Sir2 and Fun30 regulate ribosomal DNA replication timing via MCM helicase positioning and nucleosome occupancy

The association between late replication timing and low transcription rates in eukaryotic heterochromatin is well-known, yet the specific mechanisms underlying this link remain uncertain. In Saccharomyces cerevisiae, the histone deacetylase Sir2 is required for both transcriptional silencing and late replication at the repetitive ribosomal DNA arrays (rDNA). We have previously reported that in the absence of SIR2, a derepressed RNA PolII repositions MCM replicative helicases from their loading site at the ribosomal origin, where they abut well-positioned, high-occupancy nucleosomes, to an adjacent region with lower nucleosome occupancy. By developing a method that can distinguish activation of closely spaced MCM complexes, here we show that the displaced MCMs at rDNA origins have increased firing propensity compared to the nondisplaced MCMs. Furthermore, we found that both, activation of the repositioned MCMs and low occupancy of the adjacent nucleosomes critically depend on the chromatin remodeling activity of FUN30. Our study elucidates the mechanism by which Sir2 delays replication timing, and it demonstrates, for the first time, that activation of a specific replication origin in vivo relies on the nucleosome context shaped by a single chromatin remodeler. Yeast strain Strains used in the study were derived from BY4741 and W303 and are provided in Suplemental Table 1. Cells were grown in standard yeast peptone 2% dextrose media except for hydroxyurea time course study which employed synthetic media. Strains containing point mutations in FUN30 were made using a plasmid that contains CAS9 and gRNA targeting the PAM sites adjacent to the desired mutation sites in FUN30 as described. Oligos containing FUN30-targeting gRNAs were cloned into pML104. pML104 was a gift from John Wyrick (Addgene plasmid # 67638)(Laughery et al. 2015). The plasmids, which induce double strand breaks within FUN30, were co-transformed with DNA blocks containing the repair templates with the desired mutations and altered PAM sites, synthetically synthesized by IDT. FUN30 DNA was PCR amplified from the resulting transformants and sequenced to verify that the desired mutations have been introduced. Edu-incorporating yeast strains were derived from W303 and contain four Brdu vectors that express HSV-TK and hENT1 ((Viggiani and Aparicio 2006). Plasmids containing BrdU cassete obtained from Addgene (Addgene plasmid # 71789-71792), were integrated at four different auxotrophic loci (HIS3, TRP1, LEU2, and URA3). Single integration of each Brdu cassette was confirmed via PCR using plasmid-specific primer sets as described ((Viggiani and Aparicio 2006), which assured that the strains contain exactly four copies of the BrdU cassette Measurements of rDNA replication timing using S-seq S-seq experiments were carried out as previously described (Foss et al. 2017). Cells were grown to log phase in YEPD media, fixed using 70% (vol/vol) ethanol, subjected to proteinase K digestion, and their DNA stained with Sytox Green as described (Foss 2001). Cells were sorted according to DNA content on a BD Biosciences FACSAria II cell sorter into G1 and S fractions. DNA from a minimum of 1 e-6 cells from each fraction was isolated using the YeaStar Genomic DNA Kit (Zymo Research). DNA was fragmented by sonication and sequenced. MNase seq We carried out MNase-Seq as previously described (Foss 2019). Briefly, cells grown to log phase in rich medium, Yeast Peptone Agar with 2% glucose (YEPD), from an overnight 25 mL culture were synchronized with 3 μM alpha-factor for 1.5 hrs. at 30 ° C. Arrested cells were crosslinked with 1% formaldehyde for 30 min at room temperature water bath with shaking. Formaldehyde was quenched with 125 mM glycine and cells were centrifuged at 3000 rpm for 5 min. Cells were washed twice with water and resuspended in 1.5 mL Buffer Z (1 M sorbitol, 50 mM Tris-HCl pH 7.4) with 1 mM beta-mercaptoethanol (1.1 μL of 14.3 M beta-mercaptoethanol diluted 1:10 in Buffer Z) per 25 mL culture. Cells were treated