila cells. Therefore, the requirement of pre-RCs for cohesin loading that has been reported in Xenopus extracts could be a particularity of this system. Xenopus extracts recapitulate the early embryonic cycles, a quick succession of chromosome duplication and segregation events with no active transcription. In this context, the genomic positions where pre-RCs are assembled may constitute the only 'entry points' for cohesin. In addition, considering the results of this study, the loading of cohesin at pre-RC sites in Xenopus would ensure its physical presence around origins, where it would contribute to the dynamics of DNA replication (Guillou, 2010).

Cohesin can be detected at thousands of sites along the genome. While a complete genome-wide correlation between CBSs and replication origins cannot be established because of the lack of a comprehensive map of the latter, using a bioinformatics approach, an enrichment of cohesin at the origins located within the ENCODE representation of the genome has indeed been identified. When data from the cohesin ChIP-chip assay were compared with the genomic positions of origins mapped within ENCODE by nascent strand analyses in the same cell line, it became clear that origins are preferential sites for cohesin binding. This observation, further validated by cohesin ChIP assays, seems a conserved feature through evolution because it has also been reported in yeast, Drosophila (MacAlpine, 2009), and even Bacillus subtilis, and suggests a role for cohesin in origin activity. Actually, it was found that cohesin down-regulation slows down S-phase progression by a mechanism that is independent of sister chromatid cohesion, regulation of gene expression, and checkpoint responses. Instead, single-molecule analyses revealed that cohesin down-regulation reduced the number of active origins and increased the average interfork distance, without affecting fork speed. These results imply that the presence of cohesin at origins modulates their activity, providing a novel link between the DNA replication and cohesion machineries, which is independent from the reported effect of cohesin acetylation on fork progression (Terret, 2009; Guillou, 2010 and references therein).

The assembly of DNA replication factories in human cells entails the physical association of a cluster of origins and the formation of chromatin loops. This study has shown that cohesin down-regulation leads to a significant increase in the length of DNA loops in which chromatin is organized. This result, combined with the negative impact of cohesin loss on DNA replication, leads to a proposal that cohesin is required for the formation and/or stabilization of loops at replication foci. In this model, cohesin would mediate the long-range intrachromosomal interactions necessary to bring together a cluster of replication origins. Loop formation would occur at late mitosis and during G1, at the time of origin selection and licensing. In the resultant structures, origins would be located at the bases of the loops, where they are more prone to fire (Courbet, 2008). Upon cohesin down-regulation, replication foci would be structured in a different manner, with fewer origins, longer loops, and, therefore, larger replicon units. This alternative arrangement explains the S-phase phenotypes and the fact that cohesin down-regulation reduces the average intensity of each replication factory without reducing the total number of replication foci (Guillou, 2010).

Interestingly, down-regulation of CTCF neither delayed DNA replication nor affected halo size. The latter observation may seem surprising, but it could be explained because the 'DNA halo' technique allows the visualization of chromatin loops anchored to insoluble nuclear structures, such as those in replication factories, rather than DNA loops that are formed transiently to regulate transcription. In any case, it is possible that other proteins cooperate with cohesin to organize loops at replication factories, much as CTCF, the mediator complex, or tissue-specific transcription factors cooperate with cohesin to regulate gene expression in different contexts (Guillou, 2010).

The genomic landscape of cohesin-associated chromatin interactions

Cohesin is implicated in establishing tissue-specific DNA loops that target enhancers to promoters, and also localizes to sites bound by the insulator protein CTCF, which blocks enhancer-promoter communication. However, cohesin-associated interactions have not been characterized on a genome-wide scale. This study performed chromatin interaction analysis with paired-end tag sequencing (ChIA-PET) of the cohesin subunit SMC1A in developing mouse limb. 2264 SMC1A interactions were identified, of which 1491 (65%) involved sites co-occupied by CTCF. SMC1A participates in tissue-specific enhancer-promoter interactions and interactions that demarcate regions of correlated regulatory output. In contrast to previous studies, interactions between promoters and distal sites were identified that are maintained in multiple tissues but are poised in embryonic stem cells and resolve to tissue-specific activated or repressed chromatin states in the mouse embryo. These results reveal the diversity of cohesin-associated interactions in the genome and highlight their role in establishing the regulatory architecture of development (Demare, 2013).

Using ChIA-PET analysis of SMC1A, a direct view was obtained of cohesin-associated topology in the genome. The results suggest that cohesin interactions facilitate tissue-specific regulatory outcomes through several mechanisms. Consistent with previous studies of individual loci, it was found that cohesin is involved in tissue-specific looping between promoters and enhancers. In these cases, tissue-specific activation of gene expression is likely to depend on tissue-specific interaction events. However, cohesin is also associated with interactions between distal sites and promoters that are maintained across tissues, but show tissue-specific chromatin signatures and gene expression. Both the promoters and the distal sites in these interactions exhibit tissue-specific active or repressed chromatin states, suggesting in these cases that tissue-specific regulation is achieved by altering the activation state of a constitutive chromatin topology (Demare, 2013).

