Figures and data
Distinct axis-protein enrichment patterns at chromosome ends.
(a) Schematic of chromosome-end architecture in S. cerevisiae. XY′ ends contain Y′ elements; X-only ends lack Y′ elements. We define the subtelomeric domains as encompassing the last 20 kb from chromosome ends; they thus also encompass any X or Y’ elements. The adjacent EARs extend 20-120kb from ends. (b) Mean enrichment of Rec8 (light blue), Red1 (red), and Hop1 (purple) versus distance from telomeres in wild type (WT) early prophase I (T = 3h) from published data 16,19,36, normalized to a genome average of 1 (gray dashed line; see Methods: Distance from telomeres plots). Range of the subtelomeric domains (SubTel) is indicated with a solid gray line, EARs are indicated with an orange line. (c) Genome-wide bootstrap distributions of fold-enrichment (32 × 20-kb windows; n = 1,000 resamples; see Methods: Bootstrapping plots). Gray dashed line is genome average. Black lines show medians and 95% CIs; orange/red circles mark the observed mean in the last 20 kb. Two-sided empirical test with Benjamini-Hochberg (BH) correction, effect sizes via Cohen’s d (negative = depletion at ends relative to the genome-wide null): Hop1 (p = 0.001; BH = 0.0015; d = −3.51); Red1 (p < 1x10-6; BH < 1 x 10-6; d = −5.23); Rec8 (p = 0.368; BH = 0.368; d = −0.89). (d) Metaplots anchored at X elements, stratified by end class. Only fully annotated X elements were used (X-only, n = 7; XY′, n = 20). Flanks scaled to element length (X: 100% each side). Gray dashed line is genome average, vertical dotted lines mark X boundaries; shaded bands indicate 95% confidence intervals (CI; see Methods: Meta gene analyses, and meta-X and Y’ elements plots). (e) Axis protein ChIP signal (Hop1, Red1, Rec8) at X elements on X-only versus XY′ ends. Values represent the mean ChIP/input signal per X element. Box-and-whisker plots show the distribution across elements. Two-sided unpaired Student’s t-tests with BH correction; stars reflect BH-adjusted p (* ≤ 0.05; n.s., not significant). Statistics (per X element; Cohen’s d; positive = higher at X-only): Hop1 (p = 0.0186; BH = 0.0186; d = 1.13); Red1 (p = 0.0058; BH = 0.0118; d = 1.77); Rec8 (p = 0.0079; BH = 0.0118; d = 1.62). (f) Metaplot anchored at Y′ elements. Only fully annotated Y′ were analyzed and flanks were scaled to 50% of Y′ length. Blue arrow indicates Y′-ORF orientation. Gray dashed line is genome average and vertical dotted lines mark Y′ boundaries. Shaded bands are 95% CIs (see Methods: Meta gene analyses, and meta-X and Y’ elements plots). Averages of two biological replicates.
Axis-protein depletion near telomeres is encoded in cis.
(a) Schematic showing the analyzed fusion chromosomes derived either from S288c or SK1 39. The subtelomeric sequences eliminated as part of the fusion process are indicated by grey bars, and the fusion sites, X and Y’ elements are marked. (b) Red1 binding (T = 4h) along telomere-proximal arms in SK1/S288C hybrids (chrIV-R and chrI-L), using data from 39. Tracks are shown for unfused (WT) and chrIV/I fused strains homozygous (hom.) or heterozygous (het) for the fused chromosomes with either CEN1 or CEN4 deleted (colors as indicated). Vertical dashed black line indicates engineered fusion sites. The plot only includes points common to all datasets, which is why the WT unfused data in the regions between the telomeres and the fusion sites are missing. Thin lines are genome-normalized Red1 tracks; thick lines are loess-smoothed overlays for trends (span = 1). Circle indicates CEN1. The dip in signal on the right side of chrI-L is because of deletion of CEN1 in some of the strains as previously described 39. (c) Example distance-from-telomere plots showing Red1 ChIP/Input signal along 3 matching chromosome arms in WT SK1/S288c hybrid strains, carrying a haploid genome of SK1 and a haploid genome of S288c 39. SK1 and S288c sequences are sufficiently different that about 25% of reads can be assigned to one of the two genomes 36. Red1 profiles were manually aligned to show that the offset caused by differences in Y’ elements and other subtelomeric sequences does not greatly alter the relative distribution or height of Red1 peaks. (d) Coding density versus distance from telomeres overlaid on a metaplot of Red1 enrichment. Graph points show mean coding density in 10-kb bins, plotted at bin midpoints (right y-axis). Error bars are standard deviation. Line connects the dots for easier visualization of trend. Y’ elements are included and are responsible for the uptick in signal in coding density in the most telomere-proximal bin.
