Meiotic chromosome structures constrain and respond to designation of crossover sites

Abstract

Crossover recombination events between homologous chromosomes are required to form chiasmata, temporary connections between homologues that ensure their proper segregation at meiosis I. Despite this requirement for crossovers and an excess of the double-strand DNA breaks that are the initiating events for meiotic recombination, most organisms make very few crossovers per chromosome pair. Moreover, crossovers tend to inhibit the formation of other crossovers nearby on the same chromosome pair, a poorly understood phenomenon known as crossover interference. Here we show that the synaptonemal complex, a meiosis-specific structure that assembles between aligned homologous chromosomes, both constrains and is altered by crossover recombination events. Using a cytological marker of crossover sites in Caenorhabditis elegans, we show that partial depletion of the synaptonemal complex central region proteins attenuates crossover interference, increasing crossovers and reducing the effective distance over which interference operates, indicating that synaptonemal complex proteins limit crossovers. Moreover, we show that crossovers are associated with a local 0.4-0.5-micrometre increase in chromosome axis length. We propose that meiotic crossover regulation operates as a self-limiting system in which meiotic chromosome structures establish an environment that promotes crossover formation, which in turn alters chromosome structure to inhibit other crossovers at additional sites.

Figure 1. SYP-1 partial depletion increases numbers of COSA-1 foci and chiasmata

(a) Immunofluorescence (IF) images of late pachytene nuclei from control or syp-1 partial RNAi worms. Scale bar represents 5 μm. (b) Dose-response graph depicting mean numbers of GFP::COSA-1 foci formed per nucleus in response to DSBs generated by increasing doses of γ-irradiation (Rad). See Methods for numbers of nuclei used; error bars indicate s.d. At >1000 Rad, both the mean numbers of foci and s.d. were increased in syp-1 RNAi relative to control. (c) Three-dimensionally rendered images of individual diakinesis bivalents comprising the mnT12 (X;IV) fusion chromosome pair. Dashed lines (white) indicate traced HTP-3 axes, with crossing of axes indicating chiasmata. Scale bar represents 1 μm.

Figure 2. SYP-1 partial depletion attenuates crossover interference

(a) Left images, projections of individual late pachytene nuclei containing the mnT12 fusion chromosome (depicted schematically at top right), which is identified by its associated HIM-8 focus; gray dotted line indicates the traced three-dimensional path of the mnT12 chromosome axis. Right images, representative computationally straightened mnT12 used for quantitative analyses in be and Figures 3-4. *, ** indicate the straightened chromosomes from the nuclei shown. Scale bars indicate 1 μm. (b) Graph indicating the percent of mnT12 with the indicated number of COSA-1 foci for control (n= 69) and syp-1 RNAi (n=115). (c) Scatterplot showing measured distances (μm) between COSA-1 foci on control mnT12 with 2 foci, on syp-1 RNAi mnT12 with 2 foci, and on all syp-1 RNAi mnT12 with multiple foci. Horizontal lines indicate the mean; error bars indicate s.d. (d) Graphs indicating the distributions of foci among 8 evenly-spaced intervals along the mnT12 axis (schematic at top) for the subsets of control (top graph) and syp-1 RNAi (bottom graph) mnT12 with the indicated numbers of COSA-1 foci. (e) Graph depicting the distributions of spacing between adjacent pairs of foci. Using the same 8-interval scheme as in (d), separation values are defined as the number of interval boundaries crossed before encountering the next focus (i.e. adjacent foci within the same interval on the same chromosome pair have a separation of 0, adjacent foci in consecutive intervals have a separation of 1, etc.). See Methods for numbers of mnT12 chromosomes used for (c-e).

Figure 3. SYP-1 partial depletion decreases crossover interference strength

(a) Graph of Interference strength (I) values for the indicated interval pairs, where I = 1- observed/expected (Methods); schematic (top) indicates division of mnT12 into 4 intervals for this analysis. (b) Graph showing best-fit probability density function curves generated when a gamma distribution was used to model the distribution of inter-focus distances; for this analysis, distances between adjacent foci were expressed as percent of axis length. See Methods for numbers of chromosomes used.

Figure 4. Crossover designation causes a local expansion of chromosome axis length

(a,b) Graphs plotting the relationship between number of COSA-1 foci and mean length (±s.e.m.) of the mnT12 chromosome axis. (a) Data for syp-1 RNAi and control at 25°C; extrapolated linear regression line was generated using the syp-1 RNAi data. (b) Data for spo-11 and spo-11/+ control at 20°C. (c) Graph plotting the relationship between mean number of COSA-1 foci and mean axis length (± s.e.m.) for unfused autosomes in spo-11, control, and syp-1 RNAi nuclei (Methods). (d) Scatterplot showing length measurements (from control nuclei at 25°C) for the axis segment extending from left end of mnT12 to the center of the HIM-8 focus for chromosomes that either lacked or had a COSA-1 focus in this segment, as depicted in images below the graph. Horizontal lines indicate mean; error bars indicate s.d. See Methods for numbers of chromosomes used.