Cryo-EM structures of RNA polymerase II–nucleosome complexes rewrapping transcribed DNA

RNA polymerase II (RNAPII) transcribes DNA wrapped in the nucleosome by stepwise pausing, especially at nucleosomal superhelical locations −5 and −1 [SHL(-5) and SHL(-1), respectively]. In the present study, we performed cryo-electron microscopy analyses of RNAPII–nucleosome complexes paused at a major nucleosomal pausing site, SHL(-1). We determined two previously undetected structures, in which the transcribed DNA behind RNAPII is sharply kinked at the RNAPII exit tunnel and rewrapped around the nucleosomal histones in front of RNAPII by DNA looping. This DNA kink shifts the DNA orientation toward the nucleosome, and the transcribed DNA region interacts with basic amino acid residues of histones H2A, H2B, and H3 exposed by the RNAPII-mediated nucleosomal DNA peeling. The DNA loop structure was not observed in the presence of the transcription elongation factors Spt4/5 and Elf1. These RNAPII-nucleosome structures provide important information for understanding the functional relevance of DNA looping during transcription elongation in the nucleosome.

RNA polymerase II (RNAPII) transcribes DNA wrapped in the nucleosome by stepwise pausing, especially at nucleosomal superhelical locations −5 and −1 [SHL(-5) and SHL(-1), respectively].In the present study, we performed cryoelectron microscopy analyses of RNAPII-nucleosome complexes paused at a major nucleosomal pausing site, SHL(-1).We determined two previously undetected structures, in which the transcribed DNA behind RNAPII is sharply kinked at the RNAPII exit tunnel and rewrapped around the nucleosomal histones in front of RNAPII by DNA looping.This DNA kink shifts the DNA orientation toward the nucleosome, and the transcribed DNA region interacts with basic amino acid residues of histones H2A, H2B, and H3 exposed by the RNAPII-mediated nucleosomal DNA peeling.The DNA loop structure was not observed in the presence of the transcription elongation factors Spt4/5 and Elf1.These RNAPII-nucleosome structures provide important information for understanding the functional relevance of DNA looping during transcription elongation in the nucleosome.
In eukaryotes, genomic DNA is compacted as chromatin.The basic unit of chromatin is the nucleosome, comprising approximately 150 to 200 base pairs of DNA and a histone octamer containing two each of histones H2A, H2B, H3, and H4 (1,2).In chromatin, nucleosomes are connected by linker DNA segments and form a beads-on-a-string configuration (3).
In cells, transcription must occur on the DNA spooled in the nucleosome.During nucleosome transcription, RNA polymerase II (RNAPII) gradually peels the DNA from the histone surface without histone dissociation, until it reaches the center of the nucleosomal DNA (the dyad DNA) (4,5).Previous studies with a prokaryotic RNA polymerase (RNAP) revealed that the nucleosomal histones in front of RNAP are dissociated when it passes through the dyad DNA of the nucleosome (6,7).Histones are then transferred from in front to behind the transcribing RNAP, and the nucleosome is reassembled on the transcribed template DNA behind the RNAP (8).Histone transfer has also been observed during nucleosome transcription by eukaryotic RNAPII (9)(10)(11).This is consistent with the fact that the epigenetic information of the nucleosomes, such as histone post-translational modifications and histone variants, is maintained during transcription elongation.
Two mechanisms had been proposed for the RNAPIIdependent nucleosome transfer: the histone chaperonedependent and -independent pathways (8,(12)(13)(14)(15)(16)(17).We previously reported the cryo-electron microscopy (cryo-EM) structures depicting the nucleosome disassembly and reassembly processes during RNAPII transcription in the histone chaperone-dependent pathway, in the presence of FACT, Spt4/5, Spt6, Spn1, Elf1, and Paf1C (18).Conversely, in the histone chaperone-independent pathway, the histone transfer from in front to behind RNAPII is proposed to occur via the formation of a template DNA loop, in which the upstream DNA exiting RNAPII rebinds to the histones within the partially unwrapped nucleosome downstream of the RNAPII (16,17).The histone transfer mediated by DNA looping has also been suggested by nucleosome transcription studies with a prokaryotic RNAP (7,8).Interestingly, the structures of the RNAP-nucleosome complexes containing the template DNA loop have been reported for mammalian and bacterial RNAPs (19,20).