Differential recruitment of Red1 at chromosome ends by Rec8-dependent and Rec8-independent pathways.
(a) Distance-from-telomere profiles of Red1 (spike-in normalized; see Methods) in WT, rec8, hop1-phd, and hop1-phd rec8 during early prophase I (T = 3h) using published data 13,15 (see Methods: Distance from telomeres plots). Colored dashed lines indicate each strain’s genome-wide mean after spike-in scaling. Ranges of subtelomeric domains (gray) and EARs (orange) are indicated above the plot. (b) Genome-wide bootstrap distributions of fold-enrichment (32 × 20-kb windows; n = 1,000 resamples; see Methods: Bootstrapping plots). Black lines show medians and 95% CIs; orange/red circles mark the observed means in the last 20 kb. Two-sided empirical tests with BH correction; Cohen’s d (negative = depletion at ends): WT (p < 1x10-6; BH < 1x10-6; d = −5.23); hop1-phd (p < 1x10-6; BH < 1x10-6; d = −7.85); rec8 (p = 0.001; BH = 0.0013; d = 3.54); hop1-phd rec8 (p = 0.059; BH = 0.059; d = 1.87). (c-d) Meta-X and Y’ elements plots at chromosome ends. X elements were stratified by end class and only fully annotated X elements were used (X-only, n = 7; XY′, n = 20) with flanks scaled to 100% of X length. Y′ elements use flanks scaled to 50% of Y′ length. Blue arrow indicates Y′-ORF orientation. Shaded bands show two-sided 95% CIs (see Methods: Meta gene analyses, and meta-X and Y’ elements plots). Averages of two biological replicates.
Dot1 shapes axis-protein distribution at chromosome ends and chromosome interiors.
(a) Mean Red1 vs Rec8 enrichment at Rec8 peaks, split by region (terminal 20 kb, centromeres ±10 kb, interior) using published data 16. Global fit (purple) and region-specific fits (green dashes). Slope comparisons (two-sided Student’s t-tests; BH-adjusted): interior vs telomeres (p = 3.43x10-19; BH = 5.14x10-19); interior vs pericentromeres (p = 3.12x10-24; BH = 9.36×10-24); telomeres vs pericentromeres (p = 0.525; BH = 0.525). Stars denote BH-adjusted p-values (*** ≤ 0.001; n.s., not significant) (See Methods: Quantification of Red1 and Rec8 signals). (b) Mean enrichment of H4K44ac and H3K56ac 44, H3K4me3 45, and H3K79me3 versus distance from telomeres, each normalized to H3 or H4 (see Methods: Distance from telomeres plots). (c) Spike-in–normalized Red1 distance profiles in WT, dot1Δ, and set1Δ. Data from 36. See Methods: Distance from telomeres plots. Note that set1Δ mutants are somewhat less synchronous because of delays in premeiotic DNA replication. (d-e) Meta-X and Y’ elements plots of Red1 enrichment in WT (red), dot1Δ (yellow) and set1Δ mutants (purple). X elements stratified by end class and only fully annotated X elements were used (X-only, n = 7; XY′, n = 20) with flanks scaled to 100% of X length. Y′ elements use flanks scaled to 50% of Y′ length. Blue arrow indicates Y′-ORF orientation. Shaded bands show two-sided 95% CIs (see Methods: Meta gene analyses, and meta-X and Y’ elements plots). (f) Region-stratified Red1 enrichment profiles in WT (pink) and dot1Δ mutants (yellow) across intergenic (left) and genic (right) sequences versus distance from telomeres. Averages of two biological replicates.
