Sir2 Mitigates an Intrinsic Imbalance in Origin Licensing Efficiency Between Early- and Late-Replicating Euchromatin

Sir2 mitigates an intrinsic imbalance in origin licensing efficiency between early- and late-replicating euchromatin

Timothy Hoggarda, Carolin A. Müllerb, Conrad A. Nieduszynskib, Michael Weinreichc, and Catherine A. Foxa,1

aDepartment of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin–Madison, Madison, WI 53706; bSir William Dunn School of Pathology, University of Oxford, OX1 3RE Oxford, United Kingdom; and cDivision of Molecular and Cellular Biosciences, National Science Foundation, Alexandria, VA 22314

Edited by Jasper Rine, University of California, Berkeley, CA, and approved May 7, 2020 (received for review March 11, 2020) A eukaryotic chromosome relies on the function of multiple illuminate issues relevant to genome replication that would be spatially distributed DNA replication origins for its stable inheri- difficult to glean from classical approaches. It is now established tance. The spatial location of an origin is determined by the that approximately half of yeast origins, distributed broadly over chromosomal position of an MCM complex, the inactive form of the the central regions of chromosomes, act in the first portion of S DNA replicative helicase that is assembled onto DNA in G1-phase phase (early and mid origins), while the remaining origins, gen- (also known as origin licensing). While the biochemistry of origin erally located between ∼15 and 75 kb from the telomeric ends, licensing is understood, the mechanisms that promote an adequate act later (late origins). Most of the yeast genome is euchromatic, spatial distribution of MCM complexes across chromosomes are not. and its duplication requires both early and late origins (Fig. 1). We have elucidated a role for the Sir2 histone deacetylase in estab- The terminal 5 to 10 kb of yeast chromosomes exist in a het- lishing the normal distribution of MCM complexes across Saccharo- erochromatic structure. While many telomeres harbor DNA el- myces cerevisiae chromosomes. In the absence of Sir2, MCM ements with the potential to act as origins, these elements are complexes accumulated within both early-replicating euchromatin repressed by heterochromatin, making telomeres among the last and telomeric heterochromatin, and replication activity within these regions of the genome to be duplicated. This origin distribution regions was enhanced. Concomitantly, the duplication of several establishes a reproducible spatiotemporal pattern of chromo- GENETICS regions of late-replicating euchromatin were delayed. Thus, Sir2- some duplication in a proliferating yeast population. While re- mediated attenuation of origin licensing within both euchromatin cent reports have defined specific chromatin-associated proteins and telomeric heterochromatin established the normal spatial dis- enriched within the central regions of yeast chromosomes that tribution of origins across yeast chromosomes important for normal recruit a limiting S-phase kinase required for origin activation (8, genome duplication. 11), it is unclear whether and how chromatin impinges on the distribution of origin licensing. yeast | chromosomes | chromatin | Sir | origin licensing A yeast heterochromatic deacetylase, Sir2, and a nucleosome binding protein, Sir3, are components of telomeric heterochromatin. he distribution of DNA replication origins across eukaryotic Tchromosomes is important for maintaining cell proliferation Significance and genome stability through multiple cell divisions. Regions that contain a paucity of origins are linked to chromosome fra- In eukaryotes, DNA replication origins, the sites where new gility and cancer-associated deletions (1–4). Origin function re- DNA synthesis begins during the process of cell division, must lies on two distinct cell-cycle restricted reactions (5). In G1 be adequately distributed across chromosomes to maintain phase, the ORC (origin recognition complex) and Cdc6 protein normal cell proliferation and genome stability. This study de- bind DNA to direct the assembly of a stable catalytically inactive scribes a repressive chromatin-mediated mechanism that acts MCM (minichromosome maintenance) complex, also known as at the level of individual origins to attenuate the efficiency of origin licensing. In S phase, kinases and accessory proteins origin function. This attenuation is essential for achieving the convert the MCM complex into two bidirectionally oriented normal spatial distribution of origins across the chromosomes helicases that unwind the DNA to allow for new DNA synthesis, of the eukaryotic microbe Saccharomyces cerevisiae. While the also known as origin activation. Thus, the chromosomal distri- importance of chromosomal origin distribution to genome bution of two distinct reactions, MCM complex assembly (origin stability and cellular fitness is acknowledged, this study defines licensing) and MCM complex activation (origin activation), es- a chromatin modification that establishes the normal spatial tablishes the spatiotemporal distribution of origins. While recent distribution of origins across a eukaryotic genome. progress in Saccharomyces cerevisiae provides insights into the how the distribution of origin activation is regulated, little is Author contributions: T.H., M.W., and C.A.F. designed research; T.H., C.A.M., and C.A.F. known about how the chromosomal distribution of origin li- performed research; T.H., C.A.M., C.A.N., and M.W. contributed new reagents/analytic tools; T.H., C.A.N., and C.A.F. analyzed data; and T.H. and C.A.F. wrote the paper. censing is achieved (6–11). Several attributes of S. cerevisiae help address mechanisms The authors declare no competing interest. relevant to chromosome duplication. The sequence-specific This article is a PNAS Direct Submission. binding of yeast ORC and the organism’s small genome have Published under the PNAS license. allowed mapping of the ORC and MCM binding positions and Data deposition: Raw data were deposited in National Center for Biotechnology Infor- ∼ mation under BioProject ID PRJNA601998 [MCM ChIP-Seq (Input) sequencing and for the initiation sites of 400 individual yeast origins at near-nucleotide S Sort-Seq sequencing] and BioProject ID PRJNA428768 [MCM ChIP-Seq (ChIP) sequenc- resolution (12–17). Such studies have also identified origin- ing]. Reads from the S and G2 Sort-Seq experiments are available in the Gene Expression adjacent chromatin features (e.g., nucleosome positioning, modi- Omnibus under GEO accession GSE90151. fication states, nonhistone chromatin-associated proteins) and 1To whom correspondence may be addressed. Email: [email protected]. functional properties (e.g., replication time, origin efficiency, This article contains supporting information online at https://www.pnas.org/lookup/suppl/ ORC binding modes) (18–23). Thus, yeast origins can be parsed doi:10.1073/pnas.2004664117/-/DCSupplemental. by selected criteria into large cohorts whose comparisons can