These 'stable' cohesin-associated interactions appear to provide a mechanism for establishing tissue-specific promoter activation and repression through interaction with the same distal site. The interaction observed at Wnt7a involves a distal site marked by H3K27ac in cortex and H3K27me3 in limb, suggesting it may act as an enhancer of Wnt7a expression in some tissue contexts and as a repressor in others. The Wnt7a promoter also interacts with the same distal site in embryonic stem cells, where it exhibits a bivalent chromatin state. This suggests the same interaction event may maintain a poised state in ES cells and serve to activate or repress the target gene in differentiated tissues. One such distal site with dual functions is the HS5-1 enhancer at the Pcdhac1 locus: Loss of the enhancer results in reduced Pcdhac1 expression in brain and increased expression in tissues that normally exhibit very low levels of Pcdhac1 (Demare, 2013).

The mechanisms by which these stable interactions produce tissue-specific transcriptional outcomes remain to be determined. In one potential model, stable interactions are maintained across many tissues by cohesin and CTCF, irrespective of their transcriptional output. Tissue-specific transcription factors would then serve to activate or repress this constitutive regulatory topology. Alternatively, apparent 'stable' interactions may be independently specified in different tissues by CTCF in conjunction with cohesin, leading to activation or repression depending on the tissue context. In addition, although this study explicitly focused on potentially stable cohesin-associated interactions that involve CTCF in this analysis, other factors besides CTCF may also be sufficient. Conditional deletion of Ctcf from the mouse embryonic limb has been shown to result in small changes in overall gene expression. However, critical limb development genes are down-regulated, including Shh, Fgf4, Grem1, and Jag1, whereas proapoptotic Bbc3 is derepressed, potentially contributing to the degeneration of distal limb structures following loss of Ctcf. Therefore, CTCF may only be required at a subset of stable interactions, or may not be necessary for the maintenance of previously established interactions (Demare, 2013).

The results also suggest that cohesin generally establishes a stable chromatin topology in the nucleus, in addition to the specific examples discussed in this study. Considered collectively, the cohesin-associated interactions identified exhibit correlated H3K27ac and H3K27me3 chromatin states and gene expression across tissues. This is consistent with previous Hi-C and 5C studies that identified constitutive topological domains maintained across tissues and species. Stable cohesin-associated interactions may serve as a constitutive chromatin scaffold that delimits the activity of tissue-specific regulatory elements. For example, the stable interaction at Wnt7a encompasses several putative cortex enhancers identified by H3K27ac, and potentially restricts the activity of these enhancers to the Wnt7a promoter while excluding outside enhancers from influencing Wnt7a expression (Demare, 2013).

Investigating these hypotheses will require functional studies of Wnt7a and other loci that exhibit stable interaction events. For example, transgenic analysis of bacterial artificial chromosomes (BACs) spanning the Wnt7a locus, from which the distal interacting site has been removed using recombineering, may determine whether the long-range interaction is required for spatiotemporal regulation of Wnt7a. To conclusively establish that the stable interaction at Wnt7a maintains the fidelity of Wnt7a expression in vivo will ultimately require removal of the distal site from the mouse genome directly using homologous recombination in ES cells and generation of knockout mice. Such models would also potentially reveal developmental phenotypes arising from destabilization of specific long-range chromatin topologies (Demare, 2013).

Analyses of chromatin modification and transcription factor binding have produced two-dimensional regulatory maps of many mouse and human tissues, but lack the connectivity information required to assign regulatory elements to specific genes. This study obtained an initial, genome-wide view of three-dimensional cohesin-associated chromatin interactions. The results highlight the diverse roles of cohesin in establishing chromatin topology and tissue-specific gene expression and provide insight into how regulatory functions are partitioned in the genome (Demare, 2013).

Different enhancer classes in Drosophila bind distinct architectural proteins and mediate unique chromatin interactions and 3D architecture

Genome-wide studies has identified two enhancer classes in Drosophila that interact with different core promoters: housekeeping enhancers (hkCP) and developmental enhancers (dCP). It is hypothesized that the two enhancer classes are occupied by distinct architectural proteins, affecting their enhancer-promoter contacts. It was determined that both enhancer classes are enriched for RNA Polymerase II, CBP, and architectural proteins but there are also distinctions. hkCP enhancers contain H3K4me3 and exclusively bind Cap-H2, Chromator, DREF and Z4, whereas dCP enhancers contain H3K4me1 and are more enriched for Rad21 and Fs(1)h-L. Additionally, the interactions of each enhancer class were mapped utilizing a Hi-C dataset with <1 kb resolution. Results suggest that hkCP enhancers are more likely to form multi-TSS interaction networks and be associated with topologically associating domain (TAD) borders, while dCP enhancers are more often bound to one or two TSSs and are enriched at chromatin loop anchors. The data support a model suggesting that the unique architectural protein occupancy within enhancers is one contributor to enhancer-promoter interaction specificity (Cubenas-Potts, 2017).

