"Point" centromeres of Saccharomyces harbor single centromere-specific nucleosomes

Abstract

The "point" centromere of budding yeast is genetically defined by an ≈ 125-bp sequence. Recent fluorescence measurements of kinetochore clusters have suggested that this sequence specifies multiple centromere histone 3 (CenH3) nucleosomes. However, high-resolution mapping demonstrates that there is only one CenH3 nucleosome per centromere, providing biochemical confirmation of the point centromere model.

Figure 1

Single-base-pair resolution mapping of Cse4 nucleosomes. Native chromatin was extracted from nuclei of budding yeast cells grown in rich medium after MNase digestion over an eightfold range (Krassovsky et al. 2012). ChIP of FLAG-tagged Cse4 was performed on soluble chromatin (“Input”), and the resulting DNA fragments were used to prepare libraries as described for paired-end sequencing on an Illumina HiSeq 2000 instrument. Cse4 ChIP/input ratios of normalized counts over a 5-bp running average were plotted for the 400-bp interval centered over the mid-CDE. The cumulative ChIP/input data for four MNase time points (2.5, 5, 10, and 20 min) are superimposed for all 16 centromeres (colored lines). Dotted lines and arrows indicate the regions considered to be “flanks.” Paired-end reads for all eight samples used in this analysis were obtained from the eight lanes of a single flow cell with a total of 597.9 million clusters, yielding 371.4 million paired-end reads that were successfully mapped to the yeast genome.

Figure 2

Low occupancy of Cse4 over centromere-flanking regions. For each centromere, Cse4 enrichment is the ChIP/input normalized count ratio summed over each base pair in the CDE. For each centromere flank, Cse4 enrichment is the ChIP/input ratio summed over each base pair, excluding the 200-bp interval centered over the mid-CDE (between the dotted lines in Figure 1). Flank/centromere ratios are shown for individual chromosomes, and include both flanks out to 5 kb (blue squares) or 15 kb (red diamonds). The ratio for all chromosome arms (green open circles) was obtained by dividing the total Cse4 ChIP/input ratio in the sample, excluding the CDE ±100 bp, by that for all 16 CDEs. A ratio of 1 implies that there is cumulative enrichment in both flanks that is equal to that in the CDE. The hatched region represents the predicted range of the ratio based on fluorescence (4–8 for Coffman et al. (2011) and 3.5–6 for Lawrimore et al. (2011).