Epitope tag-specific differences in the detection of COSA-1 marked crossover sites in C. elegans spermatocytes

6 jpagesNascent crossover sites in C. elegans meiocytes can be cytologically detected using epitope-tagged versions of the pro-crossover protein COSA-1. In spermatocytes, differences exist between cytologically-detected and genetically-detected double crossover rates. Here, we examine nascent crossovers using both GFP- and OLLAS-tagged COSA-1. Similar to previous work, we find that most late pachytene spermatocytes display 5 COSA-1 foci, indicating one crossover per autosome bivalent. However, we detected more nuclei with >5 COSA-1 foci using OLLAS::COSA-1, reflecting some bivalents having 2 COSA-1 foci. These results demonstrate tag-specific differences in the detection of COSA-1 marked nascent crossovers in spermatocytes.This work was supported by the National Institutes of Health R35GM128890 to DEL, a Jane Coffin Childs Postdoctoral Fellowship and National Institutes of Health 1K99HD109505-01 to CKC, National Institutes of Health R35GM126964 to AMV, and a Stanford Dean’s Fellowship award to CJU. DEL is also a Searle Scholar and recipient of a March of Dimes Basil O’Connor Starter Scholar award. SIM imaging was performed at Stanford University Cell Sciences Imaging Core Facility, which is supported by Award Number 1S10OD01227601 from the National Center for Research Resources

Diagram of events in premeiotic germline with respect to autosome pairing, centromere clustering, and SC formation based on the results from Joyce et al. [2] and Christophorou et al. [1].

<p>(We thank Angela Seat for figure and editorial assistance.)</p

Multiple opposing constraints govern chromosome interactions during meiosis.

Homolog pairing and crossing over during meiosis I prophase is required for accurate chromosome segregation to form euploid gametes. The repair of Spo11-induced double-strand breaks (DSB) using a homologous chromosome template is a major driver of pairing in many species, including fungi, plants, and mammals. Inappropriate pairing and crossing over at ectopic loci can lead to chromosome rearrangements and aneuploidy. How (or if) inappropriate ectopic interactions are disrupted in favor of allelic interactions is not clear. Here we used an in vivo "collision" assay in budding yeast to test the contributions of cohesion and the organization and motion of chromosomes in the nucleus on promoting or antagonizing interactions between allelic and ectopic loci at interstitial chromosome sites. We found that deletion of the cohesin subunit Rec8, but not other chromosome axis proteins (e.g. Red1, Hop1, or Mek1), caused an increase in homolog-nonspecific chromosome interaction, even in the absence of Spo11. This effect was partially suppressed by expression of the mitotic cohesin paralog Scc1/Mdc1, implicating Rec8's role in cohesion rather than axis integrity in preventing nonspecific chromosome interactions. Disruption of telomere-led motion by treating cells with the actin polymerization inhibitor Latrunculin B (Lat B) elevated nonspecific collisions in rec8Δ spo11Δ. Next, using a visual homolog-pairing assay, we found that the delay in homolog pairing in mutants defective for telomere-led chromosome motion (ndj1Δ or csm4Δ) is enhanced in Lat B-treated cells, implicating actin in more than one process promoting homolog juxtaposition. We suggest that multiple, independent contributions of actin, cohesin, and telomere function are integrated to promote stable homolog-specific interactions and to destabilize weak nonspecific interactions by modulating the elastic spring-like properties of chromosomes

Multiple Opposing Constraints Govern Chromosome Interactions during Meiosis

<div><p>Homolog pairing and crossing over during meiosis I prophase is required for accurate chromosome segregation to form euploid gametes. The repair of Spo11-induced double-strand breaks (DSB) using a homologous chromosome template is a major driver of pairing in many species, including fungi, plants, and mammals. Inappropriate pairing and crossing over at ectopic loci can lead to chromosome rearrangements and aneuploidy. How (or if) inappropriate ectopic interactions are disrupted in favor of allelic interactions is not clear. Here we used an <em>in vivo</em> “collision” assay in budding yeast to test the contributions of cohesion and the organization and motion of chromosomes in the nucleus on promoting or antagonizing interactions between allelic and ectopic loci at interstitial chromosome sites. We found that deletion of the cohesin subunit Rec8, but not other chromosome axis proteins (e.g. Red1, Hop1, or Mek1), caused an increase in homolog-nonspecific chromosome interaction, even in the absence of Spo11. This effect was partially suppressed by expression of the mitotic cohesin paralog Scc1/Mdc1, implicating Rec8's role in cohesion rather than axis integrity in preventing nonspecific chromosome interactions. Disruption of telomere-led motion by treating cells with the actin polymerization inhibitor Latrunculin B (Lat B) elevated nonspecific collisions in <em>rec8</em>Δ <em>spo11</em>Δ. Next, using a visual homolog-pairing assay, we found that the delay in homolog pairing in mutants defective for telomere-led chromosome motion (<em>ndj1</em>Δ or <em>csm4</em>Δ) is enhanced in Lat B–treated cells, implicating actin in more than one process promoting homolog juxtaposition. We suggest that multiple, independent contributions of actin, cohesin, and telomere function are integrated to promote stable homolog-specific interactions and to destabilize weak nonspecific interactions by modulating the elastic spring-like properties of chromosomes.</p> </div

DSB–independent ectopic collision levels in mutants with defects in centromere coupling and bouquet formation.