www.pnas.org/cgi/doi/10.1073/pnas.2004664117 PNAS Latest Articles | 1of8 Downloaded by guest on September 24, 2021 origin (ori) Results 600.1600.3601 603 603.5604605 606 607 609 610 Early Origins Were Enriched among Euchromatic Origins Most G1 (x1.0) Responsive to SIR2. Origin licensing requires Cdc6. Thus, MCM ChIP-Seq signals (MCM signals) are lost at chromosomal origins S cdc6-4 early (x1.1) in cells cultured at 37 °C (25, 32). However, MCM signals are restored at many origins, including euchromatic origins, in cdc6-4 sir2Δ cells (25, 32). Not all euchromatic origins in cdc6-4 sir2Δ cells are rescued to the same extent. Because Sir2- middle (x1.5) heterochromatic regions are late replicating and inhibitory to origin function, we initially predicted that the euchromatic origins most affected by SIR2, i.e., those most rescued for MCM binding in cdc6-4 sir2Δ cells compared to cdc6-4 cells, would be late origins (24, 25, 30, 33, 34). Instead we found that the most late (x1.9) Sir2-reponsive euchromatic origins were enriched for early and depleted for late origins (Fig. 2A). The analyses in Fig. 2A used G2 (x2.0) MCM signals derived from combining data from three biological replicates and smoothing the data by binning nucleotide signals (25). We reexamined these data at nucleotide resolution as Fig. 1. Spatiotemporal progression of yeast chromosome VI replication. depicted in Fig. 2B. MCM signals, abolished in cdc6-4 cells, were Replication of yeast chromosome VI (horizontal line) in a proliferating yeast cdc6-4 sir2Δ population is shown. Origins are indicated with perpendicular lines. Cen- rescued at euchromatic origins in cells, as expected tromere is indicated by the square. Origins with the highest probability of (Fig. 2C, all). Early-euchromatic origins were rescued more activation, depicted by black or dark gray vertical lines, are used in most cell substantially than late-euchromatic origins, consistent with the cycles (efficient) and act in early S. Less efficient origins, which are used outcome in Fig. 2A. Thus, while each of the euchromatic origin in <50% of cell cycles, are depicted in light gray or gray-dotted lines. Late cohorts parsed by their replication time (Trep values) included origins, enriched for inefficient origins, usually act later in S. The majority of Sir2-responsive origins, the early origins were more likely than yeast DNA euchromatic, and origins represented by black or gray bars, are late origins to show Sir2 sensitivity. considered euchromatic origins, regardless of when or how efficiently they function. Origins within telomeres are red. Telomeres are in Sir2 hetero- Sir2-Chromatin Marks Were Higher at Early- Compared to Late-Euchromatic chromatin that represses origins (24). Thus telomeric origins do not function in wild-type cells but do in sir2Δ cells. Origins. Molecular hallmarks of Sir2-heterochromatin, Sir2-dependent depletion of H4K16ac, and Sir3 binding to nucleosomes, are present at euchromatic origins (25). While both early- and late- Recent work reveals that Sir2 and Sir3 act directly on nucleo- euchromatic origins are flanked by nucleosomes showing Sir2- somes adjacent to euchromatic origins (25), an unanticipated dependent depletion of H4K16ac and Sir3 binding (25), early- result, given the paradigm for Sirs in yeast heterochromatic euchromatic origins showed greater levels of these marks structures that inhibit both transcription and origin function compared to late-euchromatic origins (Fig. 3 and SI Appendix, (25–27). Genetic analyses reveal that molecular features of Sir2 Fig. S1). The difference was most pronounced at the nucleo- chromatin found at euchromatic origins are functionally relevant somesflankingORC.ThusSir2-chromatinlevelsweregreater (25, 28, 29), e.g., a deletion of SIR2 (sir2Δ) suppresses the at early- compared to late-euchromatic origins. temperature-sensitive growth and origin licensing defects caused SIR2 Promoted Equitable Distribution of MCM Complexes between cdc6-4 by the mutation. However, because the relevance of Sir2 Early- and Late-Euchromatic Origins. SIR2 has a profound effect in origin licensing has only been assessed in mutant yeast de- on origin licensing at euchromatic origins in cdc6-4 cells fective for this reaction, the physiological role of this Sir2 chro- (Fig. 2C), but its effect in wild-type (CDC6) cells was unclear matin at origins is unclear. (25). Therefore, MCM signals were examined at higher resolu- We used genome-wide mapping of MCM to show that Sir2 tion in CDC6 cells that differed in their SIR2 genotype (Figs. 2B promotes an equitable distribution of origin licensing between and 4). In SIR2 cells, late-euchromatic origins generated lower early- and late-euchromatic and telomeric X origins. In SIR2 median MCM signals than the early-euchromatic origins, as cells, these three distinct origin cohorts showed similar levels of reported (10). However, substantial overlap in the distributions MCM binding, whereas in sir2Δ cells, telomeric X origins and of signals for these two cohorts was observed, suggesting that the MCM binding differences between early- and late-euchromatic early-euchromatic origins gained MCM relative to late-euchromatic origins were minimal (Fig. 4A). In contrast, in sir2Δ cells, the origins. Telomeric X origins exist within Sir2 heterochromatin. difference in MCM signals between these origin cohorts was Sir2-chromatin marks were higher at early- compared to late- clear. MCM signals at >50% of the nucleotides differed between euchromatic origins. Thus, Sir2 attenuation of origin licensing late- and early-euchromatic origin cohorts in sir2Δ cells, whereas correlated with Sir2-chromatin levels. Replication assays no nucleotides differed at a comparable P value cutoff in SIR2 revealed that the function of both early-euchromatic and telo- cells (SI Appendix,Fig.S2). MCM signals were also assessed at meric X origins was enhanced in sir2Δ cells, providing evidence telomeric X origins that are repressed by Sir2-dependent hetero- that Sir2 attenuation of origin licensing helped limit origin chromatin (24). In SIR2 cells, telomeric X origins generated MCM function at these loci (24). In the absence of Sir2, several regions signals most similar to those generated by the late-euchromatic sir2Δ of late-replicating euchromatin failed to complete duplication by origins, whereas in cells, their MCM signals were similar to the end of S phase by a mechanism that was independent of the those of early origins and greater than those of late origins (Fig. 4B). Thus, Sir2 limited MCM binding at telomeric X and replication capacity of the Sir2-controlled rDNA locus that can early-euchromatic origins relative to late-euchromatic origins. alter euchromatic replication (30, 31). Thus, varying degrees of To quantify these differences, MCM signals between the −10 Sir2-mediated attenuation of origin licensing at individual origins and +100 nucleotides for each of the fragments were summed, balances the distribution of MCM complexes across chromo- and each origin was assigned a Z score to indicate how far the somes to promote complete duplication of euchromatin by the MCM signal for that origin diverged from the mean behavior of end of S phase. the entire collection of origin fragments (mean defined as “0”)

