Mechanisms of X chromosome dosage compensation have been studied extensively in a few model species representing clades of shared sex chromosome ancestry. However, the diversity within each clade as a function of sex chromosome evolution is largely unknown. Here, we anchor ourselves to the nematode Caenorhabditis elegans, for which a well-studied mechanism of dosage compensation occurs through a specialized structural maintenance of chromosomes (SMC) complex, and explore the diversity of dosage compensation in the surrounding phylogeny of nematodes. Through phylogenetic analysis of the C. elegans dosage compensation complex and a survey of its epigenetic signatures, including X-specific topologically associating domains (TADs) and X-enrichment of H4K20me1, we found that the condensin-mediated mechanism evolved recently in the lineage leading to Caenorhabditis through an SMC-4 duplication. Intriguingly, an independent duplication of SMC-4 and the presence of X-specific TADs in Pristionchus pacificus suggest that condensin-mediated dosage compensation arose more than once. mRNA-seq analyses of gene expression in several nematode species indicate that dosage compensation itself is ancestral, as expected from the ancient XO sex determination system. Indicative of the ancestral mechanism, H4K20me1 is enriched on the X chromosomes in Oscheius tipulae, which does not contain X-specific TADs or SMC-4 paralogs. Together, our results indicate that the dosage compensation system in C. elegans is surprisingly new, and condensin may have been co-opted repeatedly in nematodes, suggesting that the process of evolving a chromosome-wide gene regulatory mechanism for dosage compensation is constrained.
UNASSIGNED: X chromosome dosage compensation mechanisms evolved in response to Y chromosome degeneration during sex chromosome evolution. However, establishment of dosage compensation is not an endpoint. As sex chromosomes change, dosage compensation strategies may have also changed. In this study, we performed phylogenetic and epigenomic analyses surrounding Caenorhabditis elegans and found that the condensin-mediated dosage compensation mechanism in C. elegans is surprisingly new, and has evolved in the presence of an ancestral mechanism. Intriguingly, condensin-based dosage compensation may have evolved more than once in the nematode lineage, the other time in Pristionchus. Together, our work highlights a previously unappreciated diversity of dosage compensation mechanisms within a clade, and suggests constraints in evolving new mechanisms in the presence of an existing one.

At present, the incidence of tumours is increasing on a yearly basis, and tumourigenesis is usually associated with chromosomal instability and cell cycle dysregulation. Moreover, abnormalities in the chromosomal structure often lead to DNA damage, further exacerbating gene mutations and chromosomal rearrangements. However, the non‑SMC condensin I complex subunit G (NCAPG) of the structural maintenance of chromosomes family is known to exert a key role in tumour development. It has been shown that high expression of NCAPG is closely associated with tumour development and progression. Overexpression of NCAPG variously affects chromosome condensation and segregation during cell mitosis, influences cell cycle regulation, promotes tumour cell proliferation and invasion, and inhibits apoptosis. In addition, NCAPG has been associated with tumour cell stemness, tumour resistance and recurrence. The aim of the present review was to explore the underlying mechanisms of NCAPG during tumour development, with a view towards providing novel targets and strategies for tumour therapy, and through the elucidation of the mechanisms involved, to lay the foundation for future developments in health.

It is widely accepted that DNA replication fork stalling is a common occurrence during cell proliferation, but there are robust mechanisms to alleviate this and ensure DNA replication is completed prior to chromosome segregation. The SMC5/6 complex has consistently been implicated in the maintenance of replication fork integrity. However, the essential role of the SMC5/6 complex during DNA replication in mammalian cells has not been elucidated. In this study, we investigate the molecular consequences of SMC5/6 loss at the replication fork in mouse embryonic stem cells (mESCs), employing the auxin-inducible degron (AID) system to deplete SMC5 acutely and reversibly in the defined cellular contexts of replication fork stall and restart. In SMC5-depleted cells, we identify a defect in the restart of stalled replication forks, underpinned by excess MRE11-mediated fork resection and a perturbed localization of fork protection factors to the stalled fork. Previously, we demonstrated a physical and functional interaction of SMC5/6 with the COP9 signalosome (CSN), a cullin deneddylase that enzymatically regulates cullin ring ligase (CRL) activity. Employing a combination of DNA fiber techniques, the AID system, small-molecule inhibition assays, and immunofluorescence microscopy analyses, we show that SMC5/6 promotes the localization of fork protection factors to stalled replication forks by negatively modulating the COP9 signalosome (CSN). We propose that the SMC5/6-mediated modulation of the CSN ensures that CRL activity and their roles in DNA replication fork stabilization are maintained to allow for efficient replication fork restart when a replication fork stall is alleviated.

