Protein determinants of meiotic DNA break hot spots

Abstract

Meiotic recombination, crucial for proper chromosome segregation and genome evolution, is initiated by programmed DNA double-strand breaks (DSBs) in yeasts and likely all sexually reproducing species. In fission yeast, DSBs occur up to hundreds of times more frequently at special sites, called hot spots, than in other regions of the genome. What distinguishes hot spots from cold regions is an unsolved problem, although transcription factors determine some hot spots. We report the discovery that three coiled-coil proteins-Rec25, Rec27, and Mug20-bind essentially all hot spots with great specificity even without DSB formation. These small proteins are components of linear elements, are related to synaptonemal complex proteins, and are essential for nearly all DSBs at most hot spots. Our results indicate these hot spot determinants activate or stabilize the DSB-forming protein Rec12 (Spo11 homolog) rather than promote its binding to hot spots. We propose a paradigm for hot spot determination and crossover control by linear element proteins.

Figure 1. Pathway of Meiotic DSB Formation and Repair in S. pombe

About the time of replication, loading of meiotic cohesin subunits Rec8 and Rec11 is followed by loading of LinE proteins Rec10, Rec25, Rec27, and Mug20 and loading or activation of Rec12 and its six partner proteins. Rec12 makes DSBs, becoming covalently linked to the DNA. Removal of Rec12 allows repair of the DSB with the sister chromatid or homolog. Repair with the homolog can form a crossover, which allows proper segregation of homologs at the first meiotic division. See also Figure S5.

Figure 2. Mug20 Is a LinE Component Interacting with Rec25, Rec27, and Rec10

In A and B, cells with the indicated GFP fusion protein and rec gene were synchronously induced for meiosis, fixed 3 hr later, stained with DAPI, and examined by fluorescence microscopy. Each set of three images shows the GFP protein (green; left), DAPI-stained DNA (blue; middle), and merge (right). In C live cells from zygotic meiosis were examined for Mug20-GFP (green; left), Rec25-tdTomato (red; middle), and both (right). See also Figures S1, S2, S3, and S4. (A) Mug20 forms nuclear foci that depend on LinE components Rec10, Rec25, and Rec27 but only partially on the sister chromatid cohesin Rec8. (B) Mug20 is required for nuclear focus-formation of Rec25 and Rec27 and, partially, of Rec10. (C) Mug20 and Rec25 co-localize in live zygotic cells.

Figure 3. Rec25, Rec27, and Mug20 Bind DNA at Meiotic DSB Hotspots with High Preference, but Rec8, Rec10, Rec11, and Rec12 Bind with Little or No Preference

DNA covalently linked to Rec12-FLAG (signifying DSBs; harvested at 5 hr, when DSBs are maximal) or DNA crosslinked to the indicated GFP fusion protein (harvested at 3.5 hr, when foci are prominent). was analyzed by microarray hybridization. Data are median-normalized, smoothed using an 11-probe window, and plotted with an offset for legibility. “Input” is whole cell extract. Complete genome data are on the Lab Websites. See also Figures S6 and S7. (A) LinE proteins Rec25, Rec27, and Mug20 (plotted on left axis; offsets of 3, 2, and 1, respectively) bind preferentially to DSB hotspots (right axis), but LinE protein Rec10 (left axis) binds nearly uniformly except for modest preference at strong hotspots. Black circles beneath the traces indicate wild-type hotspots (see Supplemental Methods for peak-calling criteria). (B) Sister cohesin subunits Rec8 and Rec11 (left axis; offsets of 1 and 0, respectively) bind nearly uniformly, as does the inactive Rec12-213 (Y98F) mutant protein (left axis; offset of 2). (C) DSBs are nearly eliminated in rec10Δ and are significantly reduced at most hotspots in rec27Δ a nd rec11Δ null mutants and the rec10-109 missense mutant (left axis; offsets of 0, 5, 10, and 15, respectively). (D) LinE protein Rec27 preferentially marks the DSB hotspots remaining in rec8Δ.

Figure 4. Rec25, Rec27, and Mug20 Binding Is Highly Correlated with Genome-wide DSB Frequencies, but Rec8, Rec11, and Rec12 Binding Is Not

(A and B) Scatter plots of the genome median-normalized data for the two parameters indicated on the axes. All data points (~44,000; in black) are plotted on a log₁₀ scale (IP/input), but most are obscured by their high density. Red data are points within DSB hotspots. Pearson correlation coefficients (r) are for unsmoothed data. See also Table S2. (C) Summary of r for unsmoothed data. Dark red indicates highest correlation, dark blue the lowest, and lighter values between these extremes. (D) Scatter plots and r from two rec8Δ inductions. (E) Scatter plot and r, as above, for Rec12-213 (Y98F) and Rec12⁺ proteins (Lab Websites). The Rec12⁺ data reflect both self-linkage (DSBs) and crosslinking (binding), but a positive correlation is still observed.

Figure 5. Correlations between DSB Frequencies and Protein Abundances at DSB Hotspots Are Especially Strong

Scatter plots and r for DSBs (integral above or below median of Rec12-DNA covalent linkages across hotspots) and the indicated protein similarly integrated. Points above the line indicate protein enrichment at that hotspot; points below imply depletion. Data are in arbitrary units on linear axes.

Figure 6. Rec27 Binds to DSB Hotspots in the Absence of DSB-formation by Rec12, Which Binds to DSB Hotspots with Only Modest Preference

(A) Rec27 binding in rec12⁺ (Figure 3, experiment 2, offset of 1) or in rec12Δ (offset of 2; analyzed concurrently with rec12⁺ in experiment 2). r = 0.77 for single probes and 0.81 for 11-probe smoothing (Figure 4C and Table S2). Complete genome data are on the Lab Websites. (B) Binding of inactive Rec12-213 (Y98F) as in Figure 3 is largely independent of Rec27 (Figure S8). Binding is higher in protein-coding genes (red and blue bars for upper- and lower-strand coding) and lower between genes. Inset: 4003 genes were aligned at their transcription start sites (TSS) and transcription end sites (TES) (Lantermann et al., 2010). Rec12-Y98F binding (red line) is higher in genes (black rectangle with arrow) than between genes, whereas mean DSBs (Rec12-DNA covalent linkages; black line) is higher between genes than in genes. Note recombinant frequency in ura1 (7 kb blue bar near 740 kb) is 19 times lower than genome average (Supplemental Information), although Rec12-Y98F binding is about twice the genome median. See also Figure S9.

Figure 7. Proposal for Crossover Control by Rec25-Rec27-Mug20

Rec10 (blue balls) binds across chromosomes, enriched at DSB hotspots. Rec25-Rec27-Mug20 complex (red oval) binds hotspots and activates Rec12 to make high-frequency DSBs and biases DSB repair toward the sister, giving a low crossover:DSB ratio. In DSB cold regions repair is biased toward the homolog, giving a high crossover:DSB ratio. The result is a nearly uniform distribution of crossovers across the genome (crossover invariance; Hyppa and Smith, 2010).