H3K79-independent activity of Dot1 reduces axis proteins near chromosome ends.
(a) Distance-from-telomere profiles of Red1 enrichment (spike-in normalized) in WT, dot1Δ, and hht1/2-K79R during early prophase I (T=3h; see Methods: Distance from telomeres plots). WT and dot1Δ data are the same as in Fig. 4c. (b) Genome-wide bootstrap distributions (32 × 20-kb windows; n = 1,000 resamples) of Red1 enrichment. Black lines show medians and two-sided 95% CIs; orange/red circles mark the observed mean in the terminal 20 kb. Two-sided empirical test, with BH correction; effect sizes via Cohen’s d: WT (p = 0.0020; BH = 0.0030; d = −4.09); dot1Δ (p = 0.180; BH = 0.180; d = −1.30); hht1/2-K79R (p = 0.0010; BH = 0.0030; d = −6.24). (c-d) Meta-X and Y’ elements plots at chromosome ends. X elements stratified by end class and only fully annotated X elements were used (X-only, n = 7; XY′, n = 20) with flanks scaled to 100% of X length. Y′ elements use flanks scaled to 50% of Y′ length. Blue arrow indicates Y′-ORF orientation. Shaded bands show two-sided 95% CIs (see Methods: Meta gene analyses, and meta-X and Y’ elements plots). Averages of two biological replicates.
Effects of Dot1 on axis-protein deposition depend on Sir3.
(a) Distance-from-telomere profiles of Red1 enrichment (spike-in normalized) in WT, dot1Δ, sir3, and sir3 dot1Δ during early prophase I (T = 3h; see Methods: Distance from telomeres plots). (b) Genome-wide bootstrap distributions (32 × 20-kb windows; n = 1,000 resamples) of Red1 enrichment. Black lines show medians and two-sided 95% CIs; orange/red circles mark the observed mean in the terminal 20 kb. Two-sided empirical test, with BH correction; effect sizes via Cohen’s d: WT (p = 0.002; BH = 0.003; d = −4.09); dot1Δ (p = 0.18; BH = 0.18; d = −1.30); sir3 (p = 0.001; BH = 0.002; d = −5.87); sir3 dot1Δ (p = 0.001; BH = 0.002; d = −6.50). (c-d) Meta-X and Y’ elements plots at chromosome ends. X elements stratified by end class and only fully annotated X elements were used (X-only, n = 7; XY′, n = 20) with flanks scaled to 100% of X length. Y′ elements use flanks scaled to 50% of Y′ length. Blue arrow indicates Y′-ORF orientation. Shaded bands show two-sided 95% CIs (see Methods: Metagene analyses, and meta-X and Y’ elements plots). Averages of two biological replicates.
Sir3 occupancy relates to local transcription and MNase accessibility near chromosome ends.
