Method Details

The Strep-MBP-TEV-Rap1-Halo construct (Figure S2F) was cloned into pACEBac1 (Geneva Biotech) and baculovirus particles were generated using the Geneva Biotech system per manufacturer’s instructions.

For Rap1 expression, 1L cultures of Sf9 cells were grown to 2 - 2.5x10⁶ cells/mL. Subsequently, the cells were infected with baculovirus, and the cultures were incubated for 3 days at 27°C, before harvesting through centrifugation (1500 rcf., 4°C for 20 min). Supernatants were discarded and pellets were resuspended in PBS, containing protease inhibitors (Roche) (10 mL PBS/L of culture), flash frozen and kept at −80°C.

For a typical purification of Rap1-Halo, 12-15 g of frozen pellets were thawed at room temperature with 36 mL of lysis buffer (200 mM KCl, 2 mM DTT, 100 mM Tris-HCl (pH 7.5), 50 mM MgOAc, 0.1% NP-40, Protease inhibitor cocktail (Roche), 1mM PMSF and 20 μL DNaseI (NEB)). Pellets, were stirred with a magnetic stir bar until fully thawed and then kept on ice. The lysate was spun for 35 min at 35000 rpm at 4°C (Ti70 rotor, Beckman Coulter) and the supernatant was filtered through a 5 μm syringe filter (Millex, Millipore). The cleared lysate was loaded onto a Strep-Trap column (GE, AKTA system), pre-equilibrated with lysis buffer. The column was washed with storage buffer (200 mM KCl, 10 mM HEPES pH 7.6, 50 mM MgOAc, and 5 mM β-mercaptoethanol (βME)) and the protein was eluted with 5 x column volumes (CV) of elution buffer (storage buffer containing 2.5 mM desthiobiotin). Fractions containing Rap1 were identified by SDS-Page (Figures S2G and S2H), pooled and concentrated to ∼500 μL total volume using Amicon 10k molecular weight cut-off (MWCO) centrifugal filters. The protein concentration was determined using UV spectroscopy. The MBP tag was subsequently removed by TEV protease digestion at 4°C (Figure S2I). For labeling, Janelia Fluor-549 HaloTag (Janelia, JF-549) was added at a protein to dye ratio of 1:1.5 followed by incubation for 1h. Labeled Rap1 was finally purified by size exclusion chromatography (SEC) using a Superose6 10/300 GL column (GE healthcare) in storage buffer using a flow-rate of 0.4 mL/min (Figure S2J). Fractions were analyzed using SDS-PAGE (Figure S2K), clean fractions were pooled, concentrated (Amicon 10k MWCO filter) and protein concentrations were determined using UV spectrophotometry (at A280 and A571). Finally, labeling efficiency was calculated by using the extinction coefficients for Rap1 (107’065 mol^-1 cm^-1) and JF-549 (101’000 mol^-1 cm^-1). Typical labeling efficiency was found to be > 90%.

Histones were expressed and purified as described in Kilic et al. (2015). Briefly, individual wild-type human histones were cloned into pet15b plasmid vectors and expressed in BL21 DE3 plysS cells. Cells were grown in LB media containing 100 μg/mL ampicillin and 35 μg/mL chloramphenicol at 37°C until the OD₆₀₀ reached 0.6. Expression was induced by IPTG addition to a final concentration of 0.5 mM. After 3 h expression, cells were harvested by centrifugation and resuspended in lysis buffer (20 mM Tris pH 7.5, 1 mM EDTA, 200 mM NaCl, 1 mM βMe, Roche protease inhibitor) and frozen. Cells were lysed by freeze-thawing and sonication. Inclusion bodies were harvested by centrifugation. The inclusion body pellet was washed once with 7.5 mL of lysis buffer containing 1% Triton and once without. Inclusion body pellets were resolubilized in resolubilization buffer (6 M GdmCl, 20 mM Tris pH 7.5, 1 mM EDTA, 1 mM βMe) and dialyzed into urea buffer (7 M urea, 10 mM Tris, 1 mM EDTA, 0.1 M NaCl, 5 mM 1 mM βMe, pH 7.5). Histones were purified by cation exchange chromatography using a HiTrap SP HP 5 mL column (GE Healthcare). Fractions were analyzed by SDS-PAGE and pooled, followed by dialysis into water and lyophilization. Final purification was performed by preparative RP-HPLC. Purified histones were lyophilized and stored at −20°C until used for octamer refolding.

