A dCas9/CRISPR-based targeting system identifies a central role for Ctf19 in kinetochore-derived suppression of meiotic recombination

Abstract

27In meiosis, crossover formation between homologous chromosomes is essential for 28 faithful segregation. However, improperly controlled or placed meiotic recombination can 29 have catastrophic consequences on genome stability. Specifically, within centromeres and 30 surrounding regions (i.e. pericentromeres), crossovers are associated with chromosome 31 missegregation and developmental aneuploidy. In organisms ranging from yeast to 32 humans, crossovers are repressed within (peri)centromeric regions. We previously 33 identified a key role for the multi-subunit, kinetochore-associated Ctf19 complex (Ctf19c; 34 the budding yeast equivalent of the human CCAN) in regulating pericentromeric crossover 35 formation. Here, we develop a dCas9/CRISPR-based system that allows ectopic targeting 36 of Ctf19c-subunits to a non-centromeric locus during meiosis. Using this approach, we 37 query sufficiency in meiotic crossover suppression, and identify Ctf19 (the budding yeast 38 homologue of vertebrate CENP-P) as a central mediator of kinetochore-associated 39 crossover control. We show that the effect of Ctf19 is encoded in its NH 2 -terminal tail, and 40 depends on residues known to be important for the recruitment of the Scc2-Scc4 cohesin 41 regulator to kinetochores. We thus reveal a crucial determinant that links kinetochores to 42 meiotic recombinational control. This work provides insight into localized control of 43 meiotic recombination. Furthermore, our approach establishes a dCas9/CRISPR-based 44 experimental platform that can be utilized to investigate and locally manipulate meiotic 45 crossover control. This platform can easily be adapted in order to investigate other aspects 46 of localized chromosome biology. 47 48 49 50 Introduction 51Faithful chromosome segregation in meiosis requires physical connections between 52 initially unpaired homologous chromosomes (Petronczki, et al., 2003). Such linkages are 53 established through homologous recombination (HR) mediated repair of programmed DNA 54 double strand breaks (DSBs) (Keeney, 2001). Sequences that can act as HR repair templates for 55 DSB lesions can be found on the sister chromatid and the homologous chromosome, but only 56 repair that uses the homologous chromosome as a template can result in the reciprocal exchange 57 of flanking chromosomal arm regions of homologous chromosomes, yielding a crossover. A 58 crossover, together with cohesin that is laid down distally to the recombination site, establishes 59 the connection between homologs required for successful chromosome segregation in meiosis. 60The placement of crossovers is determined by the location of DSB activity and by repair 61 decisions after DSB formation. Certain regions in the genome represent a high risk to genome 62 stability when faced with DSB repair or CO formation, and molecular systems are in place to 63 spatially control CO placement and thereby guard genomic stability during meiosis. 64 Centromeres are the regions of the chromosomes where kinetochores are nucleated. Kinetochores 65 a...

The proteomic landscape of centromeric chromatin reveals an essential role for the Ctf19CCANcomplex in meiotic kinetochore assembly

“…Ctf19c CCAN is critical both for preventing pericentromeric crossovers (Vincenten et al, 2015), and for maintaining cohesive linkages between sister chromatids until meiosis II (Fernius and Marston, 2009;Mehta et al, 2014). Targeted Ctf19c CCANdependent cohesin loading at centromeres contributes to both pericentromeric crossover suppression (Kuhl et al, 2020) and meiosis II chromosome segregation ( Figure 3I-K). However, we find that defective pericentromeric cohesin cannot fully explain the profound meiotic chromosome segregation defects observed in Ctf19c CCAN -deficient meiosis.…”