with 100 μL 20 mg/ mL zymolyase at 30 ° C for 20–30 min. Spheroplasts were centrifuged at 5000 rpm for 10 min and resuspended in 5 mL NP buffer (1 M sorbitol, 50 mM NaCl, 10 mM Tris pH 7.4, 5 mM MgCl2, 1 mM CaCl2) supplemented with 500 μM spermidine, 1 mM beta-mercaptoethanol and 0.075% NP-40. Nuclei were aliquoted in tubes with varying concentrations of micrococcal nuclease (Worthington), mixed via tube inversion, and incubated at room temperature for 20 mins. Chromatin digested with 1.9 U– 7.5 U micrococcal nuclease per 1/5th of spheroplasts from a 25 mL culture yielded appropriate mono-, di-, tri- nucleosome protected fragments for next-generation sequencing. Digestion was stopped with freshly made 5x stop buffer (5% SDS, 50 mM EDTA) and proteinase K was added (0.2 mg/ml final concentration) for an overnight incubation at 65 ° C to reverse crosslinking. DNA was extracted with phenol/chloroform and precipitated with ethanol. Micrococcal nuclease digestion was analyzed via gel electrophoresis prior to proceeding to library preparation. Sequencing libraries were prepared as described below for ChEC. Chromatin Endogenous Cleavage (ChEC) ChEC-seq was carried out as previously described (Foss et al. 2019; Foss et al. 2021). Briefly, MNase was activated by addition of CaCl2 to cells that were permeabilized with digitonin. DNA was extracted from cells using phenol and chloroform, precipitated using salt and ethanol and used to construct sequencing libraries without size fractionation. Cells were centrifuged at 1,500 x g for 2 mins, and washed twice in cold Buffer A (15 mM Tris pH 7.5, 80 mM KCl, 0.1 mM EGTA) without additives. Washed cells were carefully resuspended in 570 μL Buffer A with additives (0.2 mM spermidine, 0.5 mM spermine, 1 mM PMSF, ½ cOmplete ULTRA protease inhibitors tablet, Roche, per 5 mL Buffer A) and permeabilized with 0.1% digitonin in 30 ° C water bath for 5 min. Permeabilized cells were cooled at room temperature for 1 min and 1/5th of cells were transferred in a tube with freshly made 2x stop buffer (400 mM NaCl, 20 mM EDTA, 4 mM EGTA)/1% SDS solution for undigested control. Micrococcal nuclease was activated with 5.5 μL of 200 mM CaCl2 at various times (5 mins, and 10 mins) and the reaction stopped with 2x stop buffer/1% SDS. Once all time points were collected, proteinase K was added to each of the collected time points and incubated at 55 ° C water bath for 30 mins. DNA was extracted using phenol/chloroform and precipitated with ethanol. Micrococcal nuclease digestion was analyzed via gel electrophoresis prior to proceeding to library preparation. Library was prepared as previously described used using total DNA, without any fragment size selection (Foss et al. 2019; Foss et al. 2021). Chromatin immunoprecipitation followed by sequencing (ChIP-seq) Cells were grown to log phase in rich medium, YEPD (1% Yeast Extract, 2% Peptone, 2% glucose), synchronized with 3 μM alpha-factor for 1.5 hrs. at 30°C. G1 arrested cells were released into 200 mM hydroxyurea (HU) YEPD and grown for 1hr. Cells were crosslinked with 1% formaldehyde for 30 min at room temperature, quenched with 125 mM glycine for 5 minutes, and centrifuged at 3000 rpm for 5 minutes. Cell pellets were washed three times with TBS (20 mM Tris-HCl, pH 7.6, 150 mM NaCl). Cell pellets were flash frozen. Chromatin Preparation The cell pellet was thawed in 300 μL Breaking Buffer (100 mM Tris- HCl, pH 8.0, 20% Glycerol) supplemented with protease inhibitors (Pierce Protease inhibitor EDTA-free tablets [Thermo Scientific A32965] and 1 mM PMSF). 