Plasmids containing recombinant DNA fragments for chromatin DNA assembly, which have been prepared previously (Kilic et al., 2018b) (recP1, recP5) or were newly generated using restriction digestion and ligation of previous fragments (recP1P2 or recP4P5, Figure S4B) were transformed into DH5α cells (for sequence information see Table S1). Cells were cultured overnight in 6L 2xTY medium and harvested by centrifugation. For alkaline lysis, the cells were resuspended in 120 mL lysis solution I (50 mM glucose, 25 mM Tris pH 8, 10 mM EDTA). 240 mL lysis solution II (0.3 M NaOH, 1% SDS) was added and mixed by stirring. 240 mL lysis solution III (4 M KAc, 2 M acetic acid) was added to neutralize the solution which was left at 4°C for 15 min. After centrifugation, the supernatant was passed through Miracloth (Merck). Plasmid DNA was collected by isopropanol precipitation: 0.52 volume equivalents of isopropanol was added followed by centrifugation at 11’000 x g for 20 min at 4°C. The DNA pellet was dissolved in TE 10/50 (10 mM Tris pH 7.5, 50 mM EDTA) in the presence of 100 units of RNase A, and digested for 2 h at 37°C. To perform SEC the buffer was adjusted to 2M KCl (10 mM Tris, 50 mM EDTA and 2 M KCl). The plasmid was then purified in the same buffer on a XK 50/30 column (GE Healthcare) containing a bed of 550 mL Sepharose 6 Fast Flow (GE Healthcare). Eluted plasmid DNA from was precipitated with isopropanol. The pellet was finally dissolved in TE 10 / 0.1 (10 mM Tris pH 7.5, 0.1 mM EDTA) and stored at −20°C.

Purified plasmid DNA was collected by isopropanol precipitation and the DNA pellet was dissolved in milliQ H₂O. For a typical reaction, either 200 units of DraIII-HF (NEB) (for recP1P2) or 200 units of BsaI-HF (NEB) (for recP4P5) or 200 units of both DraIII-HF and BsaI-HF (NEB) (for recP1 and recP5) were added to 200 pmol of plasmid DNA in 200μl 1x NEB CutSmart buffer. After 8-10 h digestion at 37°C, digestion progress was analyzed by gel electrophoresis on a 1% agarose gel (run in 1 x TBE running buffer, 100 V, for 50 min) to check completeness. If required, the digestion was pushed to completion by adding another 100 units of enzyme and incubation for further 8-10 h at 37°C. Once the digestion was complete, 100 units EcoRV-HF (NEB) was added and left 8-10 h at 37°C. Complete digestion was verified by electrophoresis as described above. If the digestion was not complete an additional 50 units of enzyme was added and left 8-10 h at 37°C. Once the digestion was complete, the desired chromatin DNA fragments were purified from the plasmid remnants through successive PEG precipitations. This involves adding 40% PEG 6000 to the digestion reactions until a final concentration of 5%–6% PEG 6000 was reached. Additionally, the NaCl concentration was adjusted to 0.5 M. The sample was then spun at 20’000 x g at 4°C for 20 min. The supernatant was collected, and PEG 6000 was added to the supernatant to increase the final PEG % by increments of 0.5%. The sample was then spun at 20’000 x g at 4°C for 20 min. This was repeated until a suitable purity was achieved. Finally, the chromatin DNA fragments were isolated using QIAquick PCR purification spin columns (QIAGEN).

Fluorescently labeled oligonucleotides were generated as described in Kilic et al. (2018b). Briefly, 5-10 nmol of single stranded oligonucleotide, containing amino modified C6 dT, was diluted in 25 μl 0.1 M sodium tetraborate, pH 8.5. 5 μl of a 5 mM stock of succinimidyl-ester modified fluorophore (Alexa 568, Alexa 647 or Cy3B) were added to the reaction mix and left shaking at room temperature for 4 – 8 hours. For a table enumerating all labeled oligonucleotides see Table S2.