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1 + Replication bits 0.75 time 0 Quartile 3 Early -200 0 +200 Position relative to ORC Median 0.50 ++ + Mid Quartile 1 - 0.25 Late Fraction of origins ChIP distribution 0.00 -- Multi-origin ChIP signal ChIP Low Mid High All -200 0 +200 -200 0 +200 (34) (46) (47) (207) C All Early Mid Late 20 (n = 336) 20 (n = 45) 20 (n = 43) 20 (n = 39)

15 15 15 15 0.83 0.74 0.67 0.6 10 10 10 10 MCM signal 5 5 5 5

0 0 0 0 -200 0 200 -200 0 200 -200 0 200 -200 0 200 Position relative to ORC WT cdc6-4 sir2Δ cdc6-4 GENETICS Fig. 2. Early origins were enriched among euchromatic origins most Sir2 responsive. (A) Sir2-responsive origins were parsed into quintiles. The first (low), third (mid), and fifth (high) quintiles had median Sir2-reponsive values of 0.4, 0.7, and 1.0, respectively (25). Euchromatic origins that generated a Trep value in ref. 21 were parsed into quintiles from earliest (early) to latest (late). Hypergeometric distribution was used to compare the enrichments (+) or depletions(−) within each Sir2-responsive group relative to “all” origins. The P values are indicated (+/−: P < 0.05; ++/−−: P < 0.01). (B) The MCM signal associated with a given nucleotide was the median value derived from three biological replicates. To derive the MCM distribution for the origins within a given cohort, the per-nucleotide distribution of MCMsignalsforalloriginsinthatcohortwasdetermined.Themedianvalue(dark lines) and first and third quartiles (shaded coloring) for each cohort are shown. (C) MCM signal distributions for wild type (WT) (CDC6 SIR2), cdc6-4,andcdc6-4 sir2Δ cells were determined for each of the indicated cohorts. The number at the Left corner of the box is the ratio for median MCM signals summed between nucleotides −100 through +150 in cdc6-4 sir2Δ to that in CDC6 SIR2 cells.

