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Volume 146, Issue 3, Pages (August 2011)

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Presentation on theme: "Volume 146, Issue 3, Pages (August 2011)"— Presentation transcript:

1 Volume 146, Issue 3, Pages 372-383 (August 2011)
Spo11-Accessory Proteins Link Double-Strand Break Sites to the Chromosome Axis in Early Meiotic Recombination  Silvia Panizza, Marco A. Mendoza, Marc Berlinger, Lingzhi Huang, Alain Nicolas, Katsuhiko Shirahige, Franz Klein  Cell  Volume 146, Issue 3, Pages (August 2011) DOI: /j.cell Copyright © 2011 Elsevier Inc. Terms and Conditions

2 Cell  , DOI: ( /j.cell ) Copyright © 2011 Elsevier Inc. Terms and Conditions

3 Figure 1 Axis Proteins Bind Identical Sites in vivo
ChIPchip profiles of Zip1 (black, FK1), Rec8-HA3 (green, FK1091), V5-Red1 (red, FK3870), and Hop1 (blue, FK3307) for chromosome 3. Black bars, DSB sites (in arbitrary scale) (Baudat and Nicolas, 1997). For all profiles in this work ChIP/whole-cell extract (WCE), signal intensity was plotted against the chromosomal position after denoising and after decile normalization (the 10% lowest values set below 1, see the Experimental Procedures). Numbers represent the result of genome-wide comparison between pairs of profiles, presented in all figures in identical format: names of the profiles; number of matching peaks/number of peaks of weaker profile and percent peak overlap; overlap between valleys (local minima) of profile 1 and peaks of profile 2; p, the probability to accept a random, hypergeometric model, and Pcorr, Pearson correlation between the two profiles. Cells were collected 4 hr after transfer to SPM (t4). A filled oval marks the centromere 3 (CEN3), and a red arrow marks a transposon. See also Table S2. Cell  , DOI: ( /j.cell ) Copyright © 2011 Elsevier Inc. Terms and Conditions

4 Figure 2 DSB-Promoting Factors Map to Cohesin Sites, Rather Than DSB Hotspots in DSB-Rich Regions (A) ChIPchip profiles of Rec114-myc13 (FK3307, red, repeat R1), Mer2-myc9 (FK3222, blue), and Mei4-HA6 (FK2385, green), all t4. Genome-wide correlation coefficients are shown in Figure S1A. Regions where qPCR was performed, centromere and transposon positions are marked. Black bars (in arbitrary scale) mark the positions of strong DSB sites (Baudat and Nicolas, 1997). (B) ChIPchip profiles of Rec8-HA3 (FK1091, t4, green), Rec114-myc13 (FK3307, t4, red, repeat R2), Hop1 (FK3307, t4, blue), and Rec104-myc9 (FK4370, t4, ochre) are shown for chromosome 3. Black bars, DSB sites (Baudat and Nicolas, 1997). (C) qPCR results of ChIP of Rec114-myc (FK3307) at four positions on chromosome 3 over a meiotic time course. Core 1 is a Rec8-binding site at 219.5 kb, ADP1 lies in a DSB-cold region (136.5 kb), and DSB1 and DSB3 are hotspots at positions 212 and kb. The mean ± standard variation (σ) for the ratios of n biological repeats is indicated. (D) The mean Rec114-myc13 (FK3307, t4), Hop1 (FK3307, t4), V5-Red1 (FK3870, t4), and CO (Mancera et al., 2008) signal per chromosome was plotted against chromosome length. r is the slope of the regression line for the 13 larger chromosomes, and p is the probability to accept that regression model for the three smallest chromosomes. (E) DSBs (Buhler et al., 2007) (blue) and Rec114-myc13 profile (FK3307, 4 hR; red) were smoothed at a bandwidth of 3 kb and plotted for chromosome II. Pcorr(10kbsw), genome-wide Pearson correlation, calculated for 10 kb regions in 5 kb steps and averaged (see the Experimental Procedures). (F) At a bandwidth of 70 kb, a positive correlation between DSBs and Rec114 is found for the whole genome. The SC component Zip1 (green) does not show a positive correlation with DSBs. Because of the large sliding window, peak matching is not appropriate. See also Figures S1, S2, and S3. Cell  , DOI: ( /j.cell ) Copyright © 2011 Elsevier Inc. Terms and Conditions

