Extended Data Fig. 6: Expression of Spo11 and Rec8 reduces levels of CenpA at functioning KTs.
From: Centromeres are dismantled by foundational meiotic proteins Spo11 and Rec8
a, b, ChIP analysis of Cnp1CenpA at centromeric central core region. Endogenously 3xFlag-tagged Cnp1CenpA was immunoprecipitated with anti-Flag antibody and analysed by Real-Time qPCR using a primer pair annealing to the central cores of chromosomes I and III. Enrichment level is normalized against that of act1+, and then with that of a control wt strain with no tag on Cnp1CenpA. Spo11 was expressed under control of nmt41 (spo11-OE1) and cells were grown under conditions that induce nmt41 (minimal media). Mitotic Cnp1CenpA enrichment at the central core is reduced upon expression of Spo11 (a) or Rec8 (b), as is the case for cells overexpressing Spo11 under the adh1 promoter, or Rec8 under adh1 control, in rich media (Fig. 4b). The values for each biological replicate are shown as grey dots. Mean enrichment and standard deviations are shown in bars. c–e, Expression of Spo11 and Rec8 confers overall reduction of KT components on properly segregating chromosomes. Dotplots of Cnp1CenpA-GFP, Swi6HP1-GFP and Ndc80-GFP intensity on segregated chromosomes (see Methods). Anaphase cells without lagging chromosomes, as exemplified in the images atop each graph, were analysed, using the same strains as in Fig. 4. Arrows indicate foci whose intensities are measured. Each dot in the plot represents a distinct centromere focus. Mean intensity and standard deviation are shown for a representative experiment of two or more performed. Spo11 or Rec8 expression reduces Cnp1CenpA and Ndc80 intensity (c, e) on properly segregated anaphase centromeres, but modestly increases Swi6HP1-GFP intensity (d). P values are determined by Kolmogorov–Smirnov tests and indicated above brackets. Scale bars, 5 μm.