This study characterize the protein occupancy, chromatin interactions and architecture profiles for the two enhancer classes found in Drosophila. Each enhancer class has distinct H3K4 methylation states, is bound by both common and distinct architectural proteins, and is involved in distinct types of chromatin interactions. First, it was established that hkCP enhancers exclusively bind CAP-H2, Chromator, DREF and Z4, while dCP enhancers do not and are preferentially enriched for but not exclusively bound by Fs(1)h-L and Rad21. In addition, hkCP enhancers are more likely than dCP enhancers to associate with multiple TSSs, which promotes a higher transcriptional output. Finally, hkCP enhancers preferentially associate with topologically associating domain (TAD) borders, whereas dCP enhancers are enriched at chromatin loop anchors present inside TADs. Interestingly, enhancers activated by both core promoters exhibit more hkCP enhancer like characteristics, indicating that the both CP enhancers may represent an intermediate among the distinctive hkCP and dCP enhancers. Altogether, these results provide strong correlative evidence, supporting a model suggesting that architectural proteins are critical regulators of enhancer-promoter interaction specificity and that the interactions between enhancers and promoters significantly contribute to the generation of 3D chromatin architecture (Cubenas-Potts, 2017).

The importance of architectural proteins in regulating enhancer-promoter interactions in Drosophila is supported by the observation that the vast majority of architectural protein sites present in the genome correspond to enhancers and promoters. Historically, architectural proteins were identified as insulators, which were functionally demonstrated to block enhancer-promoter interactions. The insulator function of architectural proteins correlates with their enrichment at TAD borders. However, several lines of evidence, including ChIA-PET analysis of CTCF- and cohesin-mediated interactions in mammals, suggest that these architectural proteins help mediate long range contacts among regulatory sequences. In Drosophila this study observed that nearly all of the Group 1 and Group 2 architectural protein sites are associated with enhancers or promoters defined by STARR-seq, TSSs or CBP peaks, suggesting that architectural proteins help mediate enhancer-promoter interactions. Notably, Group 3 architectural proteins include the classic insulator proteins CTCF, CP190, Mod(mdg4) and SuHw, and at least 25% of their peaks cannot be explained by enhancers or promoters. It is interesting to speculate that the non-enhancer-promoter sites may be involved in more classical insulator functions or contributing to the chromatin architecture of inactive regions of the genome (Cubenas-Potts, 2017).

The conclusion that architectural proteins are critical regulators of the specificity between enhancers and promoters is supported by two main lines of evidence. First, the current results demonstrate a strong correlation between each enhancer class and distinct architectural protein subcomplexes. Functional evidence supporting this conclusion comes from mutational analyses of the DRE motif in the distinct enhancer classes, which likely recruits DREF and the other hkCP enhancer associated architectural proteins. Zabidi (2015) demonstrated that the tandem DRE motif alone was sufficient to enhance expression of the housekeeping core promoter and that mutation of DRE motifs within an hkCP enhancer reduced its promoter interactions in a luciferase assay. Furthermore, addition of a DRE motif to a dCP enhancer changed its promoter specificity. Because DREF and potentially BEAF-32 bind to the DRE motif, these results strongly support a model suggesting that the differential occupancy of Cap-H2, Chromator, DREF and Z4 in the two enhancer classes is a critical regulator of their specific interactions with the core promoter types. However, the data cannot discount the notion that unique transcription factor binding at proximal TSSs also contribute to the specificity of enhancer-promoter interactions. Although hkCP enhancer identity is most highly correlated with CAP-H2, Chromator, DREF and Z4 localization, these four architectural proteins are not found in isolation within hkCP enhancers. BEAF-32 and CP190 are also strongly enriched in hkCP enhancers, which are also associated with high occupancy APBSs and TAD borders. Thus, the full architectural protein complement at hkCP enhancers is far more complex than the four hkCP-specific architectural proteins. In addition, architectural proteins that are truly unique to dCP enhancers were not detected. Because dCP enhancers exhibit higher cell type specificity, it cannot be discounted that there are additional dCP enhancers present in the Drosophila genome that were not identified by STARR-seq and thus, excluded from this analysis. From these studies, it is unclear if the enrichment of Fs(1)h-L and Rad21, particularly because Fs(1)h-L and Rad21 are present in hkCP enhancers at lower levels, or the absence of BEAF-32, CAP-H2, Chromator, CP190, DREF and Z4 truly distinguishes the architectural protein complexes found at dCP enhancers. In the future, careful biochemical analyses will be required to gain a comprehensive understanding of the complete organization of architectural protein subcomplexes associated with each enhancer class (Cubenas-Potts, 2017).