5 Figure 3 Mer2, S-Cdk, and Mei4 Recruit Rec114 to Axial Sites in DSB-Rich Regions (A and B) Profile of Rec114-myc13 in wild-type and different mutant backgrounds: (A) wild-type (FK3307, blue), mer2Δ (FK3005, red), and mei4Δ (FK3006, brown) all at t3 (A) and (wild-type (FK3307, blue, t4), spo11-Y135F (FK2993, green, t4), rec104Δ (FK2999, orange, t3), clb5Δ, clb6Δ (FK4196, black, t4) (B). A filled oval marks centromere 3. Genome-wide Pearson correlation coefficients are indicated. (C) qPCR on ChIP of Rec114-myc13 and Mei4-HA6 in MER2 mer2Δ (FK3219 and FK3214, respectively) and mer2-S30A mer2Δ cells (FK3220 and FK3215). The western blot in the right panel shows that mer2-S30A does not affect Rec114-myc levels. The mean ± σ for the ratios of n biological repeats is indicated. See also Figure S4. Cell  , DOI: ( /j.cell ) Copyright © 2011 Elsevier Inc. Terms and Conditions

6 Figure 4 Axial Element Components Hop1 and Red1 Recruit Mer2 to Axial Sites in DSB-Rich Regions (A and B) Profile of Mer2-myc9 in wild-type and various mutant backgrounds: wild-type (FK3222, blue), mei4Δ (FK3265, red), rec114Δ (FK3264, green) (A) and wild-type (FK3222, blue), mer2-S30A (FK4108, black), clb5Δ, clb6Δ (FK4099, orange), red1Δ (FK3858, lilac) (B). A filled oval marks centromere 3. Genome-wide Pearson correlation coefficients are indicated. (C) qPCR of ChIP of Mer2-myc9 of the same DNA used for ChIPchip. The ratio of the signals core 1:ADP1 is plotted for each experiment and the mean ± σ for the ratios of n biological repeats is indicated. See also Figure S5. Cell  , DOI: ( /j.cell ) Copyright © 2011 Elsevier Inc. Terms and Conditions

7 Figure 5 Cohesin Modifies the Chromosomal Distribution of Hop1 and Rec114 (A) Profile of Rec114-myc13 in wild-type (FK3307, blue) and various mutant backgrounds: rec8Δ (FK3683, green), hop1Δ (FK3016, light blue), and red1Δ (FK3466, lilac), all at t4. (B) Profile of Hop1 in wild-type (FK3307, orange) and rec8Δ (FK3683, black) at t4. The blue arrow points to a domain in which wild-type Rec114 and Hop1 signals dominate (blue and orange peaks). The green arrow points to a domain where these signals dominate in rec8Δ (green and black peaks). (C) qPCR of Rec114-myc ChIP at three positions in wild-type (left), and hop1Δ cells (right), at 3 and 4 hr in SPM. The ratio of the signals core 1:ADP1 is plotted for each experiment and the mean ± σ for the ratios of n biological repeats is indicated. (D) qPCR of Rec114-myc ChIP at two positions in wild-type (left), or rec8Δ cells (right) and of a Hop1 ChIP at two positions in wild-type (left), or rec8Δ cells (right), at 4 hr in SPM. See also Figure S6. Cell  , DOI: ( /j.cell ) Copyright © 2011 Elsevier Inc. Terms and Conditions