In the present study, we performed nucleosome transcription reactions in vitro and determined the cryo-EM structures of the RNAPII-nucleosome complexes, in which the upstream DNA region after RNAPII passage binds to the nucleosomal histones located in front of RNAPII.In these RNAPIInucleosome complex structures, the upstream DNA is rewrapped in different forms from the previously reported ones.Interestingly, the DNA loop is not detected in the presence of the transcription elongation factors Spt4/5 and Elf1, which may sterically inhibit DNA rewrapping.These structures provide important information for understanding the biological relevance of the DNA loop formed when RNAPII is stalled on the DNA in the nucleosome.

Cryo-EM structures of RNAPII-nucleosome complexes with upstream DNA looping
The nucleosome transcription reaction was conducted with purified Komagataella pastoris RNAPII in the presence of TFIIS, a transcription elongation factor that reactivates arrested or backtracked RNAPII.The nucleosome contained a 9 base-pair mismatched DNA region at the linker DNA to serve as a priming site for the RNA elongation reaction.We designed the nucleosomal DNA template in which RNAPII stalls when its catalytic center reaches the position 42 basepairs from the nucleosome entry site, by incorporating 3 0 -dATP (Fig. 1, A and B).The nucleosome reconstitution (Fig. S1, A and B) and the transcription assay were conducted as described previously (4,5), but the nucleosome: RNAPII ratio (2:1-1:1) and the NaCl concentration (0 mM to 100 mM) were changed (Fig. S1, C and D).The RNAPII-nucleosome complexes were then prepared by ultracentrifugation sedimentation with a sucrose and glutaraldehyde gradient (GraFix) for cryo-EM analysis (21) (Fig. S2, A and B).
We performed 3D reconstruction for the RNAPIInucleosome complexes, in which RNAPII has proceeded 41 base pairs from the nucleosome entry site and is paused at the nucleosomal SHL(-1) position, with about 60 DNA base-pairs peeled from the histone surface (Figs.S2C, S3-S6, and S14).We determined two novel structures in which the transcribed upstream DNA behind RNAPII binds to the DNA-peeled surface of the nucleosome (Fig. 1C).We named these structures RNAPII-nucleosome L40 and RNAPII-nucleosome L60 , which contain DNA loops with 41 and 62 base pairs of upstream DNA, respectively.The DNA loop formation may be enhanced in the presence of 100 mM NaCl, because it was not obvious under conditions without additional NaCl (4).
Comparison of the RNAPII-nucleosome L40 and RNAPIInucleosome L60 structures In both structures, the upstream DNA is sharply kinked at the DNA exit site of RNAPII, and is folded back toward the downstream nucleosome (Fig. 2A).The upstream DNA spacers between RNAPII and the nucleosome are 10 base pairs and 23 base pairs in the RNAPII-nucleosome L40 and RNAPII-nucleosome L60 structures, respectively (Fig. 2A).In the RNA-PII-nucleosome L40 structure, the nucleosomal histones are located close to RNAPII and may directly contact it (Fig. 2B).In contrast, the nucleosomal histones in the RNAPII-nucleosome L60 do not directly contact RNAPII (Fig. 2C).Therefore, the nucleosome of the RNAPII-nucleosome L60 complex may be more flexible than that of the RNAPII-nucleosome L40 complex.This structural difference between the RNAPIInucleosome L40 and RNAPII-nucleosome L60 complexes may affect subsequent histone removal by RNAPII progression.

Interactions of upstream DNA with nucleosomal histones
In both the RNAPII-nucleosome L40 and RNAPII-nucleosome L60 structures, RNAPII is paused at the SHL(-1) position, and about 60 base pairs of the downstream nucleosomal DNA is peeled away by the RNAPII progression.The DNA-binding surface of the proximal H2A, H2B, and H3, which formerly contacted the DNA, becomes accessible and captures the upstream DNA.The length of the upstream DNA that directly contacts the nucleosomal histones is approximately 30 base pairs in both structures (Fig. 3A).The upstream DNA spacer of the RNAPII-nucleosome L60 complex is 13 base pairs longer than that of the RNAPII-nucleosome L40 complex (Fig. 2A).Consequently, the nucleosome orientation relative to RNAPII differs by about 50 between the RNAPII-nucleosome L40 and RNAPII-nucleosome L60 structures (Fig. 3B).The DNAbinding path of the upstream DNA bound to the histones is similar to that of the canonical nucleosome in these novel structures ( 22) (Fig. 3A).