(SI Appendix, Fig. S1). The Z scores were displayed in alter replication dynamics. The same yeast strains used for MCM box-and-whiskers plots (Fig. 4D). In SIR2 cells, each of the origin ChIP-Seq were cultured at the permissive growth temperature cohorts generated similar MCM signals, indicating that MCM for cdc6-4 so that the effect of this mutation on replication dy- complexes were equitably distributed. In contrast, in sir2Δ cells, namics could also be examined. The number of sequence reads the median Z score of the early-origin cohort fell above the for a given region in S are normalized to the corresponding reads mean, while the median Z score for the late-origin cohort fell from G1 to generate a Sort-Seq value. Genome annotation below the mean, indicating that the distributions of MCM signals produces chromosomal replication profiles with peaks indicating for these two cohorts differed. The telomeric X origins also an active origin (SI Appendix, Fig. S4) (36). Wild-type and sir2Δ generated higher MCM Z scores in sir2Δ cells compared to late- cells generated virtually indistinguishable profiles. Therefore, euchromatic origins. Thus, in the absence of Sir2, MCM com- Sir2 effects on euchromatin replication dynamics were not ob- plexes were more efficiently distributed to early-euchromatic and servable at this resolution. In contrast, origin function in cdc6-4 telomeric X origins compared to late-euchromatic origins. The cells, regardless of SIR2 genotype, was reduced over several re- Sir2-controlled rDNA origin did not show enhanced MCM gions of the genome. Thus, only a fraction of origins functioned binding in sir2Δ cells, consistent with a published study (SI Ap- normally in cdc6-4 cells even at permissive temperature, and pendix, Fig. S3) (35). SIR2 did not affect origin preference. Thus, Sir2 did not alter the Some origins are inefficient, possibly because they are not li- origins that remained the most functional when origin licensing censed in many cells (17). To focus on origins that were licensed was compromised by cdc6-4. to some minimal extent, we repeated the analysis using only To enhance quantitative comparison of the relevant origins, origins within each cohort that generated an origin efficiency Sort-Seq values were assigned to 30-kb replicons parsed into value in a genome-scale study (17). Considering only these ori- three cohorts by their origins’ replication times (Treps) or Sir2 gins, the difference between MCM signals at the early and late responsiveness, and presented in box-and-whiskers plots origins observed in sir2Δ cells was enhanced (Fig. 4E). Thus, Sir2 (Fig. 5 A and B). The results validated the approach: the early limited MCM accumulating within heterochromatic telomeres replicons generated greater Sort-Seq values than the late repli- and early-replicating euchromatin relative to late-replicating cons, and SIR2 delayed replication of the heterochromatic euchromatin. telomeric X origins. In cdc6-4 cells, regardless of SIR2 genotype, the relative behavior of these replicons was maintained. How- Changes in MCM Distribution Correlated with Changes in Replication ever, when these replicons were parsed by Sir2 responsiveness, a Dynamics. S-phase Sort-Seq experiments were used to address cdc6-4 effect on origin usage was observed (Fig. 5B). In CDC6 whether alterations in MCM distribution had the potential to cells, regardless of SIR2 genotype, the three Sir2-responsive

Hoggard et al. PNAS Latest Articles | 3of8 Downloaded by guest on September 24, 2021 Second, the median values for the early-origin deciles (10th to 50th) in SIR2 cells were similar, whereas they continually decreased in sir2Δ cells, suggesting SIR2 imposed greater stochasticity of origin function (SI Appendix,Fig.S5). Third, in sir2Δ cells the difference in the behavior of the 60th and 70th ranked deciles became more acute, creating a gap in replication efficiency of these cohorts. Thus, SIR2 promoted the normal progression of euchromatic origin function in an unperturbed S phase.