hkCP enhancers are associated with multi-TSS chromatin interactions and TAD borders. The promoter-clustering by hkCP enhancers results in a dose-dependent increase in transcriptional output for the interacting genes. Thus, one likely molecular mechanism by which hkCP enhancers promote robust transcriptional activation is by increasing the local concentration of RNA Polymerase II and general transcription factors (GTFs) by bringing multiple TSSs into close proximity. It is interesting that the hkCP enhancers, which form promoter clusters, are associated with TAD borders. It is speculated that the hkCP enhancer interactions involve inter-TAD contacts within the A-type compartment, indicative of the formation of transcription factories (70). From this analysis, it is unclear if the hkCP enhancers alone are sufficient for the formation of the 3D interactions or the neighboring TSSs and their associated transcription factors are also contributing to these contacts. It is hypothesized that the genes recruited to the factories contain the housekeeping promoter motifs (DRE, Ohler 1, Ohler 6 and TCT) and that the hkCP enhancer residents Cap-H2, Chromator, DREF and Z4, are critical to the formation of these 3D contacts (Cubenas-Potts, 2017).

dCP enhancers are more likely to be present within TADs and are enriched on the subTAD-like chromatin loop anchors. dCP enhancers do not form promoter clusters, but are more likely to interact with individual TSSs. One possible explanation for this observation is that the genes interacting with dCP enhancers require the binding of sequence-specific transcription factors, and increasing the concentration of GTFs and RNA polymerase II is not an effective mechanism to promote transcriptional output. The chromatin loop association is consistent with dCP enhancers forming a strong contact with a single TSS. However, it is acknowledged that dCP enhancers are likely one of multiple molecular mechanisms contributing to chromatin loop formation. Surprisingly, the chromatin loops that were observed in Drosophila are distinct from the chromatin loops described in humans. A recent study reported approximately 10,000 chromatin loops in the genome of GM12878 lymphoblastoid cells, but this study detected only 458 chromatin loops in Drosophila utilizing a similar method. The reason why there are so few chromatin loops in Drosophila compared to humans is unclear. It is possible that chromatin loops represent a more precise level of architecture within TADs between specific enhancers and promoters in mammals, but because TADs are significantly smaller in flies (median size 32.5 kb compared to 880 kb in mice, the chromatin loops are not as prominent or easily detected in the Drosophila genome. Notably, it appears that the chromatin loops are generated by different architectural proteins in the two species. The chromatin loops in humans are anchored by convergent CTCF motifs, while the results presented in this study demonstrate that the chromatin loop anchors in Drosophila are depleted of CTCF. Because the chromatin loops in Drosophila show a strong enrichment for Fs(1)h-L, a Brd4 homolog, and the architectural proteins Rad21, Nup98, TFIIIC and Mod(mdg4), it is possible that a combination of transcription and architectural proteins is required for chromatin loop formation in flies, which may be different from mammals . Altogether, it is clear that dCP enhancers are involved in individual contacts with TSSs and are likely one mechanism by which chromatin loops form in Drosophila (Cubenas-Potts, 2017).

Surprisingly, only ~20% and ~12.5% of all hkCP enhancer and ~7.5% and ~8.5% of dCP enhancer interactions involve a TSS or enhancer on the opposite anchor, respectively. The biological significance of the enhancer to non-TSS association is unclear. One possible explanation is that current methods for identifying statistically significant interactions are not sufficiently robust and that many of the enhancer to non-TSS interactions are not representative of biologically significant contacts. However, it cannot be discounted that the non-TSS interactions mediated by enhancers are real and the biological significance of these contacts remains to be determined. Throughout this analysis, the patterns of TSS interactions were compared with each enhancer class instead of drawing conclusions about the absolute number of TSSs bound per enhancer, minimizing the impact of any non-specific interactions within the data. Additional molecular studies for the various type of enhancer interactions (enhancer to promoter, enhancer to non-TSS, etc.) will be required to evaluate the various biological contributions of each (Cubenas-Potts, 2017).

This study found that the functional differences between enhancers that activate housekeeping versus developmental genes are reflected in their chromatin and architectural protein composition, and in the type of interactions they mediate. hkCP enhancers are marked by H3K4me3, associate with TAD borders, and mediate large TSS-clustered interactions to promote robust transcription. This class of enhancers contain the architectural proteins CAP-H2, Chromator, DREF and Z4. In contrast, dCP enhancers are marked by H3K4me1, associate with chromatin loop anchors and are more commonly associated with single TSS-contacts. dCP enhancers are depleted of the hkCP-specific architectural proteins and show an enrichment for Fs(1)h-L and Rad21. The results support a model suggesting that the unique occupancy of architectural proteins in the distinct enhancer classes are key contributors to the types of interactions that enhancers can mediate genome-wide, ultimately affecting enhancer-promoter specificity and 3D chromatin organization. In the future, further characterization of the broadly defined housekeeping and developmental enhancers into smaller subclasses may yield additional levels of regulation and formation of unique architectural protein and transcription factor protein complexes as key mediators of long range chromatin contacts (Cubenas-Potts, 2017).