(a) Metaplots anchored at X elements, stratified by end class, and only fully annotated X elements were used (X-only, n = 7; XY′, n = 21). Flanks scaled to element length (X: 100% each side). Vertical dotted lines mark X boundaries. Shaded bands indicate 95% CIs (see Methods: Metagene analyses, and meta-X and Y’ elements plots). (b) Sir3 ChIP signal at X elements on X-only versus XY′ ends during mid-log, meiotic induction (T = 0h), and meiotic prophase (T = 3h). Values represent the mean ChIP/input signal per X element. Box-and-whisker plots show the distribution across elements. Two-sided unpaired Student’s t-tests with BH correction; stars reflect BH-adjusted p (** ≤ 0.01). Statistics (per X element; Cohen’s d; negative = lower at XY′): mid-log (p = 0.007; BH = 0.007; d = −1.22); T = 0h (p = 0.007; BH = 0.007; d = −1.32); T = 3 h (p = 0.002; BH = 0.006; d = −1.11). (c) Metaplot anchored at Y′ elements. Fully annotated Y′ only; flanks scaled to 50% of Y′ length. Vertical dotted lines mark Y′ boundaries. Blue arrow indicates Y′-ORF orientation. Shaded bands show 95% CIs (see Methods: Metagene analyses, and meta-X and Y’ elements plots). (d-e) Example Sir3 ChIP-seq tracks on chr VII-L and chr VI-L at mid-log and T = 3h. Curves show ChIP/Input. Positions of X elements, Y’ elements and ORFs are indicated (inset white triangle shows ORF orientation). (f) mRNA-seq fold change (sir3/WT, log10) at T = 3h versus average Sir3 ChIP enrichment in the 250 bp upstream of ORFs. Blue dots represent measurements within 5 kb of X elements, red dots are measurements in the rest of the genome. Pearson r values are shown. P values are based on Fisher’s r-to-z transformation and two-sided z-test. (g) MNase-seq fragment frequency (RPM/kb) at T = 3h versus distance from telomeres (0–40 kb) for WT, dot1Δ, sir3, and sir3 dot1Δ. All experiments: averages of two biological replicates.
Sir influences DSB formation near chromosome ends.
(a) Boxplots of log10 Spo11-oligo signal (hits per million) at X and Y′ elements in WT and sir2Δ, from published datasets 19,73. Points are element scores; gray lines connect matched elements across strains. Two-sided Wilcoxon rank-sum tests with BH correction; effect sizes are rank-biserial r (positive = higher in sir2Δ). X elements: (p = 7.9x10-4, BH = 1.6x10-3); r = 0.83), Y′ elements: (p = 0.127; BH = 0.127; r = 0.32). Stars reflect BH-adjusted p (** ≤ 0.01; n.s., not significant). (b) TrAEL-seq hotspot intensity per 5-kb bin as a function of distance from X elements. Log10 average peak intensity (RPM) for peaks in each bin in dmc1Δ, sir3 dmc1Δ, dot1Δ dmc1Δ, and sir3 dot1Δ dmc1Δ (see Methods: TrAEL-seq hotspot calling and quantification). Per-bin two-sided Wilcoxon rank-sum tests contrasting (sir3 dmc1Δ + sir3 dot1Δ dmc1Δ) vs (dmc1Δ + dot1Δ dmc1Δ), BH-corrected across bins; effect sizes are rank-biserial (positive = higher in sir3 mutants. Bin 1: p = 3.15 x 10-7; BH = 1.26 x 10-6; r = 0.67. Bin 2: p = 0.237; BH = 0.474; r = 0.13. Bin 3: p = 0.527; BH = 0.703; r = 0.07. Bin 4: p = 0.823; BH = 0.823; r = 0.02. Stars reflect BH-adjusted p (*** ≤ 0.001; n.s., not significant). (c) Mean hotspot counts per 5-kb bin as a function of distance from X elements for the indicated strains. Hotspots were identified from TrAEL-seq peak calls. Because most telomere-proximal bins contained only 0-2 hotspots, the data had too few discrete steps for box or violin plots. Therefore, the means of the data are shown as a bar plot (error bars: SEM). Per-bin two-sided Wilcoxon rank-sum tests contrasting (sir3 dmc1Δ + sir3 dot1Δ dmc1Δ) vs (dmc1Δ + dot1Δ dmc1Δ), BH-corrected across bins; effect sizes are rank-biserial (positive = higher in sir3 mutants. Bin 1: raw p = 5.63x10-3; BH = 2.25x10-2; r = 0.24. Bin 2: raw p = 0.376; BH = 0.752; r = 0.08. Bin 3: raw p = 0.687; BH = 0.915; r = 0.04. Bin 4: raw p = 0.920; BH = 0.920; r = 0.01. Stars reflect BH-adjusted p (* ≤ 0.05; n.s., not significant). (d) TrAEL-seq (colored) and MNase-seq (gray) tracks at a representative subtelomeric region (chrV-R) for dmc1Δ, sir3 dmc1Δ, dot1Δ dmc1Δ, and sir3 dot1Δ dmc1Δ. Black arrow indicates a cryptic DSB hotspot that becomes active in the absence of SIR3. Gray arrow indicates an unusual Y’-associated hotspot that becomes stronger in the absence of SIR3. Other Y’ elements generally do not exhibit altered TrAEL-seq signal. (e) TrAEL-seq meta-plots showing mean DSB signal (reads per million) as a function of distance from X elements in the strains indicated. Ranges of subtelomeric domains (gray) and EARs (orange) are indicated above the plot.