SIR2 Was Required for Completing Duplication of Late-Replicating Euchromatin Independent of rDNA Replication Demands. While the S Sort-Seq data differences were subtle, they revealed a Sir2 effect on euchromatic origin replication: preventing late-origin replication efficiency from falling further behind that of early origins and even trailing telomeric X origins (Fig. 5D). To challenge this outcome, we applied the Z-score approach to independent S Sort-Seq data from ref. 31 and observed similar alterations in the replication, strengthening the conclusion that normal progression of euchromatic origin function in S required SIR2 (SI Appendix, Fig. S6). Sir2 can indirectly promote euchromatic origin activity by acting directly to form heterochromatin that inhibits the function of the rDNA origin, present in hundreds of copies within the Fig. 3. Sir2-chromatin marks were higher at early- compared to late- sir2Δ euchromatic origins. (A) Early- and late-euchromatic origin cohorts were yeast rDNA repeat array (30). Thus, in cells, the rDNA examined for Sir2-chromatin marks on six origin-adjacent nucleosomes. (B) origins (r-ORIGIN) are more active, and because there are so Normalized H4K16ac assessed in SIR2 cells at early- and late-euchromatic many, the limiting origin activation factors are sequestered away origins. (C) The change in H4K16ac observed for early- and late- from euchromatic origins. This sir2Δ-caused rDNA sequestration euchromatic origin-adjacent nucleosomes in sir2Δ cells. (D) Sir3 MNase of origin activation factors contributes to the incomplete dupli- ChIP-Seq signals at early (purple)- and late (mint)-euchromatic origin co- cation, or “replication gaps,” of late-replicating regions in early − horts. The median Sir3 signal at the indicated 1 nucleosome differed G2 phase (31). To assess whether sir2Δ-induced MCM distribu- between early- and late-euchromatic origins with the indicated P value tion changes might also be linked to these replication gaps, the (Wilcoxon rank sum tests). The significance cutoff used demanded SIR2 that >90% of the nucleotide positions within the signal apexes had P val- effects of on the replication of relevant origin cohorts in G2 ues <0.05. (E) The origins in the early and late cohorts were randomly assigned to two groups and the Sir3 MNase ChIP-Seq signals from ref. 32 were determined for each randomized cohort. A SIR2 sir2Δ B SIR2 sir2Δ C 17.5 SIR2 sir2Δ 20 20 cohorts produced similar broad box-and-whiskers plots, an expected outcome given that each of these cohorts contained a 15.0 mixture of replicons duplicated at different times in S (Fig. 2A). 15 15 In contrast, in cdc6-4 cells, regardless of SIR2 genotype, the 12.5 different Sir2-responsive cohorts produced box-and-whiskers 10 10 plots similar to those produced for the cohorts parsed by their

cdc6-4 A MCM signal MCM signal S-phase replication times (compare data in Fig. 5 and 5 5 10.0 B). Thus, in cdc6-4 cells, the origin cohort containing the most Sir2-responsive origins also showed the highest replication activity. The origin cohorts most effective at binding MCM when 0 0 -200 0 200 -200 0 200 -200 0 200 -200 0 200 -10 100 origin licensing was compromised by the cdc6-4 mutation were Position relative to ORC also the cohorts that showed the highest replication function in DEEfficient origins Sir2-euchromatin 0.01 0.004 cdc6-4 cells, indicating that alterations in MCM distribution had 2 0.11 Early (n = 45 / 37*) an impact on replication dynamics. 0.23 1 Mid (n = 43 / 34*)

Sir2 Promoted the Normal Progression of Euchromatic Origin Replication 0 Late (n = 38 / 22*) in an Unperturbed S Phase. The above analyses suggested that in * n for efficient origins CDC6 cells, Sir2 did not alter euchromatic origin function, yet Sir2 -1 Sir2-heterochromatin MCM Z-score is important to completing replication of late-replicated regions by X-origins (n = 9) sir2Δ sir2Δ early-G2 phase (31). The dynamic range of the Sort-Seq analyses is SIR2 SIR2 limited (1-2n). In the spatiotemporal control of chromosome du- Fig. 4. SIR2 promoted more equitable distribution of MCM complexes be- plication, a more meaningful value is how specific genomic regions tween early- and late-euchromatic origins. (A) MCM signals were de- are duplicated relative to one another (37). Therefore, to examine termined as in Fig. 2C for the indicated origins in congenic SIR2 CDC6 and the replication behavior of origin cohorts at higher resolution and sir2Δ CDC6 cells. (B) Analyses as in A, with data for the telomeric X origins over a greater dynamic range, we assigned Z scores to origin frag- included. (C) Magnified view of the median values between nucleotides −10 C and +100 for the early- and late-euchromatic origins in A.(D) Median signals ments using their Sort-Seq ratios (Fig. 5 ), ranked the origins by − their replication time, portioned the ranked origins into deciles, and between nucleotides 10 and +100 were summed to generate an MCM area for each origin, Z scores were assigned and displayed in box-and-whiskers displayed the Z scores for each decile in box-and-whiskers plots P D plots for each indicated cohort. The values (Wilcoxon rank sum test) for the (Fig. 5 ). Effects of Sir2 on euchromatic origin replication were difference between the early- and late-euchromatic cohorts’ median MCM Z revealed. First, origin function of 50th and 60th deciles (early/mid-S scores are indicated. (E) Analysis as in D after exclusion of inefficient origins) differed more substantially in SIR2 cells than in sir2Δ cells. origins (17).