Condensin II drives large-scale folding and spatial partitioning of interphase chromosomes in Drosophila nuclei

Metazoan chromosomes are folded into discrete sub-nuclear domains, referred to as chromosome territories (CTs). The molecular mechanisms that underlie the formation and maintenance of CTs during the cell cycle remain largely unknown. This paper reports the development of high-resolution chromosome paints to investigate CT organization in Drosophila cycling cells. Large-scale chromosome folding patterns and levels of chromosome intermixing are shown to be remarkably stable across various cell types. The data also suggest that the nucleus scales to accommodate fluctuations in chromosome size throughout the cell cycle, which limits the degree of intermixing between neighboring CTs. Finally, this study shows that the cohesin and condensin complexes are required for different scales of chromosome folding, with condensin II being especially important for the size, shape, and level of intermixing between CTs in interphase. These findings suggest that large-scale chromosome folding driven by condensin II influences the extent to which chromosomes interact, which may have direct consequences for cell-type specific genome stability (Rosin, 2018).

Metazoan genomes are arranged into a nested hierarchy of structural features, ranging from small chromatin loops to larger insulated neighborhoods or topologically associated domains (TADs). TADs are believed to direct and insulate gene regulatory networks, which can engage in long-range interactions with each other, ultimately packaging chromosomes into sub-nuclear compartments termed chromosome territories (CTs) (Rosin, 2018).

CTs are a widespread feature of nuclear organization across a variety of cell types and species, as revealed by both fluorescence in situ hybridization (FISH) and chromosome-conformation-capture (3C)-based studies. Recently, several studies have implicated the ring-shaped SMC (structural maintenance of chromosomes) complexes-cohesin and condensin-in the regulation of large-scale chromatin folding and CT formation. However, the contribution of each complex to local topology, large-scale chromatin folding, and chromosome individualization at single-cell resolution has been hindered by technical limitations. The consequence of CT loss during interphase also remains unclear. This is due, in part, to both the paucity of factors known to directly influence this level of organization and the difficulty in visualizing their effects at single cell resolution. However, CT intermixing has been theorized to influence the location and frequency of translocations and the position of a gene within and between CTs seems to influence its access to the machinery responsible for specific nuclear functions, such as transcription, splicing, and DNA repair (Rosin, 2018).

This study leveraged the flexible, scalable Oligopaint FISH technology to generate high-resolution chromosome paints to the entire Drosophila genome. Combined with a custom 3D segmentation pipeline, a comprehensive picture of chromosome size, shape, and position at single-cell resolution. The results show that various cell types in Drosophila harbor spatially partitioned CTs. Interestingly, widespread somatic homolog pairing in Drosophila results in homologs sharing a single CT, suggesting that homologous and heterologous chromosomes are distinguished at the cellular level in this species. Further, this study characterize the differential roles of cohesin and condensin complexes in local chromatin compaction, large-scale chromatin folding, and CT formation. Cohesin and condensin II were shown to drive different scales of chromatin folding during interphase, with condensin II being especially important for large-scale interactions and the spatial partitioning of chromosomes. These findings indicate that condensin II-driven large-scale chromatin conformations during interphase influence the extent to which chromosomes interact, which has the potential to affect gene regulation and genome stability (Rosin, 2018). In this study, we demonstrate that Drosophila cells harbor spatially distinct CTs and found remarkably consistent levels of intermixing in a variety of cell types and throughout the cell cycle. While the vast majority of cells showed contact between all three major chromosomes, it was possible to measure that, on average, only 40% of the Drosophila genome is intermixed (not accounting for homologous chromosomes). This is strikingly similar to the estimate of 40-46% CT intermixing in human lymphocytes, possibly indicating a widespread and conserved restraint on inter-chromosomal interactions. However, it is noted that a small population of cells do exhibit >90% overlap between neighboring CTs. The fate of these cells will be important to explore in the future (Rosin, 2018).

Further, the condensin II complex was identified as an essential factor for CT formation in cycling cells. These results are consistent with those reported on condensin in yeast, tetrahymena, and post-mitotic polytene cells of Drosophila. These data are also in line with previous work showing that condensin II serves as an 'anti-pairing' factor that disrupts pairing interactions and separates homologous loci. Additionally, it was shown that condensin II overexpression can further compact chromosomes and reduce the level of CT intermixing. Together, these data highlight the highly conserved role of the condensin II complex in controlling the level of inter-chromosomal associations in eukaryotic cells (Rosin, 2018).

If condensin II has the capacity to spatially separate homologous and heterologous chromosomes, how does somatic pairing persist in Drosophila cells that have CTs? One possibility is that pairing interactions are established prior to CT formation and thus, homologous chromosomes would be folded in concert. This would be consistent with some persistence of homolog pairing through mitosis and suggests a model in which chromosomes are folded into CTs through post-mitotic condensin II activity. In addition, pairing interactions may require additional condensin activity to separate homologous versus heterologous interactions. Indeed, these studies showed that condensin II overexpression increases whole-chromosome unpairing in Kc167 and BG3 cells. It is speculated that interphase condensin II levels and thus inter-chromosomal associations are tightly regulated, and could be modified in a cell-type-specific manner. For instance, in contrast to virtually all other cell types in Drosophila, homologous chromosomes in germline stem cells remain unpaired throughout development. This separation between homologs could potentially reflect increased levels of condensin II activity and may indicate that inter-chromosomal associations are reduced to protect the stem-cell population from potentially deleterious rearrangements. Indeed, previous work has shown that different extents of chromosome intermixing correlate with translocation frequencies-both those occurring naturally in the human population and those induced experimentally in human and mouse lymphocytes. Therefore, an alteration in condensin II activity and subsequent CT intermixing levels has the potential to influence the location and frequency with which translocations occur. Intriguingly, mice carrying a hypomorphic allele of cap-H2 were recently shown to frequently develop T-cell lymphomas with highly rearranged chromosomes in the transformed cells. It will be important to determine whether this increased genome instability is associated with increased CT contact prior to the rearrangement event (Rosin, 2018).

