An ever-changing landscape in Roberts syndrome biology: Implications for macromolecular damage.

Roberts syndrome (RBS) is a rare developmental disorder that can include craniofacial abnormalities, limb malformations, missing digits, intellectual disabilities, stillbirth, and early mortality. The genetic basis for RBS is linked to autosomal recessive loss-of-function mutation of the establishment of cohesion (ESCO) 2 acetyltransferase. ESCO2 is an essential gene that targets the DNA-binding cohesin complex. ESCO2 acetylates alternate subunits of cohesin to orchestrate vital cellular processes that include sister chromatid cohesion, chromosome condensation, transcription, and DNA repair. Although significant advances were made over the last 20 years in our understanding of ESCO2 and cohesin biology, the molecular etiology of RBS remains ambiguous. In this review, we highlight current models of RBS and reflect on data that suggests a novel role for macromolecular damage in the molecular etiology of RBS.

Condensins and cohesins - one of these things is not like the other!

Condensins and cohesins are highly conserved complexes that tether together DNA loci within a single DNA molecule to produce DNA loops. Condensin and cohesin structures, however, are different, and the DNA loops produced by each underlie distinct cell processes. Condensin rods compact chromosomes during mitosis, with condensin I and II complexes producing spatially defined and nested looping in metazoan cells. Structurally adaptive cohesin rings produce loops, which organize the genome during interphase. Cohesin-mediated loops, termed topologically associating domains or TADs, antagonize the formation of epigenetically defined but untethered DNA volumes, termed compartments. While condensin complexes formed through cis-interactions must maintain chromatin compaction throughout mitosis, cohesins remain highly dynamic during interphase to allow for transcription-mediated responses to external cues and the execution of developmental programs. Here, I review differences in condensin and cohesin structures, and highlight recent advances regarding the intramolecular or cis-based tetherings through which condensins compact DNA during mitosis and cohesins organize the genome during interphase.

Of Rings and Rods: Regulating Cohesin Entrapment of DNA to Generate Intra- and Intermolecular Tethers.

The clinical relevance of cohesin in DNA repair, tumorigenesis, and severe birth defects continues to fuel efforts in understanding cohesin structure, regulation, and enzymology. Early models depicting huge cohesin rings that entrap two DNA segments within a single lumen are fading into obscurity based on contradictory findings, but elucidating cohesin structure amid a myriad of functions remains challenging. Due in large part to integrated uses of a wide range of methodologies, recent advances are beginning to cast light into the depths that previously cloaked cohesin structure. Additional efforts similarly provide new insights into cohesin enzymology: specifically, the discoveries of ATP-dependent transitions that promote cohesin binding and release from DNA. In combination, these efforts posit a new model that cohesin exists primarily as a relatively flattened structure that entraps only a single DNA molecule and that subsequent ATP hydrolysis, acetylation, and oligomeric assembly tether together individual DNA segments.

Correction: Of Rings and Rods: Regulating Cohesin Entrapment of DNA to Generate Intra- and Intermolecular Tethers.

[This corrects the article DOI: 10.1371/journal.pgen.1006337.].

Pds5 regulators segregate cohesion and condensation pathways in Saccharomyces cerevisiae.

Cohesins are required both for the tethering together of sister chromatids (termed cohesion) and subsequent condensation into discrete structures-processes fundamental for faithful chromosome segregation into daughter cells. Differentiating between cohesin roles in cohesion and condensation would provide an important advance in studying chromatin metabolism. Pds5 is a cohesin-associated factor that is essential for both cohesion maintenance and condensation. Recent studies revealed that ELG1 deletion suppresses the temperature sensitivity of pds5 mutant cells. However, the mechanisms through which Elg1 may regulate cohesion and condensation remain unknown. Here, we report that ELG1 deletion from pds5-1 mutant cells results in a significant rescue of cohesion, but not condensation, defects. Based on evidence that Elg1 unloads the DNA replication clamp PCNA from DNA, we tested whether PCNA overexpression would similarly rescue pds5-1 mutant cell cohesion defects. The results indeed reveal that elevated levels of PCNA rescue pds5-1 temperature sensitivity and cohesion defects, but do not rescue pds5-1 mutant cell condensation defects. In contrast, RAD61 deletion rescues the condensation defect, but importantly, neither the temperature sensitivity nor cohesion defects exhibited by pds5-1 mutant cells. In combination, these findings reveal that cohesion and condensation are separable pathways and regulated in nonredundant mechanisms. These results are discussed in terms of a new model through which cohesion and condensation are spatially regulated.