4of8 | www.pnas.org/cgi/doi/10.1073/pnas.2004664117 Hoggard et al. Downloaded by guest on September 24, 2021 A WT sir2Δ cdc6-4 cdc6-4 sir2Δ

Replication time

Early Mid Late

SortSeq signal Sir2-heterochromatin B X-origins Sir2-responsiveness

High Mid Low SortSeq signal C SortSeq ratio SortSeq area +500 ratios S coverage i =Σ -500 SortSeq Z-score G1 coverage 005- 0005+ Replication time D percentile 0.2 ** th ** *** 10 (n = 23) 2 th 20(n = 22) th 30 (n = 24) th 1 40(n = 22) th 50 (n = 23) th 60(n = 20) 0 th 70 (n = 22)

80th GENETICS

SortSeq Z-score (n = 23) th 90 (n = 21) -1 th 100 (n = 18) X-origins (n = 9) SIR2 sir2Δ

Fig. 5. Changes in MCM distribution coincided with changes in replication dynamics. (A) Sort-Seq values for 30-kb replicons centered on euchromatic origins parsed by their Trep values from ref. 21. Telomeric X origin replicons also shown. (B)AsinA except with replicons parsed by euchromatic origins’ assigned Sir2 responsiveness (25). (C) Assigning Sort-Seq Z scores to distinct 1,001-bp origin fragments. (D) Euchromatic origins parsed into 10 distinct cohorts by their Trep values. Z scores are plotted from the earliest 10% (10th) to the latest 10% (100th) decile in SIR2 and sir2Δ cells. Telomeric X origin Z scores are also plotted. The Wilcoxon rank sum P values for the differences between the 50th and 60th and the 60th and 70th cohorts are indicated: **< 0.01 and ***< 0.001.

cells was examined applying the Z-score approach to the data contrast, the late-euchromatic origins remained underduplicated from ref. 31 (Fig. 6A). The cohorts produced the expected even in sir2Δ r-origin* cells (Fig. 6 A and B, compare late- box-and-whiskers plots in S phase. However, in G2 phase, early- euchromatic origin cohort behavior in sir2Δ r-origin* to SIR2 and late-euchromatic origins generated indistinguishable Sort-Seq r-ORIGIN cells). Thus, the r-origin* mutant substantially re- Zscoresnear0inSIR2 cells, indicating equivalent duplication. In duced the delayed replication of telomeric X but not late- contrast, in sir2Δ cells, early- and late-euchromatic origins Z scores euchromatic origins, providing evidence that that telomeric X diverged, with the former generating Z scores above the pop- origins’ gain of MCM complexes in sir2Δ cells was relevant to ulation mean and the latter generating Z scores below the mean their enhanced replication efficiency over late-euchromatic ori- (Fig. 6A). Thus, while in SIR2 cells only the telomere X origins gins. Notably, early-euchromatic origins also gained MCM remained unduplicated in G2, in sir2Δ cells both late-euchromatic complexes relative to late-euchromatic origins (Fig. 4), and the and telomeric X origins remained unduplicated. Sort-Seq data indicated that several of these origins generated In sir2Δ cells with a cis mutation that weakens the rDNA or- higher-ranking Z scores in sir2Δ cells, indicative of enhanced igin, referred to here as r-origin*, rDNA-dependent sequestra- replication efficiency. Two-dimensional origin mapping of early- tion of origin activation factors is reduced, allowing for partial euchromatic origin, ORI922 that gained MCM signals and ac- rescue of sir2Δ-induced replication gaps (31) (SI Appendix, Fig. quired an enhanced S-phase Z score in sir2Δ cells, also revealed S3). If changes in MCM distribution contributed to the sir2Δ- that its origin activity was enhanced in sir2Δ cells (SI Appendix, induced incomplete duplication of late-euchromatic origins in Fig. S7). Thus Sir2’s direct attenuation of origin licensing at G2 (Fig. 6A), then duplication of these elements should not be telomeric X origins and early-euchromatic origins limited their rescued by the r-origin* mutation, whereas duplication of telo- origin function and prevented replication gaps from forming in meric X origins that had gained MCM in sir2Δ cells should be late-replicating euchromatin. (Fig. 4). Therefore, the cohorts were assessed in sir2Δ r-origin* cells (Fig. 6B). In sir2Δ r-origin* cells, duplication of the telo- Discussion meric X origin cohort in G2 was rescued (Fig. 6 A and B; Recent evidence reveals that the yeast Sir proteins, known for compare early-euchromatic and telomeric X origins in G2 in their roles in forming yeast heterochromatin (also known as sir2Δ r-ORIGIN cells to sir2Δ r-origin* cells), supporting the transcriptionally silent chromatin), also act directly within eu- model wherein duplication of telomeric X origins in sir2Δ cells is chromatin where they can have an impact on chromosome rep- limited by the availability of origin activation factors (31). In lication (25, 27, 38). In particular, Sir2 and Sir3 act directly on