Up to 50% of patients with severe congenital heart disease (CHD) develop life-altering neurodevelopmental disability (NDD). It has been presumed that NDD arises in CHD cases because of hypoxia before, during, or after cardiac surgery. Recent studies detected an enrichment in de novo mutations in CHD and NDD, as well as significant overlap between CHD and NDD candidate genes. However, there is limited evidence demonstrating that genes causing CHD can produce NDD independent of hypoxia. A patient with hypoplastic left heart syndrome and gross motor delay presented with a de novo mutation in SMC5. Modeling mutation of smc5 in Xenopus tropicalis embryos resulted in reduced heart size, decreased brain length, and disrupted pax6 patterning. To evaluate the cardiac development, we induced the conditional knockout (cKO) of Smc5 in mouse cardiomyocytes, which led to the depletion of mature cardiomyocytes and abnormal contractility. To test a role for Smc5 specifically in the brain, we induced cKO in the mouse central nervous system, which resulted in decreased brain volume, and diminished connectivity between areas related to motor function but did not affect vascular or brain ventricular volume. We propose that genetic factors, rather than hypoxia alone, can contribute when NDD and CHD cases occur concurrently.

Epstein-Barr virus (EBV) persistently infects people worldwide. Delivery of ∼170-kb EBV genomes to nuclei and use of nuclear membrane-less replication compartments (RCs) for their lytic cycle amplification necessitate evasion of intrinsic antiviral responses. Proteomics analysis indicates that, upon B cell infection or lytic reactivation, EBV depletes the cohesin SMC5/6, which has major roles in chromosome maintenance and DNA damage repair. The major tegument protein BNRF1 targets SMC5/6 complexes by a ubiquitin proteasome pathway dependent on calpain proteolysis and Cullin-7. In the absence of BNRF1, SMC5/6 associates with R-loop structures, including at the viral lytic origin of replication, and interferes with RC formation and encapsidation. CRISPR analysis identifies RC restriction roles of SMC5/6 components involved in DNA entrapment and SUMOylation. Our study highlights SMC5/6 as an intrinsic immune sensor and restriction factor for a human herpesvirus RC and has implications for the pathogenesis of EBV-associated cancers.

Structural maintenance of chromosomes (SMC) complexes are critical chromatin modulators. In eukaryotes, the cohesin and condensin SMC complexes organize chromatin, while the Smc5/6 complex directly regulates DNA replication and repair. The molecular basis for the distinct functions of Smc5/6 is poorly understood. Here, we report an integrative structural study of the budding yeast Smc5/6 holo-complex using electron microscopy, cross-linking mass spectrometry, and computational modeling. We show that the Smc5/6 complex possesses several unique features, while sharing some architectural characteristics with other SMC complexes. In contrast to arm-folded structures of cohesin and condensin, Smc5 and Smc6 arm regions do not fold back on themselves. Instead, these long filamentous regions interact with subunits uniquely acquired by the Smc5/6 complex, namely the Nse2 SUMO ligase and the Nse5/Nse6 subcomplex, with the latter also serving as a linchpin connecting distal parts of the complex. Our 3.0-Å resolution cryoelectron microscopy structure of the Nse5/Nse6 core further reveals a clasped-hand topology and a dimeric interface important for cell growth. Finally, we provide evidence that Nse5/Nse6 uses its SUMO-binding motifs to contribute to Nse2-mediated sumoylation. Collectively, our integrative study identifies distinct structural features of the Smc5/6 complex and functional cooperation among its coevolved unique subunits.

