(A) Red1 binding profiles in WT (green), hop1Δ mutants (blue), and rec8Δ hop1Δ mutants (purple) from ChIP-seq experiments (NCIS normalized). A small (Chromosome I) and a medium sized chromosome (Chromosome V) are shown as examples to illustrate the reduced Red1 density on chromosome V in WT but not in hop1∆ mutants (indicated schematically by the double-headed arrows). Arrowheads indicate increased Red1 binding confirmed by qPCR in (E). (B) Genome-wide Hop1 ChIP-seq profiles in red1∆ mutants (gold) (NCIS normalized). Chromosomes I and V are shown. (C) Genome-wide Rec8 ChIP-seq profiles in WT (orange) and red1∆ mutants (red) (NCIS normalized). Chromosomes I and V are shown. (D) Red1 density/20 bp (sum of Red1 signal after NCIS normalization divided by chromosomal length) plotted against chromosomal length. The three smallest chromosomes exhibit highly increased binding in WT (green dots), but this difference is largely abolished in hop1∆ mutants (blue dots). Red1 density is reduced to noise level and no longer shows biased distribution in hop1∆ rec8∆ mutants (purple dots). Horizontal lines indicate the mean of 16 chromosomes (continuous lines), plus minus twofold standard deviation (dashed lines). (E) White bars: ratio of V5-red1 ChIP-Seq signal (hop1∆/HOP1) at six cohesin peaks that decrease (a-f: chr3 219k, chr1 171k, chr1 195k, chr6 216k, chr1 95k, chr1 134k) and six cohesin peaks that increase (g-l: chr1 156k, chr5 274k, chr4 435k, chr4 712k, chr7 506k, chr16 576k) upon HOP1 deletion (data from the experiment shown in (A)). Yellow bars: ratio of V5-Red1 signals (hop1∆/HOP1) obtained from ChIP-qPCR primer pairs located at the indicated positions. Gray bars: ratio of V5 signals (untagged/V5-Red1, HOP1) obtained at the corresponding positions. The gray line separates reduction (below 1) from increase (above 1). Arrowheads indicate positions on chr1 and chr5 shown in (A). DNA from each biological repeat was evaluated both at increasing and decreasing sites to exclude systematic bias in DNA preparation. Each single repeat confirmed the direction of change at each location. n indicates the number of repeats. (F) Average densities of V5-Red1 ChIP-seq signals over all 16 yeast centromeres, aligned at their midpoints. The averaged peaks are fourfold higher in hop1∆ mutants (blue) than in HOP1 cells (green). The fold increase at centromeres exceeds the ∼twofold increase observed for the entire chromosomes (see D). (G) Rec8-HA (orange) and Rec8-HA red1∆ (red) flank the centromere at a slightly wider distance (around 345 bp) than Red1 (270 bp). (H) V5-Red1 (light green) and Rec8-HA (orange) are shown as in (G). The average transcription levels from 4 hr after meiotic induction (Brar et al., 2012a) around centromeres (forward—black, reverse—dark green) reveal a transcription free pocket, in which Red1 resides. Meiotic cohesin peaks almost coincide with the ends of transcripts. Rec8-HA and V5-Red1 are shown on two different scales.