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 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.

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.

Chromosome Organization in Early Meiotic Prophase

One of the most fascinating aspects of meiosis is the extensive reorganization of the genome at the prophase of the first meiotic division (prophase I). The first steps of this reorganization are observed with the establishment of an axis structure, that connects sister chromatids, from which emanate arrays of chromatin loops. This axis structure, called the axial element, consists of various proteins, such as cohesins, HORMA-domain proteins, and axial element proteins. In many organisms, axial elements are required to set the stage for efficient sister chromatid cohesion and meiotic recombination, necessary for the recognition of the homologous chromosomes. Here, we review the different actors involved in axial element formation in Saccharomyces cerevisiae and in mouse. We describe the current knowledge of their localization pattern during prophase I, their functional interdependence, their role in sister chromatid cohesion, loop axis formation, homolog pairing before meiotic recombination, and recombination. We also address further challenges that need to be resolved, to fully understand the interplay between the chromosome structure and the different molecular steps that take place in early prophase I, which lead to the successful outcome of meiosis I.

Towards a Structural Mechanism for Sister Chromatid Cohesion Establishment at the Eukaryotic Replication Fork

Cohesion between replicated chromosomes is essential for chromatin dynamics and equal segregation of duplicated genetic material. In the G1 phase, the ring-shaped cohesin complex is loaded onto duplex DNA, enriching at replication start sites, or “origins”. During the same phase of the cell cycle, and also at the origin sites, two MCM helicases are loaded as symmetric double hexamers around duplex DNA. During the S phase, and through the action of replication factors, cohesin switches from encircling one parental duplex DNA to topologically enclosing the two duplicated DNA filaments, which are known as sister chromatids. Despite its vital importance, the structural mechanism leading to sister chromatid cohesion establishment at the replication fork is mostly elusive. Here we review the current understanding of the molecular interactions between the replication machinery and cohesin, which support sister chromatid cohesion establishment and cohesin function. In particular, we discuss how cryo-EM is shedding light on the mechanisms of DNA replication and cohesin loading processes. We further expound how frontier cryo-EM approaches, combined with biochemistry and single-molecule fluorescence assays, can lead to understanding the molecular basis of sister chromatid cohesion establishment at the replication fork.

Induction of the ALT pathway requires loss of ATRX-DAXX in concert with genotoxic lesions at telomeres

A key requisite for indefinite growth of cancer cells is the ability to continuously elongate telomeres to circumvent the onset of senescence or apoptosis. In approximately 10 – 15% of cancers this is achieved through the Alternative Lengthening of Telomeres (ALT) pathway, a Break Induced Replication (BIR) mediated mechanism of telomere copying. ATRX has emerged as the key tumour suppressor in ALT cancers but its loss is insufficient to drive induction of the pathway. Here, we report that depletion of ATRX and/or DAXX in the presence of various genotoxic agents is sufficient to induce ALT. Moreover, co-deletion of ATRX and SETD2, commonly mutated in high grade gliomas (HGGs), elicits induction of ALT. Mechanistically, SETD2 restricts the accumulation of telomeric R-loops, which, in the absence of ATRX, leads to fork collapse and the loss of telomere sister chromatid cohesion. Cumulatively this provides a substrate for out of register BIR and telomere lengthening.