RPs are present at the PMA1 gene locus. (A) Schematic diagram of the PMA1 gene; gray bar represents the gene ORF; the PCR amplicons used for the ChIP assay are indicated by dotted lines above (numbers correspond to the primers positions relative to start codon). (B) Polyacrylamide gels showing radiolabeled PCR products produced by the PMA1-specific primer pairs (top bands) and by the pair specific for the intergenic region (bottom bands); using input DNA before ChIP. (C) PCR products as in B using ChIP-enriched DNA from the strains indicated. The relative enrichment of PMA1 DNA relative to intergenic sequence is expressed as ratio of the intensitity of the same fragments produced with the input DNA. (D) PCR analysis as in C of samples treated with RNases A and T1 prior ChIP. (E) Real-time PCR quantification using the same set of primers as above (indicated in the x-axis). Fold enrichments (y-axis) are relative to the intergenic control fragment and are calculated as ratio of ratios as in B. Gray bars show ChIP experiments not treated with RNase, black bars with RNase. Error bars represent standard deviation of three repeats.

Genome-wide association of RPs. (A) Chromosomal binding profiles of the RPs on all three chromosomes, analyzed by the MAT software and visualized with the IGB software. Each RP is shown with a different color on the graph: green (RpL7), navy blue (RpL11), and sky blue (RpL25); x-axis shows the distance from the left chromosome end in megabases (Mb); the y-axis log2 MAT enrichment score (0–4). Each plot is based on two ChIP–chip biological replicas and two control arrays hybridized with input DNA, used as standard in all experiments. Position of centromeres (cen) and telomeres (tel) are highlighted with dotted boxes. (B) Venn diagram showing the number of genes/genomic regions associated with the three RPs. (C) Pie-chart showing the proportions of the genes that are associated with all three RPs that fall within various gene classes.

Centromeric regions bind RPs. (A) ChIP-on-chip enrichment graphs for RPs at the centromeric region of each chromosome, generated with the IGB software. The map below each panel shows a schematic of fission yeast centromeres, with the three major domains labeled otr, imr, and cnt (see text for more details). Centromeric tRNA gene loci are indicated by red lines, and the ORFs of protein-coding genes by brown boxes. Two tDNA clusters in centromere 2 are highlighted by dotted boxes. Values on the y-axis show log2 enrichment scores. (B) Real-time PCR quantification of RPs enrichment at three specific centromeric repeats regions, with or without RNase digestion prior to ChIP.

Centromere and nucleolus are spatially detached. (A) Micrographs showing the subnuclear localization of the nucleolar protein Gar2-GFP (green) and the centromeric protein Cnp1 (red) in interphase S. pombe cells. 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI, blue) was used to stain the DNA. (B) Micrograph of Gar2 and Cnp1 proteins at various cell cycle stages. (C) Micrographs of a cell taken at two different focal planes (at the nucleolus or centromere). On the right is a schematic of the cell with the nucleolus in green, the DNA in blue and the centromere in red. (D) Histogram showing the distribution of cells within four different nucleolus-centromere distance classes (in microns [μm]). The quantification is based on images of 100 unsynchronized cells acquired with a Hamammatsu Orca R2 CCD camera using a 100× objective. Distances were measured with the Nikon NIS-element software from the center of the nucleolar to the center of the centromeric signal.

HA-tagged RPs are functional. (A) Agarose gel showing that the PCR products expected from correct HA tagging in all integration strains; the primers were a gene-specific forward primer corresponding to the 3′ end of the gene and a common reverse primer corresponding to the kanMX6 tagging cassette (primers sequences in Supplemental Table S2). (B) Western blotting analysis of whole-cell protein extracts of cells expressing the HA-tagged RPs indicated. (C) Polysome fractionation and Western blot visualization of the HA-tagged RPs. (D) Serial dilution colony spotting assay of the tagged strains (from left to right, ∼10⁵, 10⁴, 10³, and 10² cells/mL, 4 μL were spotted onto YES plate).

RPs are present at the ACT1 gene locus. (A) Schematic diagram of the ACT1 gene, gene ORF is represented by gray bar; dotted lines indicate PCR amplicons as in Figure 2. (B) Polyacrylamide gel with radiolabeled PCR products produced by the ACT1-specific primer pairs (top bands) and the pair corresponding to the intergenic region (bottom bands), using input DNA before ChIP. (C) PCR as in B of ChIP-enriched DNA. The relative enrichment of ACT1 fragments over the intergenic region was calculated as in Figure 2. (D) PCR of samples treated with RNases A and T1 prior ChIP as in C. (E) Quantification of ChIP-enriched DNA using real-time PCR with the primers indicated in the x-axis.

RPs' chromosomal association shows little correlation to Pol II occupancy. Scatter plot showing RPs versus Pol II occupancy at those regions that were found associated with Rps, based on published genome-wide enrichment scores for Pol II (x-axis) (Wilhelm et al. 2008) and the RPs enrichement (y-axis). A shows correlation with RpL7, B with RpL11, and C with RpL25. Pearson correlation is shown at the top right of each panel.

RPs associate with most tRNA genes. (A) Example of RPs' association at noncentromeric tRNA genes. The peaks of enrichment at tRNA loci are indicated by red vertical lines and by arrowheads at the bottom; individual tRNA are labeled (+ and − refer to genes in the upper and lower DNA strands, respectively). Values on the y-axis show log2 enrichment scores. (B) Histogram displaying the association of the RPs with all known 171 tRNA genes: All tRNA genes were classified in six classes based on the increasing degree to which they were RPs enriched (from less than onefold to more than fivefold, indicated by the color legend on the right). The heights of the bars represent cumulative percentages of the tRNAs encoded by each chromosome. (C) Schematic of the tRNA^Tyr constructs. The top panel shows the wild-type construct; below that, a derivative carrying a mutation in the promoter (B Box deletion); and at the bottom, a derivative carrying a deletion of the entire tRNA coding sequence. Graph on the bottom shows real-time PCR quantification of ChIP enrichment for RpL7-HA at the different tRNA constructs.