Reaction progress was analyzed by RP-HPLC using a gradient from solvent A (95% 0.1M triethylammonium acetate (TEAA) pH 7, 5% ACN) to solvent B (70% 0.1M TEAA pH 7, 30% ACN) on a 3 μm 4.6x150 mm InertSustain C18 column (GL sciences) over 20 min. More dye was added when required. For purification, the labeled DNA was ethanol precipitated (by the addition of 2.75 equivalents of cold ethanol, 0.3M NaOAc pH 5.2, followed by centrifugation at 20’000 x g at 4°C for 20 min) twice successively to remove excess unconjugated dye. The DNA pellet was finally dissolved in 100 μl solvent A and purified by HPLC. The purified DNA was finally ethanol precipitated and dissolved in milliQ water to a concentration of 2.5 μM.

Labeled DNA was prepared by PCR (fragments P2, P3_S1, P3_S2, P3_S2^∗, P3_S1S2, P3_Rpl30, P3_Rpl30_S1 and P4, for sequences and labeling schemes, see Tables S1, S2, and S3). For a typical reaction, 96 × 50 μL PCR reactions in 1 x ThermoPol reaction buffer (NEB) were prepared using template (0.01 ng μL^-1), forward primer (0.250 μM), reverse primer (0.250 μM), dNTPs (0.2 mM, NEB) and Taq DNA polymerase (1.25 units, NEB). A typical program included an initial step of 12 s at 94°C, followed by 30 cycles of 12 s at 94°C, 12 s annealing at 58-65°C and 12 s extension at 72°C. Final extension was also done at 72°C for 12 s. PCR reactions were subsequently purified using QIAquick PCR purification spin columns (QIAGEN).

About 0.33 nmol of PCR generated DNA (P3_S1, P3_S2, P3_S2^∗, P3_S1S2, P3_Rpl30 and P3_Rpl30_S1, Table S1) was digested in 200 μl of 1 x CutSmart buffer using 100 units of BsaI-HF (NEB) and 100 units of DraIII-HF (NEB) for 8-10h at 37°C. The progress of the digestion was analyzed on a 2% agarose gel (running conditions: 1 x TBE, 110 V for 50 min). Finally the DNA fragments were purified using QIAquick PCR purification spin columns (QIAGEN) and the concentration was determined by UV spectroscopy.

For the generation of nucleosome DNA for single-molecule experiments, a biotin containing anchor (Anchor_rev, Table S2) was annealed to its complementary strand containing a phosphorylated 5′- BsaI overhang (P3_Anchor_fwd, Table S2) and a 10-fold excess was added to 150-300 pmol (∼20-40 μg) of digested PCR generated DNA (P3_S1, P3_S2, P3_S2^∗ and P3_S1S2) in 100 μl 1x T4 ligase buffer (NEB). Upon complete ligation of digested DNA, excess biotin anchor was removed by PEG precipitation. Finally, the DNA fragments were purified using QIAquick PCR purification spin columns (QIAGEN) and the concentration was determined by UV spectroscopy.

Nucleosomes (MN_S1, MN_S2, MN_S2^∗, MN_S1S2, MN_S1_FRET, MN_S2_FRET, MN_Rpl30, MN_Rpl30_S1, MN_Rpl30_S1_FRET, Table S3) were prepared following Dyer et al. (2004). Typically, 1-5 μg of labeled and biotinylated DNA (P3_S1, P3_S2, P3_S2^∗ and P3_S1S2) was combined with purified refolded octamers at experimentally determined ratios (1:1 to 1:2, DNA to histone octamer) in 10 μl TE (10 mM Tris-HCl pH 7.5, 1 mM EDTA) supplemented with 2 M KCl. After a 30 min incubation period at room temperature, 10 μl TE was added and further incubated for 1 h. This was followed by sequential addition of 5 μl TE, 5 μl TE and finally 70 μl TE with 1 h incubation periods in between each addition, to arrive to 0.2 M KCl. Samples were then spun at 20’000 x g for 10 min at 4°C and the supernatant was kept on ice. To determine the quality of MN assemblies, 0.8% Agarose 0.25 x TB gels were run at 90 V on ice for 90 min (Figures S2A–S2E, S5C, and S5K).