300 μL acid-washed glass beads were added to the mixture. Cells were lysed using a BioSpec Mini-Bead-beater in a cold room: 5x 30-second pulses with 1-minute on ice between rounds. The supernatant was transferred to a new tube and added 600 μL FA buffer (50 mM Hepes-KOH, pH 7.6, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% Sodium deoxycholate) with protease inhibitors. The chromatin was sonicated with a F60 Sonic Dismembrator 10 times for 10 seconds at setting 4, with 1-minute rests on ice between rounds. The sonicated chromatin was centrifuged at 14,000 rpm for 15 minutes at 4°C. The supernatant was transferred and spun again at 14,000 rpm for 15 minutes at 4°C. Immunoprecipitation (IP) For IP, 20 μL of Dynabeads Protein G beads (Invitrogen 10004D) were washed three times with 500 μL PBS-T (0.8% NaCl, 0.144% Na2HPO4, 0.02% KCl, 0.024% KH2PO, 0.1% Tween 20, pH 7.1), resuspended in PBS-T, and incubated with 5 μL of FLAG M2 (Sigma F1804) antibody for 60 minutes at room temperature. The antibody-conjugated beads were washed and resuspended in 20 μL FA buffer with protease inhibitors. IP chromatin samples (1 μg) were brought up to 400 μL with FA buffer with protease inhibitors and incubated with antibody- conjugated beads for 90 minutes at room temperature. Beads were washed three times with FA buffer with protease inhibitors, twice with FA-HS buffer (50 mM Hepes-KOH, pH 7.6, 500 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% Sodium deoxycholate with protease inhibitors), and once with RIPA buffer (10 mM Tris-HCl, pH 8.0, 0.25 M LiCl, 0.5% NP-40, 0.5% Sodium deoxycholate, 5 mM EDTA with protease inhibitors). Input sample was generated by adding 0.1 μg chromatin in 40 μL FA buffer to 40 μL 2X Stop buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 20 mM EDTA, 1% SDS, 2% Tween 20) and reversed crosslinks by overnight incubation at 65°C. Elution and Crosslink Reversal Bound chromatin was eluted by incubating the beads in 80 μL 2X Stop buffer at 75°C for 10 minutes. The eluate was collected, and crosslinks were reversed by overnight incubation at 65°C. The IP and Input were treated with 4 μL of 20 mg/mL RNase A and incubated at 55°C for 1 hour, followed by 4 μL of 20 mg/mL Proteinase K and incubation at 55°C for at least 3 hours. DNA was purified using a MinElute PCR Purification Kit Paired-Ends NGS Chromatin immunoprecipitation samples were prepared for sequencing following the protocol for the Kapa Hyper Kit. For Input samples, 2 ng were processed for End Repair. For IP samples, the entire volume was used. PCR amplification was performed with 10- 14 cycles based on DNA quantity. Quantification of rDNA replication using the thymidine analogue EdU Cells were grown to log phase in rich YEPD medium from a 50 mL culture and synchronized in G1 using 3 μM alpha factor for 2 hours at 30°C. Using 500 μg/mL pronase, arrested cells were released into rich media containing 200 mM hydroxyurea and 130 μM EdU (5-Ethynyl-2’- deoxyuridine). Cells were harvested in G1 and after 60 minutes released into HU. DNA was extracted using YeaStar Genomic DNA Kit (Zymo Research) and sonicated. Separate sequencing primers were ligated to each of the samples. Samples were pooled together, and subjected to click chemistry reaction in 10 mM Tris pH 7.4, 50mM NaCl, 20 μM biotin-TEG azide (Vector Laboratories), 5mM THPTA (tris-hydroxypropyltriazolylmethylamine) (Vector Laboratories), 1mM copper sulfate, and 15 mM sodium ascorbate (total volume 200 μL). Click reaction proceeded for 30 minutes at room temperature prior to DNA ethanol precipitation. Streptavidin magnetic beads (New England Biolabs) were washed 3 times with B&W buffer I (100 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.05% Tween20, 0.5% SDS) before adding 100 μL of bead suspension to 180 μL DNA plus B&W buffer I. After a 30-minute incubation at room temperature while gently inverting tubes, bead bound DNA was washed with 200 μL B&W buffer I followed by 100 μL Stringency wash buffer (0.1% SDS, 0.1% SSC buffer) and 200 μL Wash Buffer II (100 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.05% Tween 20). DNA was eluted off the beads by denaturation at 98°C for 5 minutes, followed by cooling at 25°C. Edu-labeled DNA was PCR amplified post-pulldown and subjected to sequencing. Hydoxyurea ChEC time course experiment Cells grown in synthetic media to logarithmic phase were arrested in G1 using alpha factor for 90 minutes, centrifuged, washed in water to remove alpha factor, and released into media containing 200 mM Hydroxyurea. Cells were harvested in G1 and at indicated time points following release into HU-containing media, chilled at 4 degrees and analyzed by ChEC as described above.