How many roads lead to cohesinopathies?

Genetic mapping studies reveal that mutations in cohesion pathways are responsible for multispectrum developmental abnormalities termed cohesinopathies. These include Roberts syndrome (RBS), Cornelia de Lange Syndrome (CdLS), and Warsaw Breakage Syndrome (WABS). The cohesinopathies are characterized by overlapping phenotypes ranging from craniofacial deformities, limb defects, and mental retardation. Though these syndromes share a similar suite of phenotypes and arise due to mutations in a common cohesion pathway, the underlying mechanisms are currently believed to be distinct. Defects in mitotic failure and apoptosis i.e. trans DNA tethering events are believed to be the underlying cause of RBS, whereas the underlying cause of CdLS is largely modeled as occurring through defects in transcriptional processes i.e. cis DNA tethering events. Here, we review recent findings described primarily in zebrafish, paired with additional studies in other model systems, including human patient cells, which challenge the notion that cohesinopathies represent separate syndromes. We highlight numerous studies that illustrate the utility of zebrafish to provide novel insights into the phenotypes, genes affected and the possible mechanisms underlying cohesinopathies. We propose that transcriptional deregulation is the predominant mechanism through which cohesinopathies arise. Developmental Dynamics 246:881-888, 2017. © 2017 Wiley Periodicals, Inc.

Esco2 regulates cx43 expression during skeletal regeneration in the zebrafish fin.

BACKGROUND: Roberts syndrome (RBS) is a rare genetic disorder characterized by craniofacial abnormalities, limb malformation, and often severe mental retardation. RBS arises from mutations in ESCO2 that encodes an acetyltransferase and modifies the cohesin subunit SMC3. Mutations in SCC2/NIPBL (encodes a cohesin loader), SMC3 or other cohesin genes (SMC1, RAD21/MCD1) give rise to a related developmental malady termed Cornelia de Lange syndrome (CdLS). RBS and CdLS exhibit overlapping phenotypes, but RBS is thought to arise through mitotic failure and limited progenitor cell proliferation while CdLS arises through transcriptional dysregulation. Here, we use the zebrafish regenerating fin model to test the mechanism through which RBS-type phenotypes arise. RESULTS: esco2 is up-regulated during fin regeneration and specifically within the blastema. esco2 knockdown adversely affects both tissue and bone growth in regenerating fins-consistent with a role in skeletal morphogenesis. esco2-knockdown significantly diminishes cx43/gja1 expression which encodes the gap junction connexin subunit required for cell-cell communication. cx43 mutations cause the short fin (sof(b123) ) phenotype in zebrafish and oculodentodigital dysplasia (ODDD) in humans. Importantly, miR-133-dependent cx43 overexpression rescues esco2-dependent growth defects. CONCLUSIONS: These results conceptually link ODDD to cohesinopathies and provide evidence that ESCO2 may play a transcriptional role critical for human development.

Cohesinopathies of a feather flock together.

Roberts Syndrome (RBS) and Cornelia de Lange Syndrome (CdLS) are severe developmental maladies that present with nearly an identical suite of multi-spectrum birth defects. Not surprisingly, RBS and CdLS arise from mutations within a single pathway--here involving cohesion. Sister chromatid tethering reactions that comprise cohesion are required for high fidelity chromosome segregation, but cohesin tethers also regulate gene transcription, promote DNA repair, and impact DNA replication. Currently, RBS is thought to arise from elevated levels of apoptosis, mitotic failure, and limited progenitor cell proliferation, while CdLS is thought to arise, instead, from transcription dysregulation. Here, we review new information that implicates RBS gene mutations in altered transcription profiles. We propose that cohesin-dependent transcription dysregulation may extend to other developmental maladies; the diagnoses of which are complicated through multi-functional proteins that manifest a sliding scale of diverse and severe phenotypes. We further review evidence that cohesinopathies are more common than currently posited.