<p>A. Analysis of collisions in <i>spo11Δ, spo11Δ ndj1Δ, spo11Δ zip1Δ, spo11Δ ndj1Δ zip1Δ, spo11Δ csm4Δ</i> and <i>spo11Δ csm4Δ ndj1Δ</i> mutants with Lat B treatment. Graph parameters are as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003197#pgen-1003197-g003" target="_blank">Figure 3</a>. B. Heatmap of Sidak adjusted <i>P</i>-values from Student's t-test comparing collision levels between relevant mutants in untreated and Lat B treated cells.</p

Elevated levels of nonspecific collisions in <i>rec8</i>Δ mutants do not require recombination initiation.

<p>A. Analysis of collisions in <i>spo11</i>Δ, <i>spo11</i>Δ <i>rec8</i>Δ, <i>spo11</i>Δ <i>prec8-SCC1, spo11</i>Δ <i>ndj1</i>Δ, and <i>spo11</i>Δ <i>ndj1Δ rec8</i>Δ mutants with Lat B treatment. Allelic (blue) and ectopic (red) collision levels in untreated cultures (dark bars) and Lat B treatment (light bars). Asterisks denote significant differences as follows: (*), <i>P</i>-values between 0.05 and 0.01; (**), <i>P</i>-values between 0.01 and 0.001; (***), <i>P</i>-values <0.001 by a two-tailed Student's t-test. B. Heatmap of Sidak adjusted <i>P</i>-values from Student's t-test comparing collision levels between relevant mutants in untreated and Lat B treated cells.</p

A mechanical model for homolog pairing.

<p>A hypothetical sequence of interactions between homologous chromosomes (shown as black or red) subjected to a coupled-spring oscillator (see text). In the sequence from left to right, homolog pairing becomes progressively stabilized as weak interactions are disrupted. The positive and negative forces of actin influence both homologs, with actin-based associations shown at telomeres (green; squares for the red chromosome and circles for the black homologous chromosome) and at interstitial sites (blue; squares for the red chromosome and circles for the black homologous chromosome). Arrows around the periphery of the nucleus indicate direction of movement for the telomeres (green) and the interstitial sites (blue). Grey arrows in the interior of the nucleus show “Brownian-like” motion/unknown forces on the chromosomes <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003197#pgen.1003197-Marshall1" target="_blank">[105]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003197#pgen.1003197-Heun1" target="_blank">[119]</a>. (i) In wild-type cells, segments of chromosomes that are in closer proximity have axial segments that are more compact. (ii) Compact segments of homologous chromosomes interact. (iii) Movement of chromosome attachment points on the nuclear envelope results in stretching of segments that remove unstable interactions between chromosomes. (iv) Stable interactions between allelic loci are those achieved up to the point of dHJ resolution as established using the Cre/<i>loxP</i> assay <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003197#pgen.1003197-Peoples1" target="_blank">[21]</a>.</p

Rec8 promotes allelic collisions independent of its role in sister chromatid cohesion and prevents nonspecific collisions.

<p>A. Analysis of allelic and ectopic collision levels as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003197#pgen-1003197-g001" target="_blank">Figure 1</a>. B. Heatmap of Sidak adjusted <i>P</i>-values comparing collision levels between all allelic collision levels (left) and all ectopic collision levels (right).</p

Elevated levels of nonspecific collisions in <i>rec8</i>Δ do not require Ndj1-dependent telomere attachments to the NE.

<p>A. Analysis of allelic and ectopic collision levels in <i>rec8Δ</i>, <i>ndj1</i>Δ, <i>ndj1</i>Δ <i>rec8</i>Δ, <i>csm4</i>Δ, and <i>ndj1</i>Δ <i>csm4</i>Δ mutants with Lat B treatment. Graph parameters are as described as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003197#pgen-1003197-g003" target="_blank">Figure 3</a>. B. Heatmap of Sidak adjusted <i>P</i>-values from Student's t-test comparing collision levels between relevant mutants in untreated and Lat B treated cells.</p