Hoggard et al. PNAS Latest Articles | 5of8 Downloaded by guest on September 24, 2021 A SIR2 r-ORIGIN sir2Δ r-ORIGINB sir2Δ r-origin* Sir2 prevents early-replicating origins from being as effective at origin licensing as they could be, thus limiting the collective origin 2 activity that would otherwise be possible in early euchromatin < 1E-6 0.002 (Fig. 6D). Because MCM complexes are the substrate for the < 1E-4 < 1E-6 0.1 limiting origin activation factors, and early-replicating euchroma- 1 tin has active mechanisms for recruiting these factors, a shift in MCM complexes toward early-replicating euchromatin would be 0 expected to lead to a concomitant shift in origin activation factors, resulting in more origin activity in early-replicating euchromatin than required for efficient duplication, and a depletion of origin

SortSeq Z-score -1 activity from late-replicating euchromatin. The ultimate outcome is a reduced probability that late-replicating euchromatin will com- D S Early G2 S Early G2 S Early G2 plete duplication in a timely manner (Fig. 6 ). While this simple Replication time model helps explain the data, additional experiments will be re- Sir2-heterochromatin quired to address the relative contributions of Sir2’s distinct roles at Early Mid Late X-origins the rDNA array and in origin licensing at non-rDNA origins to the C SIR2 sir2Δ regulation of genome duplication. While Sir2-heterochromatin inhibition of origin function at Early telomeres is established, it was unknown whether this inhibition occurs at the level of origin licensing or activation (24). Thus, an Early X-originsX-origins important observation was that Sir2 attenuated origin licensing LateLate at telomeric X origins substantially, though clearly not com- X-origins sir2Δ Late pletely. The increased licensing of telomeric X origins in Probability cells provided a mechanism for them to compete more effec-

MCM activation tively than late-euchromatic origins for origin activation factors (Fig. 6D). In the absence of Sir2, both early-euchromatic and Probability MCM load telomeric X origins were more competitive for MCM complexes, which should make them more competitive for origin activation Fig. 6. SIR2 was required for completing replication of late-euchromatic A factors. Notably, the rDNA origin itself did not show a relative regions independent of rDNA replication demands. ( ) Z scores for the in- sir2Δ dicated origins from S- and early G2-phase SIR2 and sir2Δ cells derived from gain in MCM signals in cells, in agreement with an earlier raw data in ref. 35 were determined as in Fig. 5C and displayed as report (35). However, activation of a Sir2-suppressed transcrip- box-and-whiskers plots for early-, mid, and late-euchromatic origins, and tion unit within rDNA in sir2Δ cells shifts MCM complexes to telomeric X origins. (B) Analyses as in A except with data from the sir2Δ regions adjacent to the rDNA origin (35). The repeat nature of r-origin* mutant. (C) Sir2 controls the relative replication probability of the the rDNA origin and this reported transcription-dependent three indicated origin cohorts at the levels of origin licensing efficiency MCM-complex shifting limited our ability to interpret Sir2’s (probability MCM load), and, at least in part via Sir2 control of the rDNA role in rDNA origin licensing by the approach used in this study. array, availability of origin activation factors (probability of MCM activa- There is a consensus that early origins exist within the most tion). While origin licensing and activation are distinct steps, loaded MCM “ ” complex is the substrate for origin activation factors. Thus alterations in open and transcriptionally active regions of the genome (3, 8, origin licensing efficiencies affect the cohorts’ competitiveness for origin 11, 18). Thus, it was initially perplexing that Sir2 chromatin, activation factors. known for its ability to inhibit origin function, was present at higher levels and had a greater impact on origin licensing at early- compared to late-euchromatic origins. We posit that the nucleosomes adjacent to euchromatic DNA replication origins to same molecular properties that make early-euchromatic origins inhibit origin licensing (25). However, the function of this Sir2- more accessible to origin regulatory machinery also make them chromatin state has thus far only been assessed in cells where more accessible to Sir proteins diffusing through the nucleo- one of the core origin licensing proteins is defective (e.g., cdc6-4, plasm. While Sir proteins are concentrated within heterochro- orc5-1, mcm2-1 yeast) (25, 28, 29), leaving a physiological role matin domains, Sir proteins associate with these regions for Sir2 in origin licensing an open issue. dynamically, releasing and rebinding (27, 39, 40). Released Sir Here, genomic and computational approaches revealed that in proteins will have opportunities to sample other regions of the the absence of Sir2, MCM complexes shift toward early- relative genome. The greater accessibility of early euchromatin might to late-replicated euchromatin. Sir2’s negative effects on origin facilitate the more frequent sampling of early-euchromatic ori- licensing depend on its catalytic deacetylation of nucleosomes gins by “free-agent” Sir proteins (26, 41, 42). (25, 28, 29). Therefore, the repressive properties of Sir2 chro- Classic suppressor genetics uncovered Sir2 chromatin’s nega- matin (e.g., reduced DNA accessibility and nucleosome mobility) tive effect on origin licensing, a phenomenon that, like other act to attenuate origin licensing by reducing the probability that a negative forms of origin regulation, would be difficult to uncover complete reaction will be completed. Any one or several discrete without genetics (28, 43). Building on this foundation, the ap- steps (e.g., ORC binding, Cdc6 binding, Cdt1-MCM association, proaches used here to quantitatively analyze large cohorts of etc.) could be affected, and to varying degrees at individual or- precisely mapped origins benefitted from the deeply annotated igins. Higher levels of Sir2 chromatin at Sir2-responsive origins yeast genome and were essential to revealing the potential phys- indicate that the levels of this repressive state are likely one iological relevance of Sir2-mediated attenuation of origin licens- component that affects the probability of origin licensing, but ing. In a microbe-like yeast, such a genome-scale effect could other variables could also be relevant, enhancing the stochas- impinge on organismal fitness by reducing cell proliferation rates ticity of this attenuating mechanism (25). and/or genome stability, as delayed replication enhances mutation In terms of cell physiology, a key point is that small reductions frequency (44, 45). In mammalian cells, SirT1, the human ortho- in the probability of origin licensing at any one origin act cumu- log of yeast Sir2, as well as Set8-mediated chromatin compaction, latively over multiple origins to impose a genome-scale impact: limit origin function and promote genome stability (46–48). No- origin density is concentrated in early-replicating euchromatin and tably, Set8-modified chromatin limits MCM binding to chromatin. at telomeres relative to late-replicating euchromatin. In essence, Thus, the use of repressive chromatin modifiers for achieving