Differential regulation of axis protein deposition near telomeres
Schematic depicting a model of how axis protein recruitment is regulated in the chromosome interior (left) and near chromosome ends (right). Axis protein recruitment occurs in parallel through recruitment by Rec8 cohesin (likely via binding to Red1) and through nucleosome binding by the Hop1 chromatin binding region (CBR). Both recruitment pathways are active near telomeres. Dot1 downmodulates Rec8-dependent recruitment, thereby preventing a positive effect of Sir3. In addition, cis-encoded features contribute to reduced axis protein binding near telomeres.
Enhanced coverage at chromosome ends with a tailored mapping workflow.
(a) Fraction of uniquely mapping and multiple-mapping reads within Y′ elements, X elements, and the terminal 20-kb (subtelomeric domains), shown for paired-end and single-end libraries. Bars show mean across replicates; error bars, SD. (b) Fragment-length distributions for unbiased input DNA in Y′, X, terminal 20 kb, and internal regions. Violin plots show the full distribution of paired fragments. Embedded boxplots mark the median and interquartile range. (c) Input fold coverage across the same region classes, displayed as violin plots with embedded boxplots. For X, Y′, and terminal 20-kb regions, coverage was calculated as the averaged depth per element. For internal regions, we averaged depth within non-overlapping 20-kb windows defined after excluding X, Y′, and terminal 20-kb regions. The increased input signal for X elements likely reflects the missing X-element annotations on several chromosomes (Supplementary Fig. 2). Since all ChIP profiles are plotted as ChIP/input, any increase in input signal will be mirrored in the ChIP signal and thus will not affect conclusions. (d) Sir3 ChIP profiles at representative subtelomeres (chr VII-L; chr VI-L), comparing unique versus unique+multi-mapping pipelines. Shaded rectangles mark X and Y’ elements. Blue boxes and arrowheads indicate ORFs and their direction. Values are means of two independent biological replicates and were reproducible across replicates.
Axis-protein ChIP profiles across all chromosome ends.
Distance-from-telomere ChIP/Input signal for Red1, Hop1, and Rec8 at every left and right arm (32 ends) in WT early prophase I. Green blocks mark all annotated Y′ elements and blue blocks with arrowheads mark all annotated X-elements (fully and partially annotated copies shown: X, n = 32; Y′, n = 31). The partially annotated copies (marked with stars) are located more internally and are likely an artefact of the incomplete sequence conservation and the small size of X elements. These partially annotated X elements were not used for analyses shown in this study. Signals are from published datasets 1–3. The positions of the silent mating type loci near the left (HML) and right (HMR) telomeres of chrIII are also indicated.
Consistent axis-protein and Spo11-oligo depletion across chromosome ends.
(a) Heatmaps of Rec8 and Red1 ChIP enrichment (ChIP/Input) in WT early prophase I (T = 3h) from published data 2,3. Each row is a chromosome end (left/right indicated). Columns are contiguous 4-kb bins extending inwards from the telomeres. Colors show the mean signal per bin; the vertical white line marks 20 kb from the end. (b) Heatmap of Spo11-oligo density (WT; data from 4) in the same 4-kb binning method. Values are means of two independent biological replicates and were reproducible across replicates.
Axis-protein distance profiles using alternative anchors and Y′ masking.
