A dCas9-Based System Identifies a Central Role for Ctf19 in Kinetochore-Derived Suppression of Meiotic Recombination

Abstract

In meiosis, crossover formation between homologous chromosomes is essential for faithful segregation. However, misplaced meiotic recombination can have catastrophic consequences on genome stability. Within pericentromeres, crossovers are associated with meiotic chromosome missegregation. In organisms ranging from yeast to humans, pericentromeric crossovers are repressed. We previously identified a role for the kinetochore-associated Ctf19 complex (Ctf19c) in pericentromeric crossover suppression. Here, we develop a dCas9/CRISPR-based system that allows ectopic targeting of Ctf19c-subunits. Using this approach, we query sufficiency in meiotic crossover suppression, and identify Ctf19 as a mediator of kinetochore-associated crossover control. The effect of Ctf19 is encoded in its NH2-terminal tail, and depends on residues important for the recruitment of the Scc2-Scc4 cohesin regulator. This work provides insight into kinetochore-derived control of meiotic recombination. We establish an experimental platform to investigate and manipulate meiotic crossover control. This platform can easily be adapted in order to investigate other aspects of chromosome biology.

Novel mechanistic insights into the role of Mer2 as the keystone of meiotic DNA break formation

“…For Western blot analysis, protein lysates from yeast meiotic cultures were prepared using TCA precipitation and run on 8% or 10% SDS gels, transferred for 90 min at 300 mA and blotted with the selected antibodies, as described ( Kuhl et al, 2020 ). Primary antibodies with respective dilutions were used: rabbit α-Hop1 (made in-house; 1:10,000), rabbit α-Mer2 40-271 (made in-house; 1:10,000); mouse α-Pgk1 (Thermo Fisher, 1:5000); rabbit α-phospho-Histone-H3-Thr11 (Abcam, 1:1000), mouse α-HA (Biolegend, diluted 1:500), α-Myc-9E11 (Abcam, ab56, 1:1000).…”

Targeted assembly of ectopic kinetochores to induce chromosome‐specific segmental aneuploidies

“…Second, our approach could be considered an accurate model of cancer chromosome mis‐segregation, by mimicking the formation of dicentric chromosomes (Gisselsson et al , 2000 ; Mackinnon & Campbell, 2011 ), and faulty kinetochore‐microtubule attachments (Cimini et al , 2001 ; Thompson & Compton, 2008 ). Furthermore, our method provides a tuneable system to further investigate kinetochore assembly regulation, to complement previous studies (Gascoigne et al , 2011 ; Hori et al , 2013 ; Kuhl et al , 2020 ), for example allowing investigation of the impact of assembling ectopic kinetochores at differing locations or chromosomal contexts.…”

“…Nuclease‐dead CRISPR‐Cas9 (dCas9) has been extensively used for imaging purposes (Chen et al , 2013 ; Ma et al , 2015 ; Qin et al , 2017 ; Stanyte et al , 2018 ) and also for recruiting functional proteins to the genome, such as transcription factors (Tanenbaum et al , 2014 ; Joung et al , 2017 ) or chromatin remodellers (Saunderson et al , 2017 ), as well as centromere protein CENP‐B (Dumont et al , 2020 ). This prompted us to test whether dCas9 could also nucleate the formation of functional kinetochores at ectopic loci in human cells, similar to what has been shown in S. cerevisiae (Kuhl et al , 2020 ). This would create a pseudodicentric chromosome that would be more susceptible to mis‐segregation due to simultaneous attachment to both centrosomes (Fig 1A ).…”

An enChIP system for the analysis of genome functions in budding yeast

“…After deposition of 3xFLAG-dCas9/pTEF1p-CYC1t at Addgene a few years ago, the coding sequence of 3xFLAG-Sp-dCas9 in this plasmid was successfully used for enChIP and other analyses in SK1-derived budding yeast strains [ 16 ]. We also succeeded in the locus-specific isolation of a target region when an enChIP analysis was performed with a gRNA targeting the CAN1 gene expressed by transformation with the p426-SNR52p-gRNA.CAN1.Y-SUP4t plasmid [ 15 ] ( Fig.…”