rDNA structure, Chromatin Endogenous Cleavage (ChEC), and Mcm displacement in sir2.

A. The 1.4 megabase rDNA region on chromosome XII is composed of approximately one hundred and fifty 9.1 kb repeats. Each repeat encodes both the 35S and 5S ribosomal RNAs (rRNA), which are transcribed by RNA Polymerase I (Pol I) and RNA Polymerase III (Pol III), respectively. The ribosomal origin of replication (rARS) is located between the 5’ ends of these genes. The C-pro transcript, which is suppressed by Sir2, initiates approximately two hundred base pairs from the rARS. The unidirectional replication fork block, which functions to prevent collision between replication and transcription machinery, is depicted in green. The probe used in Southern blots in Figure 3 to assess licensing is marked by *. B. In ChEC-seq, micrococcal nuclease (MNase; depicted as scissors) is fused to the protein of interest, in this case Mcm2. The double-hexameric Mcm helicase complex, Mcm2-7, is depicted in purple, and nucleosomes are in blue. Cleavage is induced by addition of calcium to permeabilized cells. Due to the proximity of nucleosome entrance and exit sites, Mcm-ChEC can reveal not only the binding site of the Mcm complex but also that of the adjacent nucleosome. C. Depiction of nucleosomes and Mcm complex in wild type and sir2 in G1 and G2. Nucleosomes are numbered with respect to C-pro transcription. In WT cells, Mcm is found almost exclusively at a single position, labeled as “A”. Derepression of C-pro transcription in sir2 causes RNA PolII to displace the Mcm complex rightward to positions “B” and “C”. The three different Mcm helicase complexes depicted in the bottom right panel are intended to convey the three most prominent locations for the complex in sir2; this is not intended to indicate that there is ever more than one Mcm complex in the same rDNA repeat.

S-Seq applied to rDNA replication timing and copy number determination.

A. S-seq replication profile of Chromosome XII. The region in the middle noted as the rDNA has been collapsed. Note that the rDNA replicates much earlier in sir2 (orange) than in wild type (blue). B. rDNA replication timing in double mutants between sir2 and various chromatin remodeling enzymes. fun30-S20A S28A is non-phosphorylatable, and fun30-K603R is catalytically inactive. The S to G1 ratio values (mean±SD) were 0.90±.0.02 for WT, 1.04±0.04 for fun30sir2, 1.01±0.02 for fun30K603R and 1.21±0.03 for sir2 (p<0.001 for WT vs sir2, sir2 vs fun30sir2 and sir2 vs fun30K603R sir2 using t-test). C. Effect of sir2 and fun30 mutation on rDNA size, as determined from the fraction of G1 sequencing reads that arise from 450-470 kb on chrXII. The values (% mean±SD) were 9.4±1.6 for WT, 4.6±1.2 for fun30sir2, 4.8±0.3 for fun30K603R and 9.2±1.8 for sir2 (p<0.001 for WT vs sir2, sir2 vs fun30sir2 and sir2 vs fun30K603R sir2 using t-test)

Licensing at the rARS, as determined by Southern blot.

Activation of Mcm-MNase at the rARS with calcium will eliminate the XmnI fragment, as denoted in Figure 1. PIK1, a single-copy gene in which we detect no Mcm binding, is used as a control. Quantification of the uncut band was used to infer relative rDNA array size in sir2 fun30 mutant.

Deletion of FUN30 suppresses origin activity at rDNA and promotes it elsewhere in the genome.

A. Cells were arrested in G1 with alpha factor and then released into medium containing 200 mM HU. The ratio of bubble to y arc in 2D gels demonstrates increased rARS activity in sir2 that is suppressed by fun30. B. Activation of the rARS in sir2 is accompanied by suppression of ARS305, and this effect is likewise reversed by additional deletion of fun30. C. EdU incorporation at 111 early origins demonstrates suppression of origin activity by deletion of sir2 and suppression of this effect by fun30. Each dot represents a single origin, quantified according to EdU incorporation 1 hour after release from G1 into medium containing 200 mM HU. EdU incorporation was summed across a 5 kb window centered on the Mcm binding site within each origin. Points are plotted according to EdU signal in wild type on the x axis and in the mutant in question on the y axis. Suppression of origin activity is reflected in points dropping below the dotted line at 45°. The rARS is circled in green.

Disappearance of Mcm-ChEC signal can be used to monitor origin firing.

