Structural basis of nucleosome transcription mediated by Chd1 and FACT.

No statistical methods were used to predetermine sample size. The experiments were not randomized, and the investigators were not blinded to allocation during experiments and outcome assessment.

Molecular cloning

S. cerevisiae Spt4 and Spt5 were cloned into vectors 438-A and 438-C, respectively, using ligation-independent cloning (LIC). Vectors 438-A and 438-C were a gift from S. Gradia (UC Berkeley), Addgene plasmids #55218 and #55220. Using LIC, the two genes were combined on a single 438-series vector. The construct contained Spt5 with an N-terminal 6× His tag followed by a maltose-binding protein tag, and a tobacco etch virus protease cleavage site. Spt4 did not contain tags. Each subunit in the combined vector was preceded by a PolH promoter and followed by a SV40 terminator. S. cerevisiae Spt6 was cloned into vector 438-C using LIC. The construct contained an N-terminal 6× His tag followed by a maltose-binding protein tag and a tobacco etch virus protease cleavage site. A codon-optimized sequence of S. cerevisiae TFIIS for expression in Escherichia coli was cloned into LIC-compatible vector 1-O. Vector 1-O was a gift from S. Gradia (UC Berkeley), Addgene plasmid #29658. The construct contains an N-terminal 6× His tag followed by a Mocr solubility tag and a tobacco etch virus protease cleavage site.

Preparation of protein components S. cerevisiae Pol

II was purified as described previously 41 . S. cerevisiae Chd1 and FACT were expressed and purified as described 26 . All insect cell lines used for expression were purchased from Life Technologies (Sf9, Sf21) or from Expression Systems (Hi5) and used as identified by the vendor. Cell lines were not tested for mycoplasma contamination. S. cerevisiae Spt4 and Spt5 were co-expressed in insect cells using a similar approach as that reported previously 42 . After harvest, cell pellets were resuspended in lysis buffer 500 (500 mM NaCl, 20 mM Na-HEPES, pH 7.4, 10% (v/v) glycerol, 1 mM DTT, 30 mM imidazole, pH 8.0, 0.284 μg ml −1 leupeptin, 1.37 μg ml −1 pepstatin A, 0.17 mg ml −1 PMSF, 0.33 mg ml −1 benzamidine). Cells were lysed by sonication. The cell lysate was subjected to centrifugation (18,000 g , 4 °C, 30 min) and ultracentrifugation (235,000 g , 4 °C, 60 min). The supernatant containing Spt4/5 was subsequently filtered using 0.2-µm syringe filters (Millipore). The filtered supernatant was applied to a GE HisTrap 5 ml HP (GE Healthcare), pre-equilibrated in lysis buffer. The column was subsequently washed with three column volumes (CV) lysis buffer 500, 3 CV high-salt buffer (1000 mM NaCl, 20 mM Na-HEPES, pH 7.4, 10% (v/v) glycerol, 1 mM DTT, 30 mM imidazole, pH 8.0, 0.284 μg ml −1 leupeptin, 1.37 μg ml −1 pepstatin A, 0.17 mg ml −1 PMSF, 0.33 mg ml −1 benzamidine) and 4.5 CV lysis buffer. Bound protein was eluted by gradient over 9 CV to 100% nickel elution buffer (500 mM NaCl, 20 mM Na-HEPES, pH 7.4, 10 % (v/v) glycerol, 1 mM DTT, 500 mM imidazole, pH 8.0, 0.284 μg ml −1 leupeptin, 1.37 μg ml −1 pepstatin A, 0.17 mg ml −1 PMSF, 0.33 mg ml −1 benzamidine) over 9 CV. Fractions containing Spt4/5 were pooled and dialyzed for 16 h against dialysis buffer (300 mM NaCl, 20 mM Na-HEPES, pH 7.4, 10% (v/v) glycerol, 1 mM DTT, 30 mM imidazole, pH 8.0, 0.284 μg ml −1 leupeptin, 1.37 μg ml −1 pepstatin A, 0.17 mg ml −1 PMSF, 0.33 mg ml −1 benzamidine). The dialyzed sample was applied to a GE HisTrap 5 ml HP and GE HiTrap Q 5 ml HP column combination. After application of the sample, the HisTrap 5 ml HP was removed, and the HiTrap Q 5 ml HP was washed with 5 CV dialysis buffer. Spt4/5 was eluted using a gradient elution to 100% high-salt buffer over 9 CV. Fractions containing Spt4/5 were concentrated using an Amicon Millipore 15 ml 50,000 MWCO centrifugal concentrator and applied to a GE Superdex 200 Increase 10/300 GL size exclusion column, pre-equilibrated in gel filtration buffer (500 mM NaCl, 20 mM Na-HEPES, pH 7.4, 10% (v/v) glycerol, 1 mM DTT). Peak fractions were analyzed with SDS−PAGE. Fractions containing Spt4/5 were concentrated using an Amicon Millipore 15 ml 50,000 MWCO centrifugal concentrator to a concentration of ~20 µM, aliquoted, flash frozen and stored at −80 °C. Typical preparations yielded 300 µg of Spt4/5 from 1.2 L of insect cell culture. S. cerevisiae Spt6 was expressed in insect cells and subsequently purified with a similar protocol used for H. sapiens SPT6 (ref. 43 ) with a final concentration of ~60 µM. Typical yields are ~10 mg from 1.2 L of insect cell culture. S. cerevisiae TFIIS was expressed in E. coli BL21 (DE3) RIL cells grown in LB medium. Cells were grown to an optical density at 600 nm of 0.6 at 37 °C. Expression of TFIIS was subsequently induced with 1 mM isopropyl β- d -1-thiogalactopyranoside at 18 °C for 16 h. Cells were harvested by centrifugation and resuspended in TFIIS lysis buffer (300 mM NaCl, 20 mM Na-HEPES, pH 7.4, 10% (v/v) glycerol, 1 mM DTT, 30 mM imidazole, pH 8.0, 0.284 μg ml −1 leupeptin, 1.37 μg ml −1 pepstatin A, 0.17 mg ml −1 PMSF, 0.33 mg ml −1 benzamidine). Cells were lysed by sonication. Two rounds of centrifugation (87,000 g , 4 °C, 30 min) were used to clear the lysate. The supernatant was applied to a GE HisTrap 5 ml HP. Affinity purification, dialysis and TEV digest were performed similarly as described for Spt4/5. The dialyzed and TEV-digested sample was applied to a GE HisTrap 5 ml HP, pre-equilibrated in TFIIS lysis buffer. The flow-through containing TFIIS was collected and concentrated using an Amicon Millipore 15 ml 10,000 MWCO centrifugal concentrator and applied to a GE S75 16/600 pg size exclusion column, pre-equilibrated in TFIIS size exclusion buffer (300 mM NaCl, 20 mM Na-HEPES, pH 7.4, 10% (v/v) glycerol, 1 mM DTT). Fractions containing TFIIS were concentrated to ~300 µM, aliquoted, flash frozen and stored at −80 °C. Typical yields were 10 mg from 6 L of E. coli expression culture. Protein identity of all protein components was confirmed by MS.

