TOP-2 is differentially required for the proper maintenance of cohesin on meiotic chromosomes in C. elegans spermatogenesis and oogenesis

During meiotic prophase I, accurate segregation of homologous chromosomes requires the establishment of a chromosomes with a meiosis-specific architecture. Sister chromatid cohesins and the enzyme Topoisomerase II are important components of meiotic chromosome axes, but the relationship of these proteins in the context of meiotic chromosome segregation is poorly defined. Here, we analyzed the role of Topoisomerase II (TOP-2) in the timely release of sister chromatid cohesins during spermatogenesis and oogenesis of Caenorhabditis elegans. We show that there is a different requirement for TOP-2 in meiosis of spermatogenesis and oogenesis. The loss-of-function mutation top-2(it7) results in premature REC-8 removal in spermatogenesis, but not oogenesis. This is due to a failure to maintain the HORMA-domain proteins HTP-1 and HTP-2 (HTP-1/2) on chromosome axes at diakinesis and mislocalization of the downstream components that control sister chromatid cohesion release including Aurora B kinase. In oogenesis, top-2(it7) causes a delay in the localization of Aurora B to oocyte chromosomes but can be rescued through premature activation of the maturation promoting factor via knock-down of the inhibitor kinase WEE-1.3. The delay in Aurora B localization is associated with an increase in the length of diakinesis chromosomes and wee-1.3 RNAi mediated rescue of Auorora B localization in top-2(it7) is associated with a decrease in chromosome length. Our results imply that the sex-specific effects of Topoisomerase II on sister chromatid cohesion release are due to differences in the temporal regulation of meiosis and chromosome structure in late prophase I in spermatogenesis and oogenesis.

The histone methyltransferase NSD3 contributes to cohesin loading during mitotic exit

During the cell cycle, dynamic post-translational modifications modulate the association of the cohesin complex with chromatin. Phosphorylation / dephosphorylation and acetylation / deacetylation of histones and of cohesin components ensure correct establishment of cohesion during S phase and its proper dissolution during mitosis. In contrast, little is known about the contribution of methylation to the regulation of sister chromatid cohesion. We performed a RNA interference-mediated inactivation screen against 14 histone methyltransferases of the SET domain family that highlighted NSD3 as a factor essential for sister chromatid cohesion in mitosis. We established that NSD3 ensures proper level of the cohesin loader MAU2 and of cohesin itself onto chromatin at mitotic exit. Consistent with its implication in the loading of kollerin and cohesin complexes onto chromatin, we showed that NSD3 associates with chromatin in early anaphase prior to that of MAU2 and RAD21 and dissociates from chromatin upon cell's entry into prophase. Finally, we demonstrated that of the two NSD3 variant that exist in somatic cells, the long form that carries the methyltransferase activity is the one that acts in cohesion regulation. Taken together, these results describe a novel factor associated with histone methylation in cohesin loading.

The loader complex Scc2/4 forms co-condensates with DNA as loading sites for cohesin

The genome is organized by diverse packaging mechanisms like nucleosome formation, loop extrusion and phase separation, which all compact DNA in a dynamic manner. Phase separation additionally drives protein recruitment to condensed DNA sites and thus regulates gene transcription. The cohesin complex is a key player in chromosomal organization that extrudes loops to connect distant regions of the genome and ensures sister chromatid cohesion after S-phase. For stable loading onto the DNA and for activation, cohesin requires the loading complex Scc2/4. As the precise loading mechanism remains unclear, we investigated whether phase separation might be the initializer of the cohesin recruitment process. We found that, in absence of cohesin, budding yeast Scc2/4 forms phase separated co-condensates with DNA, which comprise liquid-like properties shown by droplet shape, fusion ability and reversibility. We reveal in DNA curtain and optical tweezer experiments that these condensates are built by DNA bridging and bending through Scc2/4. Importantly, Scc2/4-mediated condensates recruit cohesin efficiently and increase the stability of the cohesin complex. We conclude that phase separation properties of Scc2/4 enhance cohesin loading by molecular crowding, which might then provide a starting point for the recruitment of additional factors and proteins.

The Interplay of Cohesin and the Replisome at Processive and Stressed DNA Replication Forks

The cohesin complex facilitates faithful chromosome segregation by pairing the sister chromatids after DNA replication until mitosis. In addition, cohesin contributes to proficient and error-free DNA replication. Replisome progression and establishment of sister chromatid cohesion are intimately intertwined processes. Here, we review how the key factors in DNA replication and cohesion establishment cooperate in unperturbed conditions and during DNA replication stress. We discuss the detailed molecular mechanisms of cohesin recruitment and the entrapment of replicated sister chromatids at the replisome, the subsequent stabilization of sister chromatid cohesion via SMC3 acetylation, as well as the role and regulation of cohesin in the response to DNA replication stress.

