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Structural basis of the nucleosome transition during RNA polymerase II passage

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Science  02 Nov 2018:
Vol. 362, Issue 6414, pp. 595-598
DOI: 10.1126/science.aau9904

Nucleosomal DNA transcription

In eukaryotes, the basic chromatin unit nucleosome stalls RNA polymerase II (RNAPII) when it transcribes genetic information on DNA. Using cryo–electron microscopy, Kujirai et al. explored seven structures of the RNAPII-nucleosome complex, in which RNAPII pauses at four locations on the nucleosome. These serial snapshots of the RNAPII progression reveal the molecular mechanism of how RNAPII peels the nucleosomal DNA off the histone stepwise.

Science, this issue p. 595

Abstract

Genomic DNA forms chromatin, in which the nucleosome is the repeating unit. The mechanism by which RNA polymerase II (RNAPII) transcribes the nucleosomal DNA remains unclear. Here we report the cryo–electron microscopy structures of RNAPII-nucleosome complexes in which RNAPII pauses at the superhelical locations SHL(−6), SHL(−5), SHL(−2), and SHL(−1) of the nucleosome. RNAPII pauses at the major histone-DNA contact sites, and the nucleosome interactions with the RNAPII subunits stabilize the pause. These structures reveal snapshots of nucleosomal transcription, in which RNAPII gradually tears DNA from the histone surface while preserving the histone octamer. The nucleosomes in the SHL(−1) complexes are bound to a “foreign” DNA segment, which might explain the histone transfer mechanism. These results provide the foundations for understanding chromatin transcription and epigenetic regulation.

In eukaryotes, genomic DNA is packaged as chromatin, in which the nucleosome is the basic unit (1). In the nucleosome, a 145– to 147–base pair (bp) DNA segment is stably wrapped around a histone octamer, composed of two each of histone H2A-H2B and H3-H4 heterodimers (2). Because the nucleosome has a highly stable architecture, it strongly affects genomic DNA functions, such as transcription (35). RNA polymerase II (RNAPII), a multisubunit molecular machine (6), transcribes protein-coding genes, but the mechanism of nucleosome transcription remains obscure.

To address this problem, we performed cryo–electron microscopy (cryo-EM) single-particle analyses of transcribing RNAPII-nucleosome complexes. In cells, RNA elongation by RNAPII is stalled at multiple sites within a nucleosome up to the nucleosomal dyad (7). After passing through the nucleosomal dyad, RNAPII transcribes the DNA without obvious impediments (7), because the histones may be removed from the DNA ahead of the RNAPII. To avoid the histone dismantling by RNAPII, we designed a nucleosomal template with a T-less sequence region, by which transcription elongation is stalled at the superhelical location SHL(−1), a natural RNAPII pausing site located ~10 bp upstream of the nucleosomal dyad, in the presence of 3′–deoxyadenosine triphosphate (fig. S1A). Widom 601, an artificial sequence with high nucleosome positioning power, was used for the design (8). After reconstitution of the human nucleosome with the DNA, a linker DNA containing the transcription bubble was ligated to one end of the nucleosomal DNA (fig. S1, A to D). RNA elongation was performed by RNAPII from the yeast Komagataella pastoris (fig. S1E). RNAPII paused predominantly at the entry of the nucleosome [SHL(−5)], reflecting the rate-limiting step of the nucleosomal transcription in vitro (fig. S1F). By contrast, RNAPII elongated RNA until reaching SHL(−1) in the presence of the transcription elongation factor TFIIS (fig. S1E), which facilitates the nucleosomal transcription (fig. S1F) (9, 10).

The obtained transcribing complexes, containing RNAPII, nucleosomes, and transcribed RNAs, were partially purified by the sucrose-glutaraldehyde gradient ultracentrifugation (GraFix) method (11). The RNAs within the RNAPII-nucleosome complex mixture were then analyzed by denaturing polyacrylamide gel electrophoresