Monitoring meiotic entry and progression in the time course used for FAIRE. (A) To validate meiotic entry and synchrony in each biological replicate, we analyzed the transcription profiles of key meiotic progression genes by expression microarray. Time point is indicated on the x-axis, and expression relative to YPD is shown on the y. Biological replicates are plotted (1, blue; 2, red; 3, green). If only two samples are shown, the YPD probe was flagged for technical reasons, and thus no comparisons could be made. Early meiotic genes include IME1, IME2, HOP1, DMC1, RAD51, and SPO11. Middle meiotic genes include NDT80, SPS1, SPS2, CLB1, CLB6, and SMK1. (B) Comparison to meiotic transcription microarray data of Chu et al. (1998). Purple indicates positive meiotic enrichment, and green indicates negative meiotic enrichment. Transcription data is represented as a ratio between time point expression and 0 h expression. (C) Completion of meiosis as measured by spore counts. Spore counts were taken at each time point. Time point is indicated on the x-axis, and the percentage of sporulated cells is indicated on the y. Biological replicates are plotted (1, blue; 2, red; 3, green; average, purple).

Chromatin at known hotspots is dynamic. Chromosome coordinates are plotted along the x-axis, with FAIRE enrichment (z score) plotted on the y. Positive z scores indicate relatively open chromatin, and negative numbers indicate relatively closed chromatin. FAIRE data from YPD (red), 0-h (dashed green), 1.5-h (blue fine dashes), and 3-h (purple and dotted) samples are plotted. Among the 1204 sites, 151 were open in all the meiosis samples (1.5 and 3 h), but not during mitotic growth (e.g., YAT1). Conversely, 156 sites were open during mitotic growth, but were not open in any of the meiotic samples. Meiotic histone H3 ChIP-chip data from 0 h (orange), 1 h (brown), and 2 h (dark brown and dotted) are plotted (Borde et al. 2009). Hotspot regions identified by Buhler et al. (2007) (light-green bars), and by Blitzblau et al. (2007) (dark-green bars) are shown. Arrows represent the coding regions of genes, with those above the x-axis coded from the Watson strand and those below coded from the Crick strand. (A) Chromatin dynamics at the CDC19 hotspot on chromosome I, an example of a hotspot with constitutively open chromatin. (B) Chromatin dynamics at the YAT1 hotspot on chromosome I, an example of a meiosis-specific site of open chromatin. (C) Chromatin dynamics at the ARG4_DED81 hotspot on chromosome VIII (Wu and Lichten 1994; Ohta et al. 1999), an example of a DSB hotspot with no observed open chromatin in any sample. (D) Chromatin dynamics at the HIS4 hotspot on chromosome III, an example of a hotspot at which the chromatin is open in YPD and closes as meiosis progresses.

Regions proximally adjacent to the subtelomere borders exhibit meiosis-specific open chromatin. (A) Examples of meiosis-specific open chromatin at the subtelomeric borders. Distance from the subtelomeric border (coordinate 0) is plotted on the x-axis and FAIRE enrichment is plotted on the y-axis. Subtelomeres (orange bars) are shown. FAIRE data from YPD (red), 0-h (green), 1.5-h (blue dashes), and 3-h (purple dots) samples are plotted. Asterisks indicate sites significantly open (P < 0.01). (B) Analysis of telomeric regions. The x-axis reports the region analyzed and the y-axis reports the ratio of observed sites of open chromatin to the expected number from random sampling. (Red) Category I; (maroon) category II; (light-blue) category III; (dark-blue) category IV sites. The telomere and subtelomeres do not deviate from the genomic averages of expected/observed sites of open chromatin. Meiosis-specific open chromatin is most prominent in the 10 kb adjacent to the subtelomeres. In the 10–20-kb and 30–40-kb windows, only category IV sites are significantly above expected values. (C) The ratio of observed to expected number of DSB hotspots as plotted as a function from distance from the subtelomeric border. Data from Buhler et al. (2007) (blue) and Blitzblau et al. (2007) (red) are shown. (D) Recombination in the subtelomeric regions as measured by linkage mapping (Barton et al. 2008). Distance from the subtelomeric border is plotted on the x-axis and recombination rate (cM/kb) is shown on the y-axis. Recombination rates are presented as the average of the 15 reported rates closest to each position plotted. Subtelomeric regions are defined as having either >50% DNA sequence identity to another region of the genome or as having <60% nondubious ORFs using a 20-kb sliding window (Barton et al. 2008). The subtelomeric border is the point at which neither of these criteria is met. The gray line represents the genomic average recombination rate of 0.37 cM/kb (Barton et al. 2008).