Nucleosome preparation

Xenopus laevis histones were expressed and purified as described previously 26 , 44 . Histone H3 was H3K36Cme3 modified 45 . DNA fragments for nucleosome reconstitution were generated by PCR essentially as described previously 27 , 36 . A vector containing a modified Widom 601 sequence was used as a template for PCR. Super-helical locations were assigned based on previous publications 5 , 6 , 26 , 46 , assuming direction of transcription from negative to positive SHLs. Large-scale PCR reactions were performed with two PCR primers (forward primer: 5′-ACG AAG CGT AGC ATC ACT GTC TTG-3′; reverse primer: 5′-ATC AGA ATC CCG GTG CCG AGG CCG C-3′) at a scale of 50 ml. Full-length PCR product is reported in Supplementary Data . PCR products were purified using anion exchange chromatography (GE Resoure Q 6 ml) followed by ethanol precipitation. The DNA product was digested with TspRI in 1X CutSmart Buffer (NEB) overnight at 65 °C to generate the 9-nt single-stranded DNA overhang. The digestion product was again purified with anion exchange chromatography, ethanol precipitated and resuspended in water. Nucleosome core particle reconstitution was then performed using the salt-gradient dialysis method 44 . The resulting nucleosome was purified using a Model 491 Prep Cell (Bio-Rad) and subsequently concentrated to 10–20 µM using an Amicon Millipore 15 ml 50,000 MWCO centrifugal concentrator. Quantification of the purified nucleosome was achieved by measuring absorbance at 280 nm. Molar extinction coefficients at 280 nm were determined for protein and nucleic acid components and were summed to yield a molar extinction coefficient for the reconstituted extended nucleosome.