When accounting for the popular model of loop extrusion and the stabilizing function of SMC complexes, condensin II activity could potentially fold whole chromosomes into a configuration that limit their interactions with the rest of the genome. While the nature of these interactions remains unknown, they are clearly distinct from cohesin-driven interactions given that cohesin depletion does not significantly change intermixing levels in Drosophila or yeast. Consistent with this hypothesis, a recent study demonstrated that depletion of the cohesin complex in mammals eliminates chromatin looping and TAD formation but does not disrupt long-range interactions between similar chromatin states, highlighting the notion that local insulation and higher-order folding must rely on distinct molecular determinants. Combined with the current findings that large-scale configurations are stable throughout the cell cycle and require condensin II activity, it is proposed that condensin II drives long-range interactions that are established early in interphase. In this model, condensin II may act as an 'organizational bookmark' by prioritizing intra-chromosomal folding immediately following mitotic exit. As condensin II is enriched at highly active regions of the genome marked by H3K4me3, its activity could potentially allow gene regulatory networks and chromatin compartments to favor intra- versus inter-chromosomal interactions. Further studies identifying the interactions driven by condensin II in relation to cohesin will be critical for understanding how these molecular machines cooperatively guide the genome through the cell cycle and development (Rosin, 2018).

Finally, this report describes an efficient and scalable method of high-resolution chromosome painting using Oligopaint FISH technology. Combined with a custom 3D-segmentation pipeline, quantitative measurements of chromosome size, shape, position, and overlap can be analyzed in a systematic and potentially high-throughput fashion. Moreover, the ability to conduct sequential rounds of hybridization with Oligopaints permits 3D analysis of many, if not all, CTs simultaneously. It is anticipated that this technology will lead to an enhanced ability to visualize and karyotype chromosomes in a number of systems, providing a novel battery of assays to better characterize how chromatin is packaged and spatially partitioned in the nucleus (Rosin, 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).

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).

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 here 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 ct^K 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 ct^K 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 here 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 ct^53d, 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).

The Drosophila RAD21 cohesin persists at the centromere region in mitosis

'Cohesin' is a highly conserved multiprotein complex thought to be the primary effector of sister-chromatid cohesion in all eukaryotes. Cohesin complexes in budding yeast hold sister chromatids together from S phase until anaphase, but in metazoans, cohesin proteins dissociate from chromosomes and redistribute into the whole cell volume during prophase, well before sister chromatids separate. This study addressed this apparent anomaly by investigating the cell-cycle dynamics of DRAD21, the Drosophila orthologue of the Xenopus XRAD21 and Saccharomyces cerevisiae Scc1p/Mcd1p cohesins. Analysis of DRAD21 in S2 Drosophila tissue culture cells and live embryos expressing a DRAD21-green fluorescent protein (GFP) fusion revealed the presence of four distinct subcellular pools of DRAD21: a cytoplasmic pool; a chromosome-associated pool which dissociates from chromatin as chromosomes condense in prophase; a short-lived centrosome-associated pool present during metaphase-anaphase; and a centromere-proximal pool which remains bound to condensed chromosomes, is found along the junction of sister chromatids between kinetochores, and persists until the metaphase-anaphase transition. It is concluded that in Drosophila, and possibly all metazoans, a minor pool of cohesin remains binds to centromere-proximal chromatin after prophase and maintains sister-chromatid cohesion until the metaphase-anaphase transition (Warren, 2000; full text of article).

Sequential loading of cohesin subunits during the first meiotic prophase of grasshoppers

The cohesin complexes play a key role in chromosome segregation during both mitosis and meiosis. They establish sister chromatid cohesion between duplicating DNA molecules during S-phase, but they also have an important role during postreplicative double-strand break repair in mitosis, as well as during recombination between homologous chromosomes in meiosis. An additional function in meiosis is related to the sister kinetochore cohesion, so they can be pulled by microtubules to the same pole at anaphase I. Data about the dynamics of cohesin subunits during meiosis are scarce; therefore, it is of great interest to characterize how the formation of the cohesin complexes is achieved in order to understand the roles of the different subunits within them. This study investigated the spatio-temporal distribution of three different cohesin subunits in prophase I grasshopper spermatocytes. Structural maintenance of chromosome protein 3 (SMC3) appears as early as preleptotene, and its localization resembles the location of the unsynapsed axial elements, whereas radiation-sensitive mutant 21 (RAD21) (sister chromatid cohesion protein 1, SCC1) and stromal antigen protein 1 (SA1) (sister chromatid cohesion protein 3, SCC3) are not visualized until zygotene, since they are located in the synapsed regions of the bivalents. During pachytene, the distribution of the three cohesin subunits is very similar and all appear along the trajectories of the lateral elements of the autosomal synaptonemal complexes. However, whereas SMC3 also appears over the single and unsynapsed X chromosome, RAD21 and SA1 do not. It is concluded that the loading of SMC3 and the non-SMC subunits, RAD21 and SA1, occurs in different steps throughout prophase I grasshopper meiosis. These results strongly suggest the participation of SMC3 in the initial cohesin axis formation as early as preleptotene, thus contributing to sister chromatid cohesion, with a later association of both RAD21 and SA1 subunits at zygotene to reinforce and stabilize the bivalent structure. Therefore, it is speculated that more than one cohesin complex participates in the sister chromatid cohesion at prophase I (Valdeolmillos, 2007).