6of8 | www.pnas.org/cgi/doi/10.1073/pnas.2004664117 Hoggard et al. Downloaded by guest on September 24, 2021 genome-scale control of origin licensing is emerging as an im- ChIP/input ratio measured between coordinates −600 to −400. Per- portant regulatory node of eukaryotic chromosome duplication nucleotide signal distribution for an origin cohort was the per-nucleotide and stability. The tools available to budding yeast allowed us to median ± 1 quartile of the distribution of scaled ratios for all origins within show how such a mechanism could promote the normal distribu- a cohort. tion of origins across chromosomes. While the importance of origin distribution to genome stability has become clear, this study Determining H4K16ac Levels at Nucleosomes Adjacent to Euchromatic Origins. identifies a specific chromatin-mediated mechanism to establish H4K16ac ChIP/input ratios for each nucleotide within each nucleosome annotated in ref. 53 were summed. Six nucleosomes, three 5′ and three 3′ to the spatial distribution of MCM complexes across a eukaryotic A – the T-rich ORC binding site, were mapped to 1.2-kb fragments (Fig. 3 ). genome (49 51). Nucleosomes were also mapped to a control group of loci, 391 randomly selected distinct euchromatic 1.2-kb regions that were not annotated in the Materials and Methods OriDB and lacked an ORCACS match. For each control fragment, the mean Yeast strains were congenic derivatives of W303-1A and have been H4K16ac level for six nucleosomes, was used to normalize the H4K16ac levels published (25). for each nucleosome surrounding the experimental loci. S-phase Sort-Seq experiments were performed on yeast growing at the ’ Sequencing Data. Raw data were assigned the following BioProject ID s: permissive-growth temperature for cdc6-4 (23 °C) as described in ref. 36. PRJNA428768 and PRJNA601998 for reads from the MCM ChIP-Seq experiments; PRJNA601998 for reads from the S Sort-Seq experiments; and GEO Data Availability. Raw data and methods used in this study are available on accession GSE90151 for reads from the S and G2 Sort-Seq experiments (31). public databases or provided within this manuscript.

Determining Per-Nucleotide MCM ChIP-Seq Signals and Distributions. MCM ACKNOWLEDGMENTS. We thank Melissa Harrison (University of Wisconsin– ChIP-Seq data from three independent experiments were mapped to Madison) for critiquing early drafts of the manuscript, and Erika Shor (Center sacCer3 using Bowtie2 and default parameters. Duplicates were removed for Discovery and Innovation, Hakensack Meridian Health), Xiaolan Zhao using MarkDuplicates within Picard tools. Per-nucleotide coverages were (Memorial Sloan Kettering Cancer Center), and members of the C.A.F. labo- determined using Samtool’s BedCov. Coverages for each independent ChIP ratory for thoughtful discussions. Support for this work was provided by NIH were normalized for sequencing depth and breadth (52). ChIP/input ratios GM056890 to C.A.F., Biotechnology and Biological Sciences Research Council were mapped to the nucleotides within origin-containing fragments, and Grant BB/N016858 to C.A.M. and C.A.N., and Wellcome Trust Investigator internally scaled for each nucleotide by dividing each ratio by the median Award 110064/Z/15/Z to C.A.N.

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