RNA extension assays

RNA extension assays were performed on the same nucleosomal template substrate used for the structural studies. A 6-FAM 5′-labelled 11-nt RNA (5′-/56-FAM/ rUrUrA rUrCrA rCrUrG rUrC-3′) was employed to monitor the transcription reaction. TFIIS has been added to all transcription reactions to prevent formation of overextended DNA–RNA hybrids and facilitate nucleosome passage 5 , 6 . The position of Pol II pausing was assigned by indicating the position of the Pol II active site on the Widom 601 DNA. This provided an unambiguous assignment at nucleotide resolution. Therefore, our pausing sites at bp 1 and bp 42 correspond to the previously described 6 , 13 pause sites, with the Pol II leading edge at SHL −5 and SHL −1, respectively. All subsequent concentrations refer to the concentration in the final reaction. The final concentrations of buffer components were 130 mM NaCl, 20 mM Na-HEPES, pH 7.4, 3 mM MgCl 2 , 4% (v/v) glycerol, 1 mM DTT/TCEP. The final volume for each RNA extension reaction was 40 µl. RNA (80 nM), nucleosomal template (80 nM) and S. cerevisiae Pol II (100 nM) were mixed in equimolar ratios and incubated for 5 min on ice. Spt4/5 (120 nM) and additional factors (500 nM each), 10× compensation buffer and water were added to achieve final assay conditions. The sample was incubated for 3 min at 30 °C. Transcription elongation was started by the addition of ATP, CTP, GTP and UTP (1 mM each) and TFIIS (60 nM). Five microliters of the reactions were quenched after 5 min, 10 min and 30 min in 5 µl 2× stop buffer (6.4 M urea, 50 mM EDTA, pH 8.0, 1× TBE buffer) if time courses were performed. Samples were treated with 4 µg proteinase K for 15 min at 37 °C, denatured at 95 °C for 3 min, and separated by denaturing gel electrophoresis (4 µl of sample applied to an 8 M urea, 1× TBE buffer, 12% acrylamide:bis-acrylamide 19:1 gel, run in 0.5× TBE buffer at 300 V for 30 min). RNA extension products were visualized using the 6-FAM label and a Typhoon 9500 FLA imager at an excitation wavelength of 473 nm and emission wavelength range of >520 nm. Gels were subjected to linear contrast enhancement. Source data for all quantified RNA extension assays are provided in Source Data Fig. 1 . All RNA extension assays were performed independently and at least three times. Full-length RNA extension products were quantified using Fiji 1.0. The products were normalized against the total intensity of the respective reaction lane to control for errors during gel loading. Bar charts show mean values and standard deviation as error bars. The following P values were applied * P < 0.05, ** P < 0.01. A two-tailed t test was employed to determine statistical significance. Reconstitution of transcribing Pol II−nucleosome complexes Complexes for cryo-EM were formed in a final buffer containing 130 mM NaCl, 20 mM Na-HEPES, pH 7.4, 3 mM MgCl 2 , 1 mM DTT/TCEP, 4% (v/v) glycerol. RNA (480 pmol, same construct as used for RNA extension assays) and nucleosome (120 pmol) were incubated for 5 min on ice. Pol II (120 pmol), Spt4/5 (180 pmol) and Spt6 (180 pmol) were added and incubated for 5 min on ice. Water and compensation buffer were added to reach final buffer conditions, and the sample was incubated for 5 min. Transcription elongation was started by the addition of 1 mM each of GTP, CTP and UTP and 0.4 mM 3′-dATP in the case of the Pol II−Spt4/5−nucleosome−FACT complex. Instead of 3′-dATP, 1 mM ADP-BeF 3 was added to the Pol II−Spt4/5−nucleosome−Chd1−FACT complex. TFIIS (108 pmol) was immediately added after addition of NTP. After 15 min of incubation at 30 °C, Chd1 (180 pmol) and FACT (180 pmol), preincubated with H2A−H2B dimer (180 pmol), or FACT alone (180 pmol), preincubated with H2A−H2B dimer (180 pmol), were added. The transcription reactions were allowed to proceed for an additional 30 min at 30 °C and quenched with EDTA (10 mM final concentration). The samples were subsequently centrifuged and applied onto a Superose 6 3.2/300 Increase size exclusion column (GE Healthcare), equilibrated in complex buffer (100 mM NaCl, 20 mM Na-HEPES, pH 7.4, 3 mM MgCl 2 , 1 mM TCEP, 4 % (v/v) glycerol). Fractions eluted from the size exclusion column were analyzed using SDS−PAGE and denaturing-urea PAGE (8 M urea, 1× TBE buffer, 12% acrylamide:bis-acrylamide 19:1 gel). Consistent with previous observations 5 , TFIIS is lost from the elongation complex during size exclusion chromatography. Fractions containing the complex were individually crosslinked and dialyzed against dialysis buffer (100 mM NaCl, 20 mM Na-HEPES, pH 7.4, 3 mM MgCl 2 , 1 mM TCEP) for 3 h, as described previously 47 . The dialyzed complexes with an approximate concentration of 50−100 nM were applied to R2/2 gold grids, Au 200 mesh (Quantifoil). The grids were glow discharged for 100 s prior to application of 2 µl of sample to each side of the grid. After incubation of the sample for 8 s, the grid was blotted and vitrified by plunging into liquid ethane using a Vitrobot Mark IV (Thermo Fisher). The Vitrobot was operated at 4 °C and 100% humidity. A blot force of 5 and blot time between 3 and 5 s were used for the sample preparation. The grids were clipped and subsequently stored in liquid nitrogen before data acquisition.