Spindle assembly checkpoint of oocytes depends on a kinetochore structure determined by cohesin in meiosis I

Since the dissolution of sister chromatid cohesion by separase and cyclin B destruction is irreversible, it is essential to delay both until all chromosomes have bioriented on the mitotic spindle. Kinetochores that are not correctly attached to the spindle generate the mitotic checkpoint complex (MCC), which inhibits the anaphase-promoting complex/cyclosome (APC/C) and blocks anaphase onset. This process is known as the spindle assembly checkpoint (SAC). The SAC is especially important in meiosis I, where bivalents consisting of homologous chromosomes held together by chiasmata biorient. Since the first meiotic division is unaffected by rare achiasmatic chromosomes or misaligned bivalents, it is thought that several tensionless kinetochores are required to produce sufficient MCC for APC/C inhibition. Consistent with this, univalents lacking chiasmata elicit a SAC-mediated arrest in DNA repair gene Mlh1^-/- oocytes. In contrast, chromatids generated by TEV protease-induced cohesin cleavage in meiotic cohesin Rec8(TEV/TEV) oocytes merely delay APC/C activation. Since the arrest of Mlh1^-/-Rec8(TEV/TEV) oocytes is alleviated by TEV protease, even when targeted to kinetochores, it is concluded that their SAC depends on cohesin as well as dedicated kinetochore proteins. This has important implications for aging oocytes, where cohesin deterioration will induce sister kinetochore biorientation and compromise MCC production, leading to chromosome missegregation and aneuploid fetuses (Tachibana-Konwalski, 2013).

Meiosis-specific cohesin mediates homolog recognition in mouse spermatocytes

During meiosis, homologous chromosome (homolog) pairing is promoted by several layers of regulation that include dynamic chromosome movement and meiotic recombination. However, the way in which homologs recognize each other remains a fundamental issue in chromosome biology. This study shows that homolog recognition or association initiates upon entry into meiotic prophase before axis assembly and double-strand break (DSB) formation. This homolog association develops into tight pairing only during or after axis formation. Intriguingly, the ability to recognize homologs is retained in Sun1 knockout spermatocytes, in which telomere-directed chromosome movement is abolished, and this is the case even in Spo11 knockout spermatocytes, in which DSB-dependent DNA homology search is absent. Disruption of meiosis-specific cohesin RAD21L precludes the initial association of homologs as well as the subsequent pairing in spermatocytes. These findings suggest the intriguing possibility that homolog recognition is achieved primarily by searching for homology in the chromosome architecture as defined by meiosis-specific cohesin rather than in the DNA sequence itself (Ishiguro, 2014).

Casein Kinase 1 coordinates Cohesin cleavage, gametogenesis, and exit from M Phase in Meiosis I

Meiosis consists of DNA replication followed by two consecutive nuclear divisions and gametogenesis or spore formation. While meiosis I has been studied extensively, less is known about the regulation of meiosis II (see Drosophila cell cycle). This study shows that Hrr25 (see Drosophila dco), the conserved casein kinase 1δ of budding yeast, links three mutually independent key processes of meiosis II. First, Hrr25 induces nuclear division by priming centromeric cohesin for cleavage by separase. Hrr25 simultaneously phosphorylates Rec8 (see Drosophila vtd), the cleavable subunit of cohesin, and removes from centromeres the cohesin protector composed of shugoshin and the phosphatase PP2A. Second, Hrr25 initiates the sporulation program by inducing the synthesis of membranes that engulf the emerging nuclei at anaphase II. Third, Hrr25 mediates exit from meiosis II by activating pathways that trigger the destruction of M-phase-promoting kinases. Thus, Hrr25 synchronizes formation of the single-copy genome with gamete differentiation and termination of meiosis (Argüello-Miranda, 2017).