Spt4/5 and Elf1 may inhibit upstream DNA rewrapping Spt4/5 and Elf1 (DSIF and ELOF1 for mammals, respectively) are constitutive factors for efficient transcription elongation (23,24).We previously reported that Spt4/5 and Elf1 synergistically enhance the RNAPII processivity in nucleosome transcription (5).We next tested whether the upstream DNA loop structure could form in the presence of Spt4/5 and Elf1.To avoid a shortage of upstream DNA in the DNA loop formation, we extended the upstream DNA region by 30 base pairs, and conducted the nucleosome transcription in the presence of Spt4/5 and Elf1 (Figs. 4A and S1, E-H).We then determined the cryo-EM structure of the Spt4/5-Elf1-RNAPII-nucleosome complex paused at the SHL(-1) position (Figs.4B and S7-S9).This structure is essentially the same as the previously reported Spt4/5-Elf1-RNAPII-nucleosome structure paused at the SHL(-1) position with the short upstream DNA template (5).
In this complex, Spt4/5 and Elf1 bind to the RNAPII surface (Fig. 4B).Interestingly, the Spt4 and Spt5 NGN domains are located near the DNA exit tunnel and interfere with the kinking of the upstream DNA, observed in the absence of Spt4/5 and Elf1 (Fig. 4C).Consequently, the upstream DNA orientation is directed away from the downstream nucleosome.Consistent with this, we could not find any structure containing an upstream DNA loop, although the binding of trans DNA (foreign DNA) to the exposed histone surface was observed (Fig. 4, B and C).In the mammalian RNAPII and DSIF, the DNA loop can reportedly be formed by the displacement of and the NGN and KOW1 domains of SPT5 in the RNAPII-nucleosome complex, in the absence of the Elf1 homolog, ELOF1 (19).Elf1 may stabilize the Spt4/5 binding to RNAPII and contribute to the Spt4/5-mediated suppression of the upstream DNA looping.It is also possible that the formation of the DNA loop structure in the mammalian DSIF-RNAPII-nucleosome complex may be facilitated by the RNAPII progression up to the SHL(0) position, as reported previously (19).
To test whether the DNA looping is induced when RNAPII approaches the SHL(0) position, we designed a template nucleosome in which RNAPII pauses when it reaches SHL(0) (Figs.S1, I and J and S10, A and B).We performed nucleosome transcription by RNAPII in the presence of Spt4/5 and Elf1, and the RNAPII was intentionally paused when its catalytic center reached the position 54 base-pairs from the nucleosome entry site (Fig. S1, K and L).We then determined the cryo-EM structure of the Spt4/5-Elf1-RNAPII-nucleosome complex paused at the SHL(0) position (Figs.S10C and S11-S13).In this complex, the EM densities corresponding to Spt4, Spt5, and Elf1 are clearly observed, and the upstream DNA orientation is incompatible with DNA loop formation (Fig. S10C).Therefore, the DNA loop formation is unlikely to occur in the presence of Spt4/5 and Elf1, even when RNAPII is paused near the SHL(0) position of the nucleosome.

Discussion
The present study has revealed the RNAPII-nucleosome structures rewrapping the upstream transcribed DNA, when the RNAPII is paused at the position 41 base-pairs from the nucleosome entry site.In this complex, about 60 base pairs of the nucleosomal DNA are peeled away, and the leading edge of RNAPII is located at the SHL(-1) position (Figs. 1, 2, and S14).A previous cryo-EM structure of the RNAPII-nucleosome complex, stalled at the 54 base-pair position, was reported with an upstream DNA loop (19).Surprisingly, our RNAPIInucleosome L40 and RNAPII-nucleosome L60 structures are quite different from the previous structure paused at the 54 base pair position.In that structure, about 70 base pairs of the nucleosomal DNA are stripped, and the nucleosome is flipped by about 180 , as compared to our structures (Fig. S15).These facts suggest that the DNA looping can occur with a 10-bp periodicity at various SHLs, rather than a specific SHL.Two different (flipped/unflipped) types of DNA loops can be formed, probably depending on the RNAPII pausing position within the nucleosomal DNA.
In the present study, we could not obtain the RNAPIInucleosome structure containing a flipped nucleosome configuration when the RNAPII was paused at the 54 base-pair position.This may be a consequence of the stable binding of Spt4/5 and Elf1 to RNAPII, as they restrict the upstream DNA orientation to prevent the DNA from rewrapping on the nucleosome (see below).The previously reported DNA loop structure with the flipped nucleosome configuration required the detachment of SPT4 and SPT5 from the RNAPII surface (19).