Ploidy and recombination proficiency shape the evolutionary adaptation to constitutive DNA replication stress

In haploid budding yeast, evolutionary adaptation to constitutive DNA replication stress alters three genome maintenance modules: DNA replication, the DNA damage checkpoint, and sister chromatid cohesion. We asked how these trajectories depend on genomic features by comparing the adaptation in three strains: haploids, diploids, and recombination deficient haploids. In all three, adaptation happens within 1000 generations at rates that are correlated with the initial fitness defect of the ancestors. Mutations in individual genes are selected at different frequencies in populations with different genomic features, but the benefits these mutations confer are similar in the three strains, and combinations of these mutations reproduce the fitness gains of evolved populations. Despite the differences in the selected mutations, adaptation targets the same three functional modules despite differences in genomic features, revealing a common evolutionary response to constitutive DNA replication stress.

Cornelia de Lange Syndrome as Paradigm of Chromatinopathies

Chromatinopathies can be defined as a class of neurodevelopmental disorders caused by mutations affecting proteins responsible for chromatin remodeling and transcriptional regulation. The resulting dysregulation of gene expression favors the onset of a series of clinical features such as developmental delay, intellectual disability, facial dysmorphism, and behavioral disturbances. Cornelia de Lange syndrome (CdLS) is a prime example of a chromatinopathy. It is caused by mutations affecting subunits or regulators of the cohesin complex, a multisubunit protein complex involved in various molecular mechanisms such as sister chromatid cohesion, transcriptional regulation and formation of topologically associated domains. However, disease-causing variants in non-cohesin genes with overlapping functions have also been described in association with CdLS. Notably, the majority of these genes had been previously found responsible for distinct neurodevelopmental disorders that also fall within the category of chromatinopathies and are frequently considered as differential diagnosis for CdLS. In this review, we provide a systematic overview of the current literature to summarize all mutations in non-cohesin genes identified in association with CdLS phenotypes and discuss about the interconnection of proteins belonging to the chromatinopathies network.

The Cohesin Complex and Its Interplay with Non-Coding RNAs

The cohesin complex is a multi-subunit protein complex initially discovered for its role in sister chromatid cohesion. However, cohesin also has several other functions and plays important roles in transcriptional regulation, DNA double strand break repair, and chromosome architecture thereby influencing gene expression and development in organisms from yeast to man. While most of these functions rely on protein–protein interactions, post-translational protein, as well as DNA modifications, non-coding RNAs are emerging as additional players that facilitate and modulate the function or expression of cohesin and its individual components. This review provides a condensed overview about the architecture as well as the function of the cohesin complex and highlights its multifaceted interplay with both short and long non-coding RNAs.

Eco1-dependent cohesin acetylation anchors chromatin loops and cohesion to define functional meiotic chromosome domains

Cohesin organizes the genome by forming intra-chromosomal loops and inter-sister chromatid linkages. During gamete formation by meiosis, chromosomes are reshaped to support crossover recombination and two consecutive rounds of chromosome segregation. Here we show that Eco1 acetyltransferase positions both chromatin loops and sister chromatid cohesion to organize meiotic chromosomes into functional domains in budding yeast. Eco1 acetylates the Smc3 cohesin subunit in meiotic S phase to establish chromatin boundaries, independently of DNA replication. Boundary formation by Eco1 is critical for prophase exit and for the maintenance of cohesion until meiosis II, but is independent of the ability of Eco1 to antagonize the cohesin-release factor, Wpl1. Conversely, prevention of cohesin release by Wpl1 is essential for centromeric cohesion, kinetochore monoorientation and co-segregation of sister chromatids in meiosis I. Our findings establish Eco1 as a key determinant of chromatin boundaries and cohesion positioning, revealing how local chromosome structuring directs genome transmission into gametes.

Vertebrate CTF18 and DDX11 essential function in cohesion is bypassed by preventing WAPL-mediated cohesin release

The alternative PCNA loader containing CTF18-DCC1-CTF8 facilitates sister chromatid cohesion (SCC) by poorly defined mechanisms. Here we found that in DT40 cells, CTF18 acts complementarily with the Warsaw breakage syndrome DDX11 helicase in mediating SCC and proliferation. We uncover that the lethality and cohesion defects of ctf18 ddx11 mutants are associated with reduced levels of chromatin-bound cohesin and rescued by depletion of WAPL, a cohesin-removal factor. On the contrary, high levels of ESCO1/2 acetyltransferases that acetylate cohesin to establish SCC do not rescue ctf18 ddx11 phenotypes. Notably, the tight proximity of sister centromeres and increased anaphase bridges characteristic of WAPL-depleted cells are abrogated by loss of both CTF18 and DDX11. The results reveal that vertebrate CTF18 and DDX11 collaborate to provide sufficient amounts of chromatin-loaded cohesin available for SCC generation in the presence of WAPL-mediated cohesin-unloading activity. This process modulates chromosome structure and is essential for cellular proliferation in vertebrates.