Cryo-EM analysis and image processing

Cryo-EM data were collected on a Titan Krios II transmission electron microscope (FEI) operated at 300 keV. A K3 summit direct detector (GATAN) with a GIF Quantum Filter with a slit width of 20 eV was used for the data acquisition. Data acquisition was performed using SerialEM at a nominal magnification of 81,000×, corresponding to a pixel size of 1.05 Å per pixel in nanoprobe EF-TEM mode. For the Pol II−Spt4/5−nucleosome−Chd1 dataset, image stacks of 40 frames were collected in counting mode over 2.2 s. The dose rate was ~18.12 e − per Å 2 per s for a total dose of 39.87 e − per Å 2 . For the Pol II−Spt4/5−nucleosome−FACT dataset, image stacks of 40 frames were collected in counting mode over 2.2 s. The dose rate was ~18.30 e− per Å 2 per s for a total dose of 40.25 e − per Å 2 . Micrographs were stacked and processed using Warp 48 . CTF estimation and motion correction was performed using Warp 48 . Particles were picked using an in-house trained instance of the neural network BoxNet2 as implemented in Warp, yielding 3,755,390 particles for the Pol II−Spt4/5−nucleosome−Chd1 dataset and 5,227,093 particles for the Pol II−Spt4/5−nucleosome−FACT dataset. Particles were extracted with a box size of 400 pixels and normalized. Further image processing was performed with cryoSPARC 49 and RELION 3.0.7. For the Pol II−Spt4/5−nucleosome−Chd1 dataset, particles were separated into two subsets and subsequently 3D classified using cryoSPARC 49 . Particles were selected for the presence of a nucleosome-like density. Selected particles were imported into RELION 50 . Two subsequent rounds of 3D classification resulted in 247,604 particles with clear nucleosomal density. A 3D refinement of these particles resulted in an overall model of 2.6 Å. The particles were CTF refined, and Bayesian polishing was conducted. A mask encompassing the nucleosome and additional density at SHL +2 was applied to two rounds of 3D classification to select for particles that contain Chd1. This resulted in 30,876 particles with clear density for Chd1 bound to the partially unwrapped nucleosome. Particles were then subsequently 3D refined resulting in map A (EMD- 12666 ) with a resolution of 2.9 Å (FSC threshold 0.143 criterion). The map showed excellent density for Pol II, but the nucleosome−Chd1 part of the map showed flexibility. Therefore, Pol II with Spt4/5 and the nucleosome−Chd1 parts of the maps were individually refined using a mask for Pol II−Spt4/5 or nucleosome−Chd1, respectively. Signal subtraction was applied. This resulted in two masked refinements (Pol II−Spt4/5, map B, EMD- 12667 ; nucleosome−Chd1 with signal subtraction, map C, EMD- 12668 ) with overall improved density. These two masked refinements were combined using the Frankenmap and Noise2map tool set of Warp, resulting in the final composite map (map D, EMD- 12449 ). For the Pol II−Spt4/5−nucleosome−FACT dataset, particles were separated into two subsets and subsequently 3D classified using cryoSPARC and RELION. Particles were selected for the presence of nucleosome-like densities. The selection resulted in 603,550 particles with clear nucleosome-like density. The selected particles were 3D refined using an angular sampling rate of 7.5° and subjected to further classifications to select for particles with bound FACT. This ultimately resulted in 48,718 particles. These particles were again subjected to 3D classification. After 3D refinement, CTF refinement and Bayesian polishing, the remaining 47,138 particles resulted in a final refinement (map 1, EMD- 12669 ) with an overall resolution of 3.1 Å (FSC threshold 0.143 criterion). To improve densities for the Pol II−Spt4/5 and nucleosome−FACT parts of the map, the particles were subjected to masked refinements (maps 2, EMD- 12670 and map 3, EMD- 12671 ). Similar to the Chd1 dataset, the masked refinements were combined using the Frankenmap and Noise2map tools included in Warp, yielding the final map (composite map 4, EMD- 12450 ). Local resolutions of the composite maps were estimated using the RELION built-in tool for the determination of local resolutions.