EMSAs to determine Rap1 binding to DNA were done in single-molecule imaging buffer (IB, 50 mM HEPES pH 7.5, 130 mM KCl, 10% v/v glycerol, 0.005% v/v Tween 20, 2 mM Trolox, 3.2% w/v glucose), in the presence of 50 ng/μl poly-d(I-C) (Roche) and with 20 μL total volume. Typically, 200 nmol stocks of DNA and 3 μM stocks of Rap1-Halo were prepared and serially diluted to desired concentrations. Reactions were mixed by pipetting and left for 10 min at room temperature. Sucrose was added to a final concentration of 8% and reactions were loaded onto 5% Polyacrylamide gels run in 0.5 x TBE at 100 V for 60 min. Images were taken using ChemiDoc MP (Biorad) (Figures S1D and S1E). For densitometry quantifications, ImageLab (Biorad) software was used for band quantification of bound and unbound fraction of DNA. The data was analyzed in Origin (OriginLab) by non-linear curve-fitting using a sigmoidal function to determine K_d.

Singly-labeled and biotinylated 12x601 DNA was produced as shown in Figure S4A. Typically, 50-60 pmol of PEG purified restriction enzyme digested recP1P2 and an excess 1x601 P3 (P3_S1 and P3_S2) (between 20%–30% excess) were added to 200 μL 1 x T4 ligase buffer containing 400 units of T4 ligase. The reaction was followed using 1% agarose gels (Figure S4C). Upon completion, P1P2P3 was PEG purified (Figure S6F) and added to excess P4P5 and biotin labeled anchor (20%–30% excess P4P5 and 10-fold excess biotin anchor). The reaction was followed using 1% Agarose gels (Figure S4D). Upon completion, the complete DNA P1P2P3P4P5A was PEG purified and subsequently purified using Qiaquick PCR purification spin columns (QIAGEN), the concentration was determined by UV spectrophotometer (Figure S4E).

The FRET pair Cy3B and Alexa647, were site-specifically introduced respectively on P2 and P4 at the 39-base-pair position relative to the dyad in the 601 sequence (Figure S6A). About 30 pmol of each piece was used for 5-piece convergent DNA ligation to produce two intermediate 6 × 601 pieces as followed: recP1 was ligated to Cy3B-labeled P2 in 20% excess for 2 h using T4 DNA ligase, then unlabeled P3 in 20% excess relative to P2 was added and left to ligate another 15 h. Similarly, recP5 was ligated to 20% excess Alexa647-labeled P4 for 15 h (Figure S6D). Singly-labeled 6x601 intermediate fragments P1-3 and P4-5 were PEG purified from individual pieces. Pellets containing enriched fragments were collected, dissolved in 50 μL TE buffer (10 mM Tris, pH 8.0, 0.1 mM EDTA), and used for the final ligation (Figure S6E). A biotinylated anchor was added into the final ligation of 2 intermediate 6x601, and the reaction was proceeded for 15 h at room temperature. PEG precipitation was performed similarly to previous step, and the enriched final products were collected and purified using Qiaquick PCR purification spin columns (QIAGEN), the concentration was determined by UV spectrophotometer.

Chromatin fibers (CH_S1, CH_S2, CH_NS, CH_NS_FRET and CH_S2_FRET, Table S3) were reconstituted from singly/doubly-labeled and biotinylated 12x601 DNA and wild-type recombinantly purified human histone octamers. In a typical dialysis, 200-300 pM 12x601 DNA, 0.5-1 equivalents of MMTV DNA and reconstituted octamers (using experimentally determined DNA:octamer ratios) were added to a micro-dialysis unit (Thermo Scientific, Slide-A-Lyzer – 10’000 MWCO), then dialyzed in TE buffer (10 mM Tris pH 7.5, 0.1 mM EDTA pH 8.0) with a linear gradient from 2 M to 10 mM KCl for 16-18 h, and finally kept in TEK10 buffer (10 mM Tris pH 7.5, 0.1 mM EDTA pH 8.0, 10 mM KCl) for another 1 h. Chromatin assemblies were centrifuged at 21’000 x g for 10 min at 4°C, the supernatant was then transferred to a fresh tube. The concentration and volume of the chromatin assemblies was determined using UV spectrophotometer. Chromatin assembly quality was controlled by the appearance of MMTV nucleosomes and ScaI digestion of 12x assemblies. Digestion reactions were analyzed on a 0.8% agarose gel and 5% TBE polyacrylamide gel electrophoresis. All experiments were carried out at 4°C (Figures S4F–S4H, S6F, and S6G).