Cohesin is a multi-unit protein complex from the structural maintenance of chromosomes (SMC) family, required for holding sister chromatids together during mitosis and meiosis. In yeast, the cohesin complex entraps sister DNAs within tripartite rings created by pairwise interactions between the central ring units SMC1 and SMC3 and subunits such as the α-kleisin SCC1 (REC8/SYN1 in meiosis). The complex is an indispensable regulator of meiotic recombination in eukaryotes. In Arabidopsis and maize, the SMC1/SMC3 heterodimer is a key determinant of meiosis. In Arabidopsis, several kleisin proteins are also essential: SYN1/REC8 is meiosis-specific and is essential for double-strand break repair, whereas AtSCC2 is a subunit of the cohesin SCC2/SCC4 loading complex that is important for synapsis and segregation. Other important meiotic subunits are the cohesin EXTRA SPINDLE POLES (AESP1) separase, the acetylase ESTABLISHMENT OF COHESION 1/CHROMOSOME TRANSMISSION FIDELITY 7 (ECO1/CTF7), the cohesion release factor WINGS APART-LIKE PROTEIN 1 (WAPL) in Arabidopsis (AtWAPL1/AtWAPL2), and the WAPL antagonist AtSWITCH1/DYAD (AtSWI1). Other important complexes are the SMC5/SMC6 complex, which is required for homologous DNA recombination during the S-phase and for proper meiotic synapsis, and the condensin complexes, featuring SMC2/SMC4 that regulate proper clustering of rDNA arrays during interphase. Meiotic recombination is the key to enrich desirable traits in commercial plant breeding. In this review, I highlight critical advances in understanding plant chromatid cohesion in the model plant Arabidopsis and crop plants and suggest how manipulation of crossover formation during meiosis, somatic DNA repair and chromosome folding may facilitate transmission of desirable alleles, tolerance to radiation, and enhanced transcription of alleles that regulate sexual development. I hope that these findings highlight opportunities for crop breeding.

Mutations of SMC5/6 components cause developmental defects, including primary microcephaly. To model neurodevelopmental defects, we engineered a mouse wherein Smc5 is conditionally knocked out (cKO) in the developing neocortex. Smc5 cKO mice exhibited neurodevelopmental defects due to neural progenitor cell (NPC) apoptosis, which led to reduction in cortical layer neurons. Smc5 cKO NPCs formed DNA bridges during mitosis and underwent chromosome missegregation. SMC5/6 depletion triggers a CHEK2-p53 DNA damage response, as concomitant deletion of the Trp53 tumor suppressor or Chek2 DNA damage checkpoint kinase rescued Smc5 cKO neurodevelopmental defects. Further assessment using Smc5 cKO and auxin-inducible degron systems demonstrated that absence of SMC5/6 leads to DNA replication stress at late-replicating regions such as pericentromeric heterochromatin. In summary, SMC5/6 is important for completion of DNA replication prior to entering mitosis, which ensures accurate chromosome segregation. Thus, SMC5/6 functions are critical in highly proliferative stem cells during organism development.

OBJECTIVE: Structural maintenance of chromosomes (SMC) proteins are key players in chromosome dynamics in all types of organisms. The so-called condensin subfamily is essential for chromosome condensation in eukaryotic cells, as is the bacterial SMC complex (called MukBEF in Escherichia coli). We expressed the Bacillus subtilis Smc protein and its two complex partners ScpA and ScpB in E. coli cells, and monitored effects on chromosome compaction by DNA staining of live cells using epifluorescence microscopy.
UNASSIGNED: We show that expression of BsSmc leads to strong chromosome compaction, while expression of ScpAB does not show any effect. Chromosome compaction by Smc was also found for mutant versions lacking ATP binding or ability for head engagement, and was counteracted by concomitant expression of ScpAB. Our findings show that the SMC complex can act as autonomous condensation system in a heterologous bacterial host system, for which neither ATP binding nor ATP hydrolysis are required. Our investigation suggests that the negative effect on compaction activity of Smc exerted by ScpAB in vivo does not involve an effect on ATPase activity, but more likely a stabilization of the engagement of head domains, which in turn may affect ATPase activity.

The multi-subunit structural maintenance of chromosomes (SMC) 5/6 complex includes SMC6 and non-SMC element (NSE)3. SMC5/6 is essential for homologous recombination DNA repair and functions as an antiviral factor during hepatitis B (HBV) and herpes simplex-1 (HSV-1) viral infections. Intriguingly, SMC5/6 has been found to associate with high-risk human papillomavirus (HPV) E2 regulatory proteins, but the functions of this interaction and its role during HPV infection remain unclear. Here, we further characterize SMC5/6 interactions with HPV-31 E2 and its role in the HPV life cycle. Co-immunoprecipitation (co-IP) revealed that SMC6 interactions with HPV-31 E2 require the E2 transactivation domain, implying that SMC5/6 interacts with full-length E2. Using chromatin immunoprecipitation, we found that SMC6 is present on HPV-31 episomes at E2 binding sites. Th depletion of SMC6 and NSE3 increased viral replication and transcription in keratinocytes maintaining episomal HPV-31, indicating that SMC5/6 restricts the viral replicative program. SMC6 interactions with E2 were reduced in the presence of HPV-31 E1, suggesting that SMC6 and E1 compete for E2 binding. Our findings demonstrate SMC5/6 functions as a repressor of the viral replicative program and this may involve inhibiting the initiation of viral replication.