Cohesin codes - interpreting chromatin architecture and the many facets of cohesin function.

Sister chromatid tethering is maintained by cohesin complexes that minimally contain Smc1, Smc3, Mcd1 and Scc3. During S-phase, chromatin-associated cohesins are modified by the Eco1/Ctf7 family of acetyltransferases. Eco1 proteins function during S phase in the context of replicated sister chromatids to convert chromatin-bound cohesins to a tethering-competent state, but also during G2 and M phases in response to double-stranded breaks to promote error-free DNA repair. Cohesins regulate transcription and are essential for ribosome biogenesis and complete chromosome condensation. Little is known, however, regarding the mechanisms through which cohesin functions are directed. Recent findings reveal that Eco1-mediated acetylation of different lysine residues in Smc3 during S phase promote either cohesion or condensation. Phosphorylation and SUMOylation additionally impact cohesin functions. Here, we posit the existence of a cohesin code, analogous to the histone code introduced over a decade ago, and speculate that there is a symphony of post-translational modifications that direct cohesins to function across a myriad of cellular processes. We also discuss evidence that outdate the notion that cohesion defects are singularly responsible for cohesion-mutant-cell inviability. We conclude by proposing that cohesion establishment is linked to chromatin formation.

Sticking a fork in cohesin--it's not done yet!

To identify the products of chromosome replication (termed sister chromatids) from S-phase through M-phase of the cell cycle, each sister pair becomes tethered together by specialized protein complexes termed cohesins. To participate in sister tethering reactions, chromatin-bound cohesins become modified by establishment factors that function during S-phase and bind to DNA replication-fork components. Early models posited that establishment factors might move with replication forks, but that fork progression takes place independently of cohesion pathways. Recent studies now suggest that progression of the replication fork and/or S-phase are slowed in cohesion-deficient cells. These findings have led to speculations that cohesin ring-like structures normally hinder fork progression but coordinate origin firing during replication. Neither model, however, fully explains the diverse effects of cohesion mutation on replication kinetics. I discuss these challenges and then offer alternative views that include cohesin-independent mechanisms for replication-fork destabilization and transcription-based effects on S-phase progression.

Cohesin: an emerging master regulator at the heart of cardiac development.

Cohesins are ATPase complexes that play central roles in cellular processes such as chromosome division, DNA repair, and gene expression. Cohesinopathies arise from mutations in cohesin proteins or cohesin complex regulators and encompass a family of related developmental disorders that present with a range of severe birth defects, affect many different physiological systems, and often lead to embryonic fatality. Treatments for cohesinopathies are limited, in large part due to the lack of understanding of cohesin biology. Thus, characterizing the signaling networks that lie upstream and downstream of cohesin-dependent pathways remains clinically relevant. Here, we highlight alterations in cohesins and cohesin regulators that result in cohesinopathies, with a focus on cardiac defects. In addition, we suggest a novel and more unifying view regarding the mechanisms through which cohesinopathy-based heart defects may arise.

G1-Cyclin2 (Cln2) promotes chromosome hypercondensation in eco1/ctf7 rad61 null cells during hyperthermic stress in Saccharomyces cerevisiae.

Eco1/Ctf7 is a highly conserved acetyltransferase that activates cohesin complexes and is critical for sister chromatid cohesion, chromosome condensation, DNA damage repair, nucleolar integrity, and gene transcription. Mutations in the human homolog of ECO1 (ESCO2/EFO2), or in genes that encode cohesin subunits, result in severe developmental abnormalities and intellectual disabilities referred to as Roberts syndrome and Cornelia de Lange syndrome, respectively. In yeast, deletion of ECO1 results in cell inviability. Codeletion of RAD61 (WAPL in humans), however, produces viable yeast cells. These eco1 rad61 double mutants, however, exhibit a severe temperature-sensitive growth defect, suggesting that Eco1 or cohesins respond to hyperthermic stress through a mechanism that occurs independent of Rad61. Here, we report that deletion of the G1 cyclin CLN2 rescues the temperature-sensitive lethality otherwise exhibited by eco1 rad61 mutant cells, such that the triple mutant cells exhibit robust growth over a broad range of temperatures. While Cln1, Cln2, and Cln3 are functionally redundant G1 cyclins, neither CLN1 nor CLN3 deletions rescue the temperature-sensitive growth defects otherwise exhibited by eco1 rad61 double mutants. We further provide evidence that CLN2 deletion rescues hyperthermic growth defects independent of START and impacts the state of chromosome condensation. These findings reveal novel roles for Cln2 that are unique among the G1 cyclin family and appear critical for cohesin regulation during hyperthermic stress.

