Topoisomerases modulate the timing of meiotic DNA breakage and chromosome morphogenesis inSaccharomyces cerevisiae

Abstract

During meiotic prophase, concurrent transcription, recombination, and changes in chromosome morphology, including chromosome synapsis, place substantial topological strain on chromosomal DNA. However, the roles of topoisomerases in this context remain poorly defined. Here, we show that meiotic Saccharomyces cerevisiae chromosomes accumulate topoisomerases primarily in promoter-containing intergenic regions (IGRs) of actively transcribing genes. Wide IGRs exhibit the highest level of meiotic transcription and the strongest topoisomerase buildup. Topoisomerase binding partially overlaps with double-strand break (DSB) hotspots, where the topoisomeraselike enzyme Spo11 initiates meiotic recombination. We show that TOP1 disruption delays DSB induction and shortens the window of DSB accumulation. By contrast, inactivation of TOP2 causes delayed DSB turnover. Furthermore, persistent binding of catalytically inactive Top2, as observed in top2-1 mutants, uncouples chromosome synapsis from Pch2/TRIP13-dependent chromosome remodeling. Thus, topoisomerases function at multiple points to modulate the timing of meiotic recombination.

Active transcription and Orc1 drive chromatin association of the AAA+ ATPase Pch2 during meiotic G2/prophase

“…[63], which might influence large-scale topological organization of loop-axis structures, and Pch2 589 localization. Indeed, previous work has identified a connection between topoisomerase II function, 590 meiotic chromosome reorganization and Pch2 recruitment [23,64]. It will be interesting to further 591 understand the link between transcription, chromosome topology and organization and Pch2 recruitment.…”

“…All peaks were manually verified prior to analysis 209 aiming to exclude those which were not present in at least two of the three technical replicates. For RNA-210 seq data analyses shown in Supplementary Figure 1C and D, reads obtained from (GSE131994, 3 hours in 211 meiosis) [23], were mapped to the SK1 genome background (SGRP) and analyzed using the RNA-seq 212 platform on CLC-genomic workbench. For analysis shown in figure Supplementary Figure 2A, peak 213 calling was performed by subtracting NLS-GFP ChIP-seq data (SRR2029413) [37] from the Pch2 ChIP-214 seq dataset.…”

“…Venn diagrams were generated using the web-based Venn diagram generator from 229 http://jura.wi.mit.edu/bioc/tools/venn.php. Pearson correlation was performed with datasets from the 230 CLC-genomics peak shape score for each Pch2 binding gene and their specific expression values obtained 231 from analysis of RNA-seq data [23] (Supplementary Figure 1D). To determine whether the Pch2 binding 232 genes were enriched in certain functional categories, gene ontology analysis was conducted using the 233 SGD GO term finder (molecular function) (https://www.yeastgenome.org/goTermFinder) at a p-value 234 cutoff of 0,01.…”

“…Analysis of genome-wide transcriptome (i.e. total RNA-317 seq) datasets from cells that synchronously progress through meiosis [23] showed that all of the genes 318 occupied by Pch2 are transcribed during meiosis, suggesting that transcription is involved in the 319 recruitment of Pch2 to these CDSs. To test if transcriptional "strength" of defined genes was predictive of 320…”

“…Inhibition of global transcription reduced Pch2 association to GAP1 gene 368 by ~50% compared to mock control (Supplementary Figure 3D), consistent with a role for transcription in 369 promoting the recruitment/association of Pch2 to regions of active transcription. To achieve a more 370 complete and specific inhibition of RNAPII, we employed the anchor-away technique [32], which has 371 been used to successfully deplete chromosomal proteins during meiosis [14,23,38,44]. This technique is 372 based on an inducible dimerization system that rapidly depletes nuclear proteins based on ribosomal flux, 373 with the aid of a tagged anchor-protein, Rpl13A (Rpl13a-2XFKBP12).…”