Model building and refinement

For the Pol II−Spt4/5−nucleosome−Chd1 structure, structures of S. cerevisiae Pol II (PDB 3PO2 ), X. laevis nucleosome (PDB 3LZ0 ), Chd1 with ADP-BeF 3 (PDB 5O9G ) and Spt4/5 (PDB 2EXU ) were rigid-body docked into map D and refined using Coot 51 .

DNA from the elongation complex and nucleosomal

DNA were connected using Coot. Density in the active site of Pol II allowed unambiguous assignment of DNA register. Surprisingly, the complex had transcribed over the T-less cassette that should stall further elongation. The ADP (Sigma-Aldrich) used in the formation of ADP-BeF 3 is reported to be contaminated with up to 2.76% ATP 52 , possibly providing the required substrate to transcribe past the end of the T-less cassette. Identification of DNA−RNA register was aided by map B. For the Pol II−Spt4/5−nucleosome−FACT structure, the refined Pol II part of the Pol II−Spt4/5−nucleosome−Chd1 structure was rigid-body docked into the density. Additionally, X. laevis nucleosome (PDB 3LZ0 ), H. sapiens FACT (PDB 6UPK ), and Spt4/5 (PDB 2EXU ) were rigid-body docked into map 4 using Coot. S. cerevisiae FACT structures (Spt16, PDB 4IOY ; Pob3, PDB 4PQ0 ) were then superposed onto the docked H. sapiens FACT structure. The dimerization domain of Spt16 and Pob3 were generated using PHYRE2 (ref. 53 ) and superposed onto the docked FACT structure without any additional manual manipulation. The CTD of Spt16 was modeled de novo as a polyalanine extension of Spt16 with no sequence assignment.

DNA from the Pol

II part of the structure and the nucleosomal DNA were connected in Coot. The density in the active site allowed for unambiguous assignment of the DNA register, and the nucleic acid sequence was adjusted accordingly. Identification of DNA−RNA register was aided by map 2. Both atomic models were real-space refined using PHENIX 54 , with secondary structure restraints against map D (Pol II−Spt4/5−nucleosome−Chd1 model) and map 4 (Pol II−Spt4/5−nucleosome−FACT model).

Figure generation

Figures for structural models were generated using PyMol (version 2.3.4; https://pymol.org/ ), UCSF Chimera 55 and UCSF ChimeraX 56 . Gel quantification was performed using Fiji, and graphs were generated using GraphPad Prism. Reporting Summary Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Online content Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41594-021-00578-6.

Supplementary information Supplementary Information Sequence of nucleosomal construct. Reporting Summary Supplementary Video 1 RNA polymerase II−Spt4/5−nucleosome−Chd1 structure and density (map D). Overall densities and densities for the Rpb1 funnel helices, Chd1 and one H3−H4 dimer are shown. Supplementary Video 2 RNA polymerase II−Spt4/5−nucleosome−FACT structure and density (map 4). Overall densities and densities for the Rpb1, funnel helices, Pol II active site and FACT Spt16 CTD are shown. Supplementary Data 1 Raw intensity values of transcription assay quantifications.

Supplementary information The online version contains supplementary material available at 10.1038/s41594-021-00578-6.