Cleaning, silanization and PEGylation of coverslips and glass slides was done described previously in Kilic et al. (2015). Briefly, coverslips (24 × 40 mm, 1.5 mm thickness) and glass slides (76 × 26 mm with 2 rows of 4 holes drilled) were sonicated for 20 min in 10% Aconox, rinsed with milliQ water and the procedure repeated sequentially with acetone and ethanol. Both coverslips and glass slides were then placed in piranha etching solution (25% v/v 30% H₂O₂ and 75% v/v H₂SO₄) for minimum 2 h. After thorough washing with milliQ H₂O, coverslips and slides were sonicated in acetone for 10 min, then incubated with 2% v/v aminopropyltriethylsilane (APTES) in acetone for 15 min, and dried. Flow-chambers were assembled from one glass slide and one coverslip separated by double-sided 0.12 mm tape (Grace Bio-labs) positioned between each hole in the glass slide, and the open ends were sealed with epoxy glue. Pipette tips were fitted in each of the 2 × 4 holes on each side of the silanized glass flow chambers as inlet reservoir and outlet sources and glued in place with epoxy glue. The glue was allowed to solidify for 30-40 min. Subsequently, 350 μL of 0.1 M tetraborate buffer at pH 8.5 was used to dissolve ∼1 mg of biotin-mPEG(5000 kDa)-SVA, and 350 μL from this was transferred to 20 mg mPEG (5000kDa)-SVA. This was centrifuged and mixed to homogeneity with a pipette before 40-45 μL aliquots were loaded into each of the four channels in the flow chamber. The PEGylation reaction was allowed to continue for the next 2½-4 h after which the solution was washed out with degassed ultra-pure water (Romil).

Measurements were done according to Kilic et al. (2015). Objective-type smTIRF was performed using a fully automated Nikon Ti-E inverted fluorescence microscope, equipped with an ANDOR iXon EMCCD camera and a TIRF illuminator arm, controlled by NIS-elements and equipped with a CFI Apo TIRF 100x oil immersion objective (NA 1.49), resulting in a pixel size corresponding to 160 nm. Laser excitation was realized using a Coherent OBIS 640LX laser (640 nm, 40 mW) and coherent OBIS 532LS laser (532 nm, 50 mW) on a custom setup laser bench. Wavelength selection and power modulation was done using an acousto-optical tunable filter (AOTF) controlled by NIS-elements. Typical laser intensities in the objective used for measurements were 0.8 mW for both 532 nm and 640 nm laser lines. For all smTIRF experiments, flow channels were washed with 500 μL degassed ultrapure water (Romil), followed by 500 μL 1 x T50 (10 mM Tris pH 8, 50 mM NaCl) and background fluorescence was recorded with both 532 nm and 640 nm excitation. 50 μL of 0.2 mg/mL neutravidin was then injected and incubated for 5 min, and washed using 500 μL 1xT50. 50 pM of Alexa647 labeled DNA/mononuceosomes/12-mer chromatin assemblies were then flowed in for immobilization in T50 with 2 mg/mL bovine serum albumin (BSA, Carlroth) (25 × 50 μm imaging area was monitored using 640 nm excitation to check for sufficient coverage). 500 μL 1 x T50 was used to wash out unbound Alexa647 labeled DNA/mononuceosomes/12-mer chromatin assemblies. 50-100 pM JF-549 labeled Rap1-Halo (see table below for details) was flowed in using imaging buffer (50 mM HEPES pH 7.5, 130 mM KCl, 10% v/v glycerol, 0.005% v/v Tween 20, 2 mM Trolox, 3.2% w/v glucose, 1x glucose oxidase/catalase oxygen scavenging system and 2 mg/mL BSA). Images were recorded using the following parameters:

Here t_on denotes the camera integration time, whereas t_off indicates interspersed time intervals of camera inactivity.

Each experiment was repeated several times (see Table S4 for number of repeats), using at least two independently produced chromatin preparations on two different days.