8 Figure 6 Model of Axis Tethering of the DSB Machinery
(A) After premeiotic DNA replication, cohesin (brown rings), axial element components Hop1 and Red1 (gray and green filled circles), and the pre-DSB recombinosome subunits Mer2, Rec114, and Mei4 (red, blue, and violet filled circles) bind to axis sites (blue dotted lines). One particular site may be occupied by a subset of these components at a given time. Hotspots (blue stars) are located between axis sites. Mer2 recruits Rec114 and Mei4 upon phosphorylation of its S30 by S-Cdk. (B) Ordered, linear arrays of loops (blue) emerge after condensation and sister chromatids are conjoined in the developing axis. Spo11-containing pre-DSB recombinosome is anchored at the axis and interacts for cleavage with one of the surrounding hotspots. Two preconditions will lead to competition between neighboring hotspots. First, based on this work, the pre-DSB recombinosome is not freely diffusible and becomes a locally limited resource, so that only a single hotspot can be cleaved at a time. Second, because of the presence of DNA damage response mediators, inactivation of the cleavage activity (dotted line) may quickly follow the first DSB, likely inhibiting further cleavage of the same and other hotspots. This effect would exacerbate competition between surrounding hotspots. A strict limitation to a single cleavage by the DSB recombinosome will also protect the sister-hotspot from being cleaved at the same time. Cell  , DOI: ( /j.cell ) Copyright © 2011 Elsevier Inc. Terms and Conditions

9 Figure S1 Binding Sites of DSB-Promoting Factors Correlate with Cohesin Sites, but Not with Hotspots, Related to Figure 2 (A) Pair-wise genome-wide comparisons between profiles shown in Figures 2A and 2B. Rec114 R1 and R2 represent two independent experiments. (B) qPCR on Mer2-myc9 (FK3222) ChIP over a meiotic time-course. Primer positions are shown in Figure 2A. (C) ChIPchip profiles of Rec8-HA (FK1091, green), Rec102-myc (FK3837, orange) and Rec104-myc (FK4370, black), all t4. DSB positions according to Baudat (gray bars) are added, to demonstrate that also these components bind to cohesin sites, rather than to hotspots. Complete datasets for all supplemental datasets are available online. Cell  , DOI: ( /j.cell ) Copyright © 2011 Elsevier Inc. Terms and Conditions

10 Figure S2 Sites of Covalently Attached Spo11 in rad50S Correlate with DSB Sites at Different Resolutions, and Hop1 and Rec114 Sites Correlate at 500bp Resolution, Related to Figure 2 (A and B) as in (Figures 2E and 2F), but showing Spo11-myc in rad50S (FK1371, t6, red) pulled down in the absence of cross-linking instead of Rec114-myc13. The resolution is selected by varying the sliding window. (C) ChIPchip profiles of Hop1 and Rec114 from the same experiment as described in Figure 2B, but after much less smoothing (500 bp sliding window) for seven chromosomes are displayed. Cell  , DOI: ( /j.cell ) Copyright © 2011 Elsevier Inc. Terms and Conditions

11 Figure S3 Rec114 Foci Are Distinct from Early Foci of Zip1, Related to Figure 2 (A) Co-staining of Rec114 (red) and Zip1 (green) on a leptotene nucleus (FK3307). (B) The number of Rec114 foci peaks in late leptotene. pM premeiotic, L, eL, mL, lL: early-, medium- and late leptotene, Z, eZ, lZ: early- and late zygotene. n is the number of nuclei analyzed, error bars correspond to the mean +/− σ. (C) Example of two nuclei with partial synapsis (late zygotene), showing that Rec114 staining is predominant in unsynapsed regions (red arrowhead). White arrowhead marks ribosomal DNA. Cell  , DOI: ( /j.cell ) Copyright © 2011 Elsevier Inc. Terms and Conditions