A 15-bp motif is over-represented in meiosis-specific open chromatin surrounding tRNA genes. (A) The 15-bp motif identified from FAIRE-enriched sites isolated from any meiotic sample, but not YPD, and FAIRE-enriched sites isolated from all meiotic samples, but not YPD, are represented (Crooks et al. 2004). (B) Open chromatin increases during meiosis at sites associated with tRNA genes. The position relative to tRNA annotated start sites (base pair) is plotted on the x-axis, and FAIRE enrichment is plotted on the y-axis. Cytoplasmic tRNA genes associated with a site of open chromatin were aligned at their annotated transcription start. Shown is a moving average of the average FAIRE-enrichment z score (window size = 15 probes). FAIRE data from YPD (red), 0-h (green dashes), 1.5-h (blue dashes), and 3-h (purple dots) samples are plotted. Regions corresponding to the functional tRNA have a median length of 72 bp (minimum of 70 bp and a maximum of 132 bp). The graph includes the 98 tRNA-associated sequences that overlap a site of open chromatin (P < 0.01).

The distribution of open chromatin and DSB hotspots across a representative 110-kb genomic region containing 53 genes. Chromosome coordinates are plotted along the x-axis, with FAIRE enrichment (z score) plotted on the y. FAIRE data were loaded into the UCSC Genome Browser along with published DSB hotspots (black and dark gray) (Blitzblau et al. 2007; Buhler et al. 2007). FAIRE data from YPD (red), 0-h (green), 1.5-h (blue), and 3-h (purple) samples are plotted.

The degree of chromatin openness is predictive of DSB hotspots. (A) ROC curve analysis of the five categories of open chromatin. True-positives and false-positives are defined with respect to DSB hotspots (Buhler et al. 2007) (Methods). The FAIRE sites plotted are ordered such that those to the left are most open according to FAIRE (lowest P-value), and those to right, while still significantly open, have higher P-values. Data from category I (solid red), category II (maroon dashes), category III (light-blue dashes), category IV (dark-blue dots), and category V sites (solid green) are plotted. AUC values are listed next to the sample name, and the minimum and maximum AUC values of the random permutations follow in parentheses. (B) ROC curve analysis of sites of open chromatin in individual time points. Data from YPD (red), 0-h (green dashes), 1.5-h (light-blue dashes), and 3-h (purple dots) samples are plotted. (C) DSB predictive value of individual probes with FAIRE z scores above the indicated threshold, expressed as likelihood ratios. Values in the table are TPF/FPF at decreasing z score thresholds, listed vertically on the left. Time points are organized on the horizontal axis. Predictive value is expressed with respect to two DSB hotspot datasets (Blitzblau et al. 2007; Buhler et al. 2007). Because this analysis is probe based, multiple true-positives may be derived from a single DSB hotspot.

Centromeres and pericentromeric regions exhibit constitutively open chromatin. (A) FAIRE profiles of each of the 16 S. cerevisiae centromeres (CEN). Asterisks indicate sites significantly open at P < 0.01. Chromosomal coordinates are plotted on the x-axis and FAIRE enrichment is plotted on the y-axis. FAIRE data from YPD (red line), 0-h (green dashes), 1.5-h (blue dashes), and 3-h (purple dots) samples are plotted. (●) Centromeres. (B) Analysis of centromere-associated regions. The x-axis reports the region analyzed and the y-axis reports the ratio of observed sites of open chromatin to the expected number from random sampling. (Red) Category I; (maroon) category II; (light-blue) category III; (dark-blue) category IV sites.