Search PubMed for articles about Drosophila Rad21

Argüello-Miranda, O., Zagoriy, I., Mengoli, V., Rojas, J., Jonak, K., Oz, T., Graf, P. and Zachariae, W. (2017). Casein Kinase 1 coordinates Cohesin cleavage, gametogenesis, and exit from M Phase in Meiosis II. Dev Cell 40: 37-52. PubMed ID: 28017619

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Bernard, P., et al. (2006). A screen for cohesion mutants uncovers Ssl3, the fission yeast counterpart of the cohesin loading factor Scc4. Curr. Biol. 16: 875-881. PubMed ID: 16682348

Coelho, P. A., Queiroz-Machado, J. and Sunkel, C. E. (2003). Condensin-dependent localisation of topoisomerase II to an axial chromosomal structure is required for sister chromatid resolution during mitosis. J. Cell Sci. 116: 4763-4776. PubMed ID: 14600262

Courbet, S., et al. (2008). Replication fork movement sets chromatin loop size and origin choice in mammalian cells. Nature 455: 557-560. PubMed ID: 18716622

Cubenas-Potts, C., Rowley, M. J., Lyu, X., Li, G., Lei, E. P. and Corces, V. G. (2017). Different enhancer classes in Drosophila bind distinct architectural proteins and mediate unique chromatin interactions and 3D architecture. Nucleic Acids Res 45(4): 1714-1730. PubMed ID: 27899590

Deardorff, M. A., et al. (2007). Mutations in cohesin complex members SMC3 and SMC1A cause a mild variant of cornelia de Lange syndrome with predominant mental retardation. Am. J. Hum. Genet. 80: 485-494. PubMed ID: 17273969

Demare, L. E., Leng, J., Cotney, J., Reilly, S. K., Yin, J., Sarro, R. and Noonan, J. P. (2013). The genomic landscape of cohesin-associated chromatin interactions. Genome Res 23: 1224-1234. PubMed ID: 23704192

Dorsett, D., et al. (2005). Effects of sister chromatid cohesion proteins on cut gene expression during wing development in Drosophila. Development 132(21): 4743-53. PubMed ID: 16207752

Dorsett, D. (2007). Roles of the sister chromatid cohesion apparatus in gene expression, development, and human syndromes. Chromosoma 116(1): 1-13. PubMed ID: 16819604

Fay, A., Misulovin, Z., Li, J., Schaaf, C. A., Gause, M., Gilmour, D. S. and Dorsett, D. (2011). Cohesin selectively binds and regulates genes with paused RNA polymerase. Curr Biol 21: 1624-1634. PubMed ID: 21962715

Fasulo, B., Deuring, R., Murawska, M., Gause, M., Dorighi, K. M., Schaaf, C. A., Dorsett, D., Brehm, A. and Tamkun, J. W. (2012). The Drosophila MI-2 chromatin-remodeling factor regulates higher-order chromatin structure and cohesin dynamics in vivo. PLoS Genet 8: e1002878. Pubmed: 22912596

Felsenfeld, A. L. and Kennison, J. A. (1995). Positional signaling by hedgehog in Drosophila imaginal disc development. Development 121: 1-10. PubMed ID: 7867491

Gandhi, R., Gillespie, P. J. and Hirano, T. (2006). Human Wapl is a cohesin-binding protein that promotes sister-chromatid resolution in mitotic prophase. Curr. Biol. 16: 2406-2417. PubMed ID: 17112726

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

Guillou, E., et al. (2010). Cohesin organizes chromatin loops at DNA replication factories. Genes Dev. 24(24): 2812-22. PubMed ID: 21159821

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Haering, C. H. Lowe, J. Hochwagen, A. and Nasmyth, K. (2002). Molecular architecture of SMC proteins and the yeast cohesin complex. Mol. Cell 9: 773-788. PubMed ID: 11983169

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

Hirano, T. (2006). At the heart of the chromosome: SMC proteins in action. Nat. Rev. Mol. Cell Biol. 7: 311-322. PubMed ID: 16633335

Horsfield, J. A., et al. (2007). Cohesin-dependent regulation of Runx genes. Development 134: 2639-2649. PubMed ID: 17567667

Ishiguro, K., Kim, J., Shibuya, H., Hernandez-Hernandez, A., Suzuki, A., Fukagawa, T., Shioi, G., Kiyonari, H., Li, X. C., Schimenti, J., Hoog, C. and Watanabe, Y. (2014). Meiosis-specific cohesin mediates homolog recognition in mouse spermatocytes. Genes Dev 28: 594-607. PubMed ID: 24589552

Kaur, M., et al. (2005). Precocious sister chromatid separation (PSCS) in Cornelia de Lange syndrome. Am. J. Med. Genet. A 138: 27-31. PubMed ID: 16100726

Kennison, J. A. and Tamkun, J. W. (1988). Dosage-dependent modifiers of Polycomb and antennapedia mutations in Drosophila. Proc. Natl. Acad. Sci. 85: 8136-8140. PubMed ID: 3141923

Krantz, I. D., et al. (2004). Cornelia de Lange syndrome is caused by mutations in NIPBL, the human homolog of Drosophila melanogaster Nipped-B. Nat. Genet. 36: 631-635. PubMed ID: 15146186

Kueng, S., et al. (2006). Wapl controls the dynamic association of cohesin with chromatin. Cell 127: 955-967. PubMed ID: 17113138

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date revised: 15 August 2023

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