Slides were prepared as described in the preceding sections. However, no BSA was added to imaging buffer (50 mM HEPES pH 7.5, 130 mM KCl, 10% v/v glycerol, 0.005% v/v Tween 20, 2 mM Trolox, 3.2% w/v glucose, 1x glucose oxidase/catalase oxygen scavenging system). JF-549 labeled Rap1-Halo was flown into the channel and nonspecifically adsorbed on the glass surface. Movies were recorded using continuous 532 nm illumination (t_on 50 msec and t_off 0.3 msec) using the indicated excitation laser powers (Figure S3A). Absolute laser power was determined using a laser power meter at the objective.

All measurements were performed using a Fluorolog®-3 Horiba Jobin Yvon spectrofluorometer, in T50 buffer (10 mM Tris pH 8, 50 mM NaCl) 60 μl total volume. Nucleosomes (final concentration of 25-30 nM) and Rap1 (0, 1, 2, 5, 10 equivalents) were mixed by pipetting in T50 buffer and left for 10 min room temperature to bind. Fluorescence emission spectra are taken from 585 nm to 700 nm (1 nm increments) using 578 nm as excitation wavelength. Spectra for DNA only, T50 only and donor only samples were taken. For a given sample, NaCl was added to 800 mM to observe nucleosome disassembly. FRET efficiency was calculated from donor emission:

with F_DA denoting donor emission in the presence of acceptor, and F_D denoting donor emission in the donor-only sample. Additionally, reactions were loaded onto 0.5x TBE 5% polyacrylamide gels to check binding.

Flow cell preparation and chromatin loading was performed as described in Kilic et al. (2018b) and the preceding paragraphs. Experiments were performed in FRET imaging buffer (40 mM KCl, 50 mM Tris pH 7.5, 2 mM Trolox, 2 mM nitrobenzyl alcohol (NBA), 2 mM cyclooctatetraene (COT), 10% glycerol and 3.2% glucose) supplemented with GODCAT (100x stock solution: 165 U/mL glucose oxidase, 2170 U/mL catalase). Experiments on chromatin remodeling effect of Rap1 were performed with imaging buffer containing 150 mM KCl, and 0.1 mg/mL of BSA was added to prevent nonspecific binding of Rap1 to glass surface. For Rap1 titration, unlabeled Rap1-Halo was used.

smFRET data acquisition was carried out with a micro-mirror TIRF system (MadCityLabs) using Coherent Obis Laser lines at 405 nm, 488 nm, 532 nm and 640 nm, a 100x NA 1.49 Nikon CFI Apochromat TIRF objective (Nikon) as well as an iXon Ultra EMCCD camera (Andor), operated by custom-made Labview (National Instruments) software.

For general smFRET imaging, a programmed sequence was employed to switch the field of view to a new area followed by adjusting the focus. The camera (at 500 EM gain) was triggered to acquire 1950 frames with 532 nm excitation and 100 ms time-resolution followed by a final change to 640 nm excitation.

Each experiment was repeated several times (see Tables S5 and S6 for number of repeats), using at least two independently produced chromatin preparations on two different days.