(a) Mean enrichment of Rec8, Red1, and Hop1 (ChIP/Input) in WT early prophase I (T = 3h) from published datasets 1–3, normalized to a genome average of 1 (gray dashed line). Left panel: distance from telomeres; right panel: distance from the annotated X-element anchor. Curves were computed as described in Methods: Distance-from-telomere profiling. (b) Distance-from-telomere profiles stratified by end class: XY′ ends (n = 21) versus X-only ends (n = 11) for Rec8, Red1, and Hop1. (c) Distance-from-telomere profiles with Y′ elements included (solid) versus masked (dashed). For masking, overlapping annotated Y′ elements were set to NA prior to averaging; all other processing was identical. All values are averages of two biological replicates and were reproducible across replicates. (d) Relationship between coding density and mean Red1 enrichment. Scatter plots show non-overlapping 20-kb bins at dots. Black lines are least-squares fits, with 95% CIs shown in gray. Pearson r values are indicated. P values are based on two-sided t-tests on the slope. “Y′ masked” means Y′ sequence is excluded from both measurements (we mask Y′ bases when tallying coding density and when averaging Red1; bins that are entirely Y′ are excluded). Including the Y’ elements eliminated the correlation in the last 20 kb (r = -0.174; p = 0.341).
Red1 distribution near chromosome ends in SK1 and S288c.
Distance-from-telomere ChIP/Input signal for Red1 (T = 4h) measured in WT SK1/S288c hybrid strains carrying a haploid genome of SK1 and a haploid genome of S288c 5. The sequences between SK1 and S288c are sufficiently different that about 25% of reads can be assigned to one of the two genomes 3. The corresponding ends for SK1 and S288c are shown juxtaposed for all 32 ends. Green blocks mark all annotated Y′ elements and blue blocks with arrowheads mark all annotated X-elements. Partially annotated X-elements are marked with stars.
Chromatin features and the role of histone methyltransferases at chromosome ends and centromeres.
(a) Meta-ORF profiles of H3K79me3 (normalized to H3) in WT at early prophase I (T = 3h). Curves show mean ChIP/Input with shaded 95% CIs for genes in the terminal 20 kb (pink) versus the rest of the genome (blue; see Methods: Meta gene analyses, and meta-X and Y’ elements plots). (b) Mean Red1 signal per chromosome end (terminal 20 kb) in WT, dot1Δ, and set1Δ using published datasets 2,3. Points are individual ends. Gray lines connect matched ends across strains. Two-sided Student’s t-tests with BH correction; effect sizes are Cohen’s d (positive = higher than WT): dot1Δ vs WT (p = 0.0020, BH = 0.0038, d = 0.82); set1Δ vs WT (p = 0.181, BH = 0.181, d = 0.34). (c) Genome-wide bootstrap distributions of Red1 enrichment (32 × 20-kb windows; n = 1,000 resamples). Black lines mark medians and two-sided 95% CIs; orange/red circles mark the observed mean in the terminal 20 kb (see Methods: Bootstrap test and violin plots). Two-sided empirical tests with BH correction; Cohen’s d (negative = depletion at ends): WT (p = 0.001, BH = 0.0015, d = −4.15); dot1Δ (p = 0.163, BH = 0.163, d = −1.34); set1Δ (p < 1×10⁻⁶, BH < 1×10⁻⁶, d = −4.86). (d) Red1 distance-from-centromere profiles (± 50 kb) in WT, dot1Δ, and hht1/2-K79R (spike-in normalized). Dashed line marks the centromere midpoint. Signals were extracted in 100-bp bins around each centromere. (e) Bootstrap test centered on centromeres (± 10 kb; 16 × 20-kb genome windows). Two-sided empirical tests with BH correction; Cohen’s d (negative = depletion at chromosome ends): WT (p = 0.679, BH = 0.679, d = 0.43); dot1Δ (p = 0.020, BH = 0.060, d = −2.44); set1Δ (p = 0.253, BH = 0.379, d = −1.12); hht1/2-K79R (p = 0.520, BH = 0.679, d = 0.65). (f-g) MNase-seq fragment frequency (RPM/kb) versus distance from telomeres (f) or centromeres (g) in WT and dot1Δ. All values are averages of two biological replicates and were reproducible across replicates.
Effects of Dot1 on axis protein deposition depend on Sir3 at centromeres.