The DNA loop has been proposed to be an intermediate of histone transfer during transcription elongation (8,10,16,17,19).However, in the present study, we found that in the Spt4/5-Elf1-RNAPII-nucleosome structure, Spt4, the Spt5 NGN domain, and Elf1 bind near the DNA exit tunnel of RNAPII, and interfere with the DNA kinking.Consequently, the binding of Spt4/5 and Elf1 moves the upstream DNA away from the nucleosome and prevents the DNA loop formation.Alternatively, a previous report with mammalian DSIF (Spt4/5 homolog) and RNAPII showed that the DNA kinking at the DNA exit tunnel causes displacement of SPT4 and SPT5 from the RNAPII surface (19).These observations suggest that the DNA loop formation is incompatible with the binding of the elongation factors.Therefore, the DNA loop-mediated histone transfer may occur during the transcription elongation by RNAPII without elongation factors or concomitantly with the SPT4/5 detachment.Further studies are awaited to solve this issue.
We previously determined the snapshot cryo-EM structures of RNAPII-nucleosome complexes, in which Spt6, Spn1, and the Paf1 complex were bound to the RNAPII together with Spt4/5 and Elf1, and explained the mechanism by which the RNAPII elongation complex promotes nucleosome disassembly and reassembly with the aid of the histone chaperone FACT (18).In this series of structures, we did not find the complex with the DNA loop.Therefore, the DNA looping pathway described in the present and previous studies may be an alternative pathway to the nucleosome transfer by RNAPII with transcription elongation factors (18,19).Histone chaperones, such as FACT, may be required in both pathways.
Previous low-resolution images obtained by conventional EM with a bacterial RNAP and nucleosome suggested that DNA damage in the nucleosome induces the DNA loop formation to stall RNAP on the damaged nucleosome (20).This finding implies that the DNA looping may function as a part of transcription-coupled DNA repair, in which RNAPII plays an essential role to detect DNA lesions in chromatin (25).In the future, it will be intriguing to study the functional consequences of DNA looping in the RNAPII-nucleosome complexes during the histone transfer in transcription elongation and/or DNA lesion recognition, in the course of transcriptioncoupled DNA repair processes.
The nucleosome was reconstituted with the histone octamer and the DNA fragment by the salt-dialysis method (26).The 42 and 72 bp bubble DNA fragments containing the 9 base-pair mismatched region were ligated to the cohesive end of the nucleosomal DNA by T4 DNA ligase (NIPPON GENE), as described previously (4).The sequences of the bubble DNA fragments are as follows: 42 bp bubble DNA fragment nontemplate strand: 5 0 -GCTTACGTCAGTCTGGCCATCTTTGTGTTTGGT GTGTTTGGGTGG -3 0  42 bp bubble DNA fragment template strand: 5 0 -CCCAAACACACCAAACACAAGAGCTAATTGACT GACGTAAGC -3 0 72 bp bubble DNA fragment nontemplate strand: 5 0 -CCTCTGCCTTTAAAGCAATAGGAGGTCCACGCT TACGTCAGTCTGGCCATCTTTGTGTTTGGTGTGTTTG GGTGG -3 0 72 bp bubble DNA fragment template strand: The nucleosome with a linker DNA containing a bubble region was purified by preparative non-denaturing PAGE, using a Prep Cell apparatus (Bio-Rad).The purified nucleosome was concentrated using Amicon Ultra 30K centrifugal filters (Millipore) and stored at −80 C.
The RNAPII-nucleosome L40 and RNAPII-nucleosome L60 particles were picked using RELION auto-picking based on a Laplacian of Gaussian filter and extracted with a binning factor of 2 (pixel size of 2.12 Å/pixel).These particles were subjected to 2D classification, RNAPII-nucleosome complex class selection, and 3D classification using a low-pass filtered map (EMDB-9713) as the reference model ( 5).The RNAPII-nucleosome complex class that forms the DNA loop was selected as a reference for Topaz picking (34).Particles were repicked using Topaz and subjected to 2D classification to remove junk particles.After the 3D classification, 51,652 and 97,768 particles were selected as the RNAPII-nucleosome L40 and the RNAPII-nucleosome L60 , respectively.Each class was re-extracted with a binning factor of 1 (pixel size of 1.06 Å/pixel).The RNAPII-nucleosome L40 and RNAPII-nucleosome L60 particles were subjected to 3D refinement, followed by CTF refinement and Bayesian polishing.Focused refinements of both RNAPII and nucleosome were separately performed, using specific masks.These focusedrefined maps were combined using phenix.combine_focu-sed_maps in the Phenix software package (35).