12 Figure S4 ChIP of Rec114-myc13 in Several Mutants, Related to Figure 3
qPCR of Rec114-myc13 ChIP in three different mutants over meiotic time-courses. Primer positions on chromosome 3 are shown in Figure 2A. Time-points are indicated by numbers and/or color-code. Average and error bars representing biological repeats (+/−σ) were added to bars, if available. (A) The enrichment (ratio between a binding site (core1) and the negative control (cold)) is plotted. Wild-type (blue, t4, FK3307), mer2Δ (shades of red indicating different time-points, FK3005), rec104Δ (shades of red, FK2999). (B) The absolute values of precipitated DNA in spo11Y135F cells (FK2993)at three different sites are plotted separately. A wild-type analyzed in parallel is shown in Figure 4C. Cell  , DOI: ( /j.cell ) Copyright © 2011 Elsevier Inc. Terms and Conditions

13 Figure S5 Mer2 Requires Axial Element Components but Not Replication or Rec114 to Bind to Axial Sites, Related to Figure 4 (A and B) confirm and extend results shown in Figure 5, using a different tag (Mer2-HA3, see extended experimental procedures). (A) Profile of Mer2-HA3 in wild-type (FK3775, blue) and various mutant backgrounds: rec114Δ (FK4005, green), hop1Δ (FK4006, orange), red1 (FK3972, lilac) at t4. Centromere 3, the transposon and hotspots (Baudat) are indicated. (B) qPCR analysis of the experiments shown in (A). The enrichment (ratio between a strong binding site (core 1) and the negative control (cold)) is plotted. n is the number of biological repeats, error bars correspond to the mean +/− σ of the ratios. (C) Comparisons between profiles of t3 and t4 of wild-type (FK3222, blue, black), mer2-S30A-myc9 (FK4108, light-, dark green), clb5Δ, clb6Δ (FK4099, light-, dark red), vertical green lines indicate identical peaks between mer2-S30A-myc9 and wild-type, vertical red lines between and clb5Δ, clb6Δ wild-type. All major peaks are unaltered between 3 and 4h time points and in the absence or presence of replication (clb5Δ, clb6Δ). Centromere 3, positions of delta elements (deltas) and hotspots (Baudat) are indicated. Cell  , DOI: ( /j.cell ) Copyright © 2011 Elsevier Inc. Terms and Conditions

14 Figure S6 A Subset of Cohesin-Binding Sites Are Recognized by Hop1 and Rec114 in the Absence of Rec8, Related to Figure 5 (A) Profiles of Hop1 and Rec114-myc from the same experiments as in Figure 6, for chromosome 3. Hop1 (FK3307, orange), Hop1 rec8Δ (FK3683, red), Rec114-myc (FK3307, blue), Rec114-myc rec8Δ (FK3683, green), all at t4. Matching peaks between wild-type and rec8Δ are indicated by vertical lines, red for Hop1 and blue for Rec114. (B) Profiles and overlap of Rec114 and Hop1 peaks on Chromosome 3 in rec8Δ cells from two independent experiments (R1 and R2 mark repeats 1 and 2). Matching peaks are again indicated by vertical lines. Genome-wide comparison statistics are provided. The numbers of detected peaks are indicated for all four profiles. Note that in the slightly weaker profile (Rec114, R2) 150 peaks are lost, because they fall below the threshold. Pcorr does not suffer from this “threshold effect” and better reflects the high similarity (Pcorr = 0.96). (C) The enrichment (ratio between a binding site (core1) and the negative control (cold)) of qPCR upon ChIP of Rec114 and Hop1 is plotted. n is the number of biological repeats, error bars correspond to the mean +/− σ of the ratios. Primer positions shown in Figure 2A. Left panel: qPCR of ChIP of untagged Rec114 using anti-Rec114 antibody. wt (FK1, blue), red1 (FK4503, brown), hop1 (FK21, black), rec8 (FK1067, green), mer2S30A (FK4500, lilac), all at t4. Right part: qPCR of ChIP of Hop1. wt (FK1, orange), rec8Δ (FK1067, red), all at t4. Note that Hop1 and Rec114 IPs have different scales. Cell  , DOI: ( /j.cell ) Copyright © 2011 Elsevier Inc. Terms and Conditions


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