(a) Distance-from-centromere meta-profiles (±50 kb) of Red1 ChIP in WT, dot1Δ, sir3, and sir3 dot1Δ (spike-in normalized). WT and dot1Δ are from published datasets 2,3. Dashed line marks the centromere midpoint. Signals were extracted in 100-bp bins around each centromere. (b) Genome-wide bootstrap distributions of fold-enrichment (16 × 20-kb windows; n = 1,000 resamples; see Methods: Bootstrapping plots). Black lines show medians and 95% CIs; orange/red circles mark the observed mean around the centromeres. Two-sided empirical test, effect sizes via Cohen’s d (negative = depletion at centromeres relative to the genome-wide null): WT (p = 0.679, BH = 0.797, d = +0.43); dot1Δ (p = 0.020, BH = 0.080, d = −2.44); sir3 (p = 0.186, BH = 0.372, d = −1.29); sir3 dot1Δ (p = 0.797, BH = 0.797, d = −0.25). All values are averages of two biological replicates and were reproducible across replicates.
Sir3 ChIP profiles across all chromosome ends.
Distance-from-telomere Sir3 ChIP/Input signal is shown for every left and right arm (32 ends) in vegetative cells (mid-log phase), at meiotic induction (T=0), and in early prophase I (T = 3h). Green blocks mark all annotated Y′ elements and blue blocks mark X elements (fully and partially annotated copies shown: X, n = 32; Y′, n = 31). The positions of the silent mating type loci near the left (HML) and right (HMR) telomere of chrIII are also indicated. Values represent the mean of two biological replicates and were reproducible across replicates.
Sir3 spreading and Y′ transcription near chromosome ends.
(a) Sir3 spreading from the X element, quantified per chromosome end from Sir3 ChIP–seq (ChIP/Input) in early prophase I (see Methods: Quantification of Sir3 spreading from chromosome ends). The consistent outlier point indicates the spreading to HML on chr III-L. (b) Example illustrating the range of Sir3 spreading as quantified in (a). (c) Y′-element mRNA abundance at T = 3h after meiotic induction in WT and sir3 (both ndt80Δ). Expression is log10(TPM) for annotated Y′ ORFs. Points are individual Y′ ORFs and gray lines connect the same ORF across strains. Two-sided Wilcoxon rank-sum test with rank-biserial effect size (positive = higher in sir3) (p = 0.037, r = 0.40). Values represent the mean of two biological replicates and were reproducible across replicates.
Relationships among DSB changes, Sir3 occupancy, and transcription in promoters.
(a) For each annotated gene, the change in Spo11-oligo signal in sir2Δ versus WT within the promoter (250 bp upstream of the TSS) is plotted against the change in mRNA in sir3 versus WT (log10 fold change). Blue: genes within 5 kb of an X element; red: all other genes. Lines are least-squares fits with shaded 95% CIs; Pearson r values are shown. P values are based on Fisher’s r-to-z transformation and two-sided z-test. Spo11 datasets are from published work 1,4 (see Methods: Promoter correlation plots). (b) For the same promoters, DSB change (sir2Δ/WT, log10) is plotted versus Sir3 binding in WT promoters (log10 ChIP/Input). Color scheme and statistics as in (a).
Distinct and combined roles of Dot1 and Sir3 in shaping DSB landscapes around centromeres and the rDNA locus.
(a) TrAEL-seq meta-plots showing DSB signal as a function of distance from centromeres plotted for dmc1Δ, sir3 dmc1Δ, dot1Δ dmc1Δ, and sir3 dot1Δ dmc1Δ strains. Mean TrAEL-seq signal (reads per million) plotted for the four strains within ± 50 kb of centromeres. (b) TrAEL-seq signal versus distance from the rDNA array on chr XII; the black bar denotes the rDNA repeat region. The same four strains are shown. Purple shading marks intervals with elevated DSB signal in dot1Δ. Values are means of two independent biological replicates and were reproducible between replicates.
SK1 yeast strains utilized in this study
ChIP-seq datasets utilized in this study