A. Cells were synchronized in G1 phase using alpha factor for 1.5 hours before being released into media supplemented with hydroxyurea (HU). Cells were harvested at various time points post-release (15-90 minutes) and subjected to Mcm-ChEC analysis. B. Decrease in Mcm-ChEC signal in wild type for 111 early (orange) and 101 late (blue) origins. Mcm-ChEC signal was quantified over 200 base pair windows centered on the Mcm binding site within each origin. Each point is plotted according to Mcm-ChEC signal at the time point in question on the y axis and the corresponding signal in G1 on the x axis, thus decrease in signal appears as a drop below the 45° diagonal. C. Mcm signal at the rDNA. Mcm-ChEC signal in G1 appears predominantly at the location indicated by the blue rectangle in wild type, whereas it is spread across both the blue and orange rectangles in the absence of Sir2. The displaced Mcm-ChEC signal in sir2 disappears more rapidly than its non-displaced counterpart, and this effect is suppressed by fun30. Decrease of Mcm-ChEC signal at the rARS is accompanied by increased signal to the right.

Nucleosome occupancy assessed by MNase-seq and Mcm-ChEC.

A. Analysis of nucleosome occupancy at rDNA origins using MNase-seq revealed a consistent high occupancy of the +1 nucleosome across all four strain backgrounds, which served as our normalization reference. Occupancy at the +2 (green box) and +3 (red box) positions was increased by deletion of FUN30 in both sir2 and SIR2. B. Analysis of nucleosome occupancy with Mcm-ChEC (see Figure 1B) reveals nucleosome occupancy in that subset of cells and rDNA repeats in which Mcm is present. Deletion of FUN30 leads to increased occupancy at the +2 and +3 positions in a sir2 background.

Model for relationship between Mcm location and replication timing at the rDNA.

Mcm helicase complex (purple ovals) in wild type abuts the +1 nucleosome (blue cylinder) in G1, making it relatively resistant to activation. Deletion of SIR2 derepresses C-pro transcription (arrow pointing to the right), and RNA PolII pushes Mcm complex to a nucleosome free area, where it is more prone to activation. Deletion of FUN30 in a sir2 mutant leads to increased nucleosome occupancy at the +2 position, adjacent to Mcm complex, making this complex resistant to activation. Short red stretch of DNA (e.g. between +2 and +3 nucleosomes in top row) indicates ACS.

Computer-generated visualization of non-displaced and displaced Mcm complexes, as determined by Mcm-ChEC.

Sequencing reads from G1 cultures processed for Mcm-ChEC for wild type (left) and sir2 (right) were plotted according to genomic location (x axis) and library insert size (y axis), with read depths indicated by color intensity. Note that these images are not agarose gels. The signal generated from inserts in the 50-100 base pair range (y axis) reflects the Mcm complexes, whereas the signal from inserts in the 150-200 base pair range reflects the +1 nucleosome (see main text). Derepression of C-pro transcription in sir2 causes RNA PolII to push the Mcm helicase complex from its normal location (arrow labeled “A”) to the right, with the most prominent signals arising at the sites indicated by arrows labeled “B” and “C”. Note that the presence of multiple Mcm footprints in these composite images does not indicate that the presence of multiple Mcm complexes in any individual repeat.

rDNA size as determined by qPCR.

rDNA size for multiple isolates from each genotype were determined by qPCR, as described (Materials and Methods). Control strains with 35 and 180 copies of the rDNA were generated in fob1 backgrounds to ensure copy number stability and used as standards. Copy numbers were 160±7, 156±7, 38±3 and 147±7 for WT, sir2, sir2fun30 and fun30, respectively (t-test p<0.001 for sir2 vs sir2 fun30).

Deletion of FUN30 suppresses activation of the displaced Mcm complex at the rDNA (replica of results in Figure 5C).

Each panel shows Mcm-ChEC data for cultures that were arrested in G1 and then released into medium containing 200 mM HU. Activation of Mcm complexes is inferred from disappearance of Mcm-ChEC signal. Non-displaced and displaced Mcm complexes are indicated by blue and orange rectangles, respectively.

Displaced Mcm complex is activated early in wild type.

Enlarged region from figure 5C shows that, although only a small proportion of Mcm complexes are displaced in WT, this displaced population, indicated by arrows, is activated early, as is the case with in sir2.

C-pro transcript levels.

C-pro transcript levels in G1-arrested cells were measured in triplicate using qPCR and expressed relative to WT. *** denotes p<0.001 for comparison with WT using t-test.

Yeast strains

Primers used