Integrating Sister Chromatid Cohesion Establishment to DNA Replication.

The intersection through which two fundamental processes meet provides a unique vantage point from which to view cellular regulation. On the one hand, DNA replication is at the heart of cell division, generating duplicate chromosomes that allow each daughter cell to inherit a complete copy of the parental genome. Among other factors, the PCNA (proliferating cell nuclear antigen) sliding clamp ensures processive DNA replication during S phase and is essential for cell viability. On the other hand, the process of chromosome segregation during M phase-an act that occurs long after DNA replication-is equally fundamental to a successful cell division. Eco1/Ctf7 ensures that chromosomes faithfully segregate during mitosis, but functions during DNA replication to activate cohesins and thereby establish cohesion between sister chromatids. To achieve this, Eco1 binds PCNA and numerous other DNA replication fork factors that include MCM helicase, Chl1 helicase, and the Rtt101-Mms1-Mms22 E3 ubiquitin ligase. Here, we review the multi-faceted coordination between cohesion establishment and DNA replication. SUMMARY STATEMENT: New findings provide important insights into the mechanisms through which DNA replication and the establishment of sister chromatid cohesion are coupled.

Genetically induced redox stress occurs in a yeast model for Roberts syndrome.

Roberts syndrome (RBS) is a multispectrum developmental disorder characterized by severe limb, craniofacial, and organ abnormalities and often intellectual disabilities. The genetic basis of RBS is rooted in loss-of-function mutations in the essential N-acetyltransferase ESCO2 which is conserved from yeast (Eco1/Ctf7) to humans. ESCO2/Eco1 regulate many cellular processes that impact chromatin structure, chromosome transmission, gene expression, and repair of the genome. The etiology of RBS remains contentious with current models that include transcriptional dysregulation or mitotic failure. Here, we report evidence that supports an emerging model rooted in defective DNA damage responses. First, the results reveal that redox stress is elevated in both eco1 and cohesion factor Saccharomyces cerevisiae mutant cells. Second, we provide evidence that Eco1 and cohesion factors are required for the repair of oxidative DNA damage such that ECO1 and cohesin gene mutations result in reduced cell viability and hyperactivation of DNA damage checkpoints that occur in response to oxidative stress. Moreover, we show that mutation of ECO1 is solely sufficient to induce endogenous redox stress and sensitizes mutant cells to exogenous genotoxic challenges. Remarkably, antioxidant treatment desensitizes eco1 mutant cells to a range of DNA damaging agents, raising the possibility that modulating the cellular redox state may represent an important avenue of treatment for RBS and tumors that bear ESCO2 mutations.

Esco2 and cohesin regulate CRL4 ubiquitin ligase ddb1 expression and thalidomide teratogenicity.

Cornelia de Lange syndrome (CdLS) and Roberts syndrome (RBS) are severe developmental maladies that arise from mutation of cohesin (including SMC3, CdLS) and ESCO2 (RBS). Though ESCO2 activates cohesin, CdLS and RBS etiologies are currently considered non-synonymous and for which pharmacological treatments are unavailable. Here, we identify a unifying mechanism that integrates these genetic maladies to pharmacologically-induced teratogenicity via thalidomide. Our results reveal that Esco2 and cohesin co-regulate the transcription of a component of CRL4 ubiquitin ligase through which thalidomide exerts teratogenic effects. These findings are the first to link RBS and CdLS to thalidomide teratogenicity and offer new insights into treatments.

Unzipped and loaded: the role of DNA helicases and RFC clamp-loading complexes in sister chromatid cohesion.

It is well known that the products of chromosome replication are paired to ensure that the sisters segregate away from each other during mitosis. A key issue is how cells pair sister chromatids but preclude the catastrophic pairing of nonsister chromatids. The identification of both replication factor C and DNA helicases as critical for sister chromatid pairing has brought new insights into this fundamental process.