Purified RSC and recombinant yNap1 were used (for the purification, see Kurat et al., 2017). All reactions were performed in reaction buffer (10 mM Tris pH 7.4, 150 mM KCl, 3 mM MgCl₂, 0.1 mg/mL BSA) and a total volume of 50 μl. The following components were added in sequential order MNs (to give a 20 nM final concentration), yNap1 (10 eq. yNap1: 1 eq. MNs), if required Rap1 (10 eq. Rap1: 1 eq. MNs), RSC complex (0.2 eq. RSC: 1 eq. MNs) and finally ATP (1mM). Reactions were placed at 30°C and 10 μl were taken for each time point, to which was added a 3-fold molar excess of plasmid DNA (compared to nucleosomes) containing a Rap1 binding site and returned to 30°C for 5 min. Reactions were then placed on ice until glucose was added to make 8% final concentration and loaded onto commercial Criterion Precast Gel (Biorad) 5% TBE, 1mm, run in 1x TBE at 200 V for 35-45 min on ice. Gels were stained in GelRed and imaged using ChemiDoc MP (Biorad) Figures S7A, S7B, and S7G). Leaving out Nap1 from the reaction did not affect RSC remodeling (Figure S7C). Remodeling assays using MNs containing fluorescently labeled octamers were also performed (Figure S7D) using the same conditions as described above. To model the RSC displaced nucleosome, an asymmetric PCR generated P3_S12_remodelled (Table S2) DNA fragment was used. This DNA was reconstituted into a nucleosome and incubated with Rap1 for 10 min at 30°C in reaction buffer (10 mM Tris pH 7.4, 150 mM KCl, 3 mM MgCl₂, 0.1 mg/mL BSA), total volume of 10 μl. A 3-fold excess of plasmid DNA (compared to nucleosomes) containing a Rap1 binding site was added and returned to 30°C for 5 min. Reactions were then placed on ice until glucose was added to make 8% final concentration and loaded onto 5% polyacrylamide 0.5x TBE, 1.5 mm, run in 0.5x TBE at 120 V for 55-60 min on ice. Gels were stained in Gelred and imaged using ChemiDoc MP (Biorad) (Figure S7E). For the sequential remodeling experiment, nucleosomes were incubated with RSC and Nap1 for 90 min as described above. At 90 min, the RSC reaction was stopped by the addition of 30 mM EDTA pH 8.0. Then, Rap1 was added for 5 min at 30°C, followed by analysis on native PAGE (Figure S7F).

RSC sliding reactions were performed in reaction buffer (10 mM Tris pH7.4, 150 mM KCl, 3 mM MgCl₂, 0.1 mg/mL BSA) and a total volume of 70 μl. The following components were added in sequential order MNs (to make 20nM final concentration), Nap1 (10 Nap1: 1 MN ratio), Rap1 (10 Rap1: 1 MN ratio, for w/o Rap1 MQ water was used as substitute), RSC complex (0.2 RSC: 1 MN ratio) and finally ATP (1 mM). Reactions were placed at 30°C for 90 min after which 10 μL was taken and glucose was added to make 8% final concentration and loaded onto commercial Criterion Precast Gel (Biorad) 5% acrylamide, 1mm, run in 1xTBE at 200 V for 35-45 min on ice. Gels were stained in Gelred and imaged using ChemiDoc MP (Biorad) (Figure S7G). To the remaining 60 μl, 60 μL 50mM Tris-HCl pH 8 and 10x NEB MNase buffer (M0247S) (to make final 1x) was added. This 120 μL total sample was split into 3 × 40 μL aliquots and to each either 6 units, 3 units or 1 unit of Mnase (M0247S) was added respectively and left to digest for 5 min at 37°C. To stop the reaction an equal volume of stop buffer was added (200 mM NaCl, 30 mM EDTA pH 8.0, 1% SDS) and left on ice for 5 min. Finally, 10 μg of Proteinase K (Sigma P2308) was added and left for 1h at 60°C and DNA fragments were isolated using QIAquick PCR purification spin columns (QIAGEN). For nucleosome only samples (t0), reactions were performed directly in 1x NEB Mnase buffer (M0247S), Mnase and Proteinase K digestion as well as DNA fragment purification was performed as described for RSC assay nucleosomes.

Following MNase digestion, DNA was purified using MinElute PCR Purification Kit (QIAGEN). The libraries were prepared using TruSeq ChIP Sample Preparation Kit (Illumina, Catalog IDs: IP-202-1012, IP-202-1024) according to manufacturer’s instructions. The libraries were sequenced on a HiSeq 4000 machine in 100 bp paired-end mode at the Genomics Platform of the University of Geneva (https://ige3.genomics.unige.ch/). Mapping of the sequencing data to the corresponding sequences was performed using Bowtie2 (sensitive end-to-end mode) on Galaxy (https://usegalaxy.org/). All densities were derived from read counts normalized to the total number of reads for each sample and BAM files was converted to bigWig files using bamCoverage and bigWig files converted to BedGraph format on Galaxy.