For the Spt4/5-Elf1-RNAPII-nucleosome complex (SHL(-1) stop), the image processing to 3D classification was the same as described above.The selected classes from 3D classification were re-extracted with a binning factor of 1 (pixel size of 1.06 Å/ pixel).Particles were subtracted with a mask for Spt4/5, followed by 3D classification.The selected classes containing Spt4/ 5 were reverted, refined, and resubtracted with a nucleosome mask.After the 3D classification with the nucleosome, 74,079 particles were selected as the Spt4/5-Elf1-RNAPII-nucleosome.The map of the nucleosome was refined and postprocessed.Subtracted particles were reverted, followed by refinement and postprocessing for the overall structure.The subtraction with an RNAPII mask was applied to the overall structure and then refined and postprocessed as the RNAPII structure.This focused-refined map was combined using phenix.combine_fo-cused_maps in the Phenix software package (35).
For the Spt4/5-Elf1-RNAPII-nucleosome complex (SHL(0) stop), particles were picked using RELION auto-picking based on a Laplacian of Gaussian filter, and extracted with a binning factor of 4 (pixel size of 4.24 Å/pixel).These particles were subjected to 2D classification, RNAPII-nucleosome complex class selection, and 3D classification using a low-pass filtered map (EMDB-6747) as the reference model (23).After the 3D classification, selected particles were subtracted with a mask for the whole RNAPII-Spt4/5-Elf1 complex.Subtracted particles were re-extracted with a binning factor of 2 (pixel size of 2.12 Å/pixel), refined, and classified as without alignment 3D classification with a mask covering both Spt4/5 and Elf1.The selected classes containing Spt4/5 and Elf1 were reverted, refined, and classified as with alignment 3D classification.The Spt4/5 and Elf1 binding classes were selected and resubtracted with a nucleosome mask made from another class with wellobserved nucleosome density.Resubtracted classes from 3D classification were re-extracted with a binning factor of 1 (pixel size of 1.06 Å/pixel), refined, and classified with alignment 3D classification.The class that contained nucleosome density (12,176 particles) was selected as the Spt4/5-Elf1-RNAPIInucleosome complex (SHL(0) stop), and these particles were subjected to 3D refinement.

Figure 1 .
Figure 1.Overall structures of RNAPII-nucleosome L40 and RNAPII-nucleosome L60 .A, design of the nucleosome DNA template.B, scheme of the transcription reaction for the cryo-EM sample preparation.C, overall structures of RNAPII-nucleosome L40 and RNAPII-nucleosome L60 .The figures show PDB models displayed as ribbons.Histones H2A, H2B, H3, and H4 are colored purple, pink, blue, and light blue, respectively.Upstream and downstream DNAs are colored green and yellow, respectively.RNAPII is colored gray.RNAPII, RNA polymerase II.

Figure 2 .
Figure 2. Structural comparison of RNAPII-nucleosome L40 and RNAPII-nucleosome L60 .A, details of the DNA paths of the RNAPII-nucleosome L40 and the RNAPII-nucleosome L60 .The DNA and nucleosomes are shown as ribbon models, and the magnesium ions in the catalytic center are highlighted with red circles on the transparent composite maps.The colors of histones, DNA, and RNAPII are the same as in Figure 1.B and C, interactions between RNAPII and nucleosome in the RNAPII-nucleosome L40 (B) and RNAPII-nucleosome L60 (C) complexes.Two views are presented with close-ups (right panels).The colors of histones, DNA, and RNAPII are the same as in Figure 1.In the lower right panel, the upstream DNA (green) is omitted to render the RNAPII-histone interface visible.RNAPII, RNA polymerase II.

Figure 3 .
Figure 3.The nucleosome structure with the upstream DNA loop.A, structural comparison of the nucleosome region of the RNAPII-nucleosome L40 , RNAPII-nucleosome L60 , and the crystal structure of the H2A-H3.3nucleosome (PDB: 5X7X) (22).The colors of histones are the same as in Figure 1.B, structural comparison of the upstream DNA regions of RNAPII-nucleosome L40 (magenta) and RNAPII-nucleosome L60 (cyan).The upstream DNA of the RNAPII-nucleosome L40 is kinked by 50 degrees relative to that of the RNAPII-nucleosome L60 .RNAPII, RNA polymerase II.