A multi-step pathway for the establishment of sister chromatid cohesion.

The cohesion of sister chromatids is mediated by cohesin, a protein complex containing members of the structural maintenance of chromosome (Smc) family. How cohesins tether sister chromatids is not yet understood. Here, we mutate SMC1, the gene encoding a cohesin subunit of budding yeast, by random insertion dominant negative mutagenesis to generate alleles that are highly informative for cohesin assembly and function. Cohesins mutated in the Hinge or Loop1 regions of Smc1 bind chromatin by a mechanism similar to wild-type cohesin, but fail to enrich at cohesin-associated regions (CARs) and pericentric regions. Hence, the Hinge and Loop1 regions of Smc1 are essential for the specific chromatin binding of cohesin. This specific binding and a subsequent Ctf7/Eco1-dependent step are both required for the establishment of cohesion. We propose that a cohesin or cohesin oligomer tethers the sister chromatids through two chromatin-binding events that are regulated spatially by CAR binding and temporally by Ctf7 activation, to ensure cohesins crosslink only sister chromatids.

Promotion of Hyperthermic-Induced rDNA Hypercondensation in Saccharomyces cerevisiae.

Ribosome biogenesis is tightly regulated through stress-sensing pathways that impact genome stability, aging and senescence. In Saccharomyces cerevisiae, ribosomal RNAs are transcribed from rDNA located on the right arm of chromosome XII. Numerous studies reveal that rDNA decondenses into a puff-like structure during interphase, and condenses into a tight loop-like structure during mitosis. Intriguingly, a novel and additional mechanism of increased mitotic rDNA compaction (termed hypercondensation) was recently discovered that occurs in response to temperature stress (hyperthermic-induced) and is rapidly reversible. Here, we report that neither changes in condensin binding or release of DNA during mitosis, nor mutation of factors that regulate cohesin binding and release, appear to play a critical role in hyperthermic-induced rDNA hypercondensation. A candidate genetic approach revealed that deletion of either HSP82 or HSC82 (Hsp90 encoding heat shock paralogs) result in significantly reduced hyperthermic-induced rDNA hypercondensation. Intriguingly, Hsp inhibitors do not impact rDNA hypercondensation. In combination, these findings suggest that Hsp90 either stabilizes client proteins, which are sensitive to very transient thermic challenges, or directly promotes rDNA hypercondensation during preanaphase. Our findings further reveal that the high mobility group protein Hmo1 is a negative regulator of mitotic rDNA condensation, distinct from its role in promoting premature condensation of rDNA during interphase upon nutrient starvation.

Correction: Chl1 DNA helicase and Scc2 function in chromosome condensation through cohesin deposition.

[This corrects the article DOI: 10.1371/journal.pone.0188739.].

PCNA promotes context-specific sister chromatid cohesion establishment separate from that of chromatin condensation.

Cellular genomes undergo various structural changes that include cis tethering (the tethering together of two loci within a single DNA molecule), which promotes chromosome condensation and transcriptional activation, and trans tethering (the tethering together of two DNA molecules), which promotes sister chromatid cohesion and DNA repair. The protein complex termed cohesin promotes both cis and trans forms of DNA tethering, but the extent to which these cohesin functions occur in temporally or spatially defined contexts remains largely unknown. Prior studies indicate that DNA polymerase sliding clamp PCNA recruits cohesin acetyltransferase Eco1, suggesting that sister chromatid cohesion is established in the context of the DNA replication fork. In support of this model, elevated levels of PCNA rescue the temperature growth and cohesion defects exhibited by eco1 mutant cells. Here, we test whether Eco1-dependent chromatin condensation is also promoted in the context of this DNA replication fork component. Our results reveal that overexpressed PCNA does not promote DNA condensation in eco1 mutant cells, even though Smc3 acetylation levels are increased. We further provide evidence that replication fork-associated E3 ligase impacts on Eco1 are more complex that previously described. In combination, the data suggests that Eco1 acetylates Smc3 and thus promotes sister chromatid cohesion in context of the DNA replication fork, whereas a distinct cohesin population participates in chromatin condensation outside the context of the DNA replication fork.