The pRS313-GALL plasmid was constructed by subcloning of SacI and XbaI fragment from pRS416-GALL plasmid and inserted into pRS313 for construction of plasmid expressing RAP1 under the control of GALL promoter. The RAP1 coding region was amplified using primers 5′-CATGTCTAGAATGTCTAGTCCAGATGATTTTGAAAC-3′ (Forward) and 5′-CATGCCCGGGTCATAACAGG TCCTTCTCAAAAAATC-3′ (Reverse) containing XbaI and SmaI sites and inserted into pRS313-GALL construct, digested with XbaI and SmaI. To construct pLR10-RPL30 plasmids, first RPL30 WT and RPL30-m1, RPL30-m2, and RPL30-m1/m2 mutants were cloned into pUC18 plasmid between SphI and SacI sites using primers 5′-ATGCGCATGCCTGCGTATATTGATTAATTGAA-3′ (Forward) and 5′-ATGCGAGC TCATATCATGCAGTACATTGACAGTATATCA-3′ (Reverse). Corresponding regions were then amplified by PCR using primers 5′-ATGCGTCGACATATCATGCAGTACATTGACAGTATATCA-3′ (Forward) and 5′-ATGC GCATGCCTGCGTATATTGATTAATTGAA-3′ (Reverse), and cloned into pLR10 plasmid just upstream of the YFP reporter gene at SphI and SalI sites. The yeast RAP1 anchor away strain HHY168 RAP1(1-134)-FRB1-RAP1(136-827)-LEU2 (YJB26) was co-transformed with the pRS415-GALL-RAP1 and pLR10-RPL30 plasmids.

The yeast cells, transformed with pRS313-GALL-RAP1 and pLR10-RPL30 plasmids, were grown overnight in SC-His-Ura containing 2% raffinose. Overnight cultures were diluted to OD600 0.1, grown at 30°C to OD600 0.3-0.4, and then treated with either vehicle (90% ethanol/10% Tween) or, for anchor-away, with rapamycin (1 mg/mL of 90% ethanol/10% Tween stock solution) at a final concentration of 1 μg/ml (Haruki et al., 2008) (1 μg/mL) for 1 hr to deplete FRB-tagged RAP1 protein. Following the rapamycin treatment, the strains were grown in medium containing 2% galactose for 2 hr to induce expression of RAP1 or 2% raffinose.

MNase digestion was performed as described (Kubik et al., 2015). Briefly, yeast cells were grown at 30°C for o/n in SC-His-Ura media containing 2% raffinose to OD₆₀₀ 0.3-0.4, crosslinked with 1% formaldehyde for 5 min and quenched by the addition of 125 mM glycine for 5 min at room temperature. The cell pellets were resuspended in spheroplasting buffer (1 M sorbitol, 1 mM β-mercaptoethanol, 10 mg/mL zymolyase) after harvesting and incubated for 8 min at room temperature. Spheroplasts were washed twice using 1 mL of 1 M sorbitol and treated with different concentrations of MNase, ranging from 0.05 to 1.0 units. The samples were incubated at 37°C for 45 min in MNase digestion buffer (1M Sorbitol, 50 mM NaCl, 10 mM Tris pH 7.4, 5 mM MgCl₂, 1 mM CaCl₂, 1mM β-mercaptoethanol, 0.5 mM spermidine and 0.075% NP-40). Digestion reactions were stopped by the addition of EDTA (30 mM), the crosslinks were reversed with SDS (0.5%) and proteinase K (0.5 mg/mL) and incubated at 37°C for 1 h and then transferred to 65°C for at least 2 h. The DNA was isolated by phenol/chloroform/isoamyl alcohol (25:24:1) extraction, concentrated with ethanol and treated with RNase at 37°C for 1 h for monitoring on agarose gel (2%). MNase profiles were determined by qPCR of chromatin samples (previously digested with 0.5 units MNase) using a set of nested primer pairs covering the RPL30 promoter region ∼561 bp upstream of the ATG.

Flow cytometry analysis was performed to detect the expression of a YFP reporter driven by RPL30 promoter and its variants in different conditions. Yeast transformants were grown to stationary phase overnight in appropriate media, the cells were diluted to OD600 0.1 the next day and grown to exponential phase at OD600 0.3-0.4. Upon flow cytometry, the cells were diluted 10-fold into SC-His-Ura media and immediately processed on Beckman Coulter Gallios Flow Cytometer. YFP-expressing cells were sorted by fluorescence-activated cell sorting (FACS) analyses using excitation lasers at 488 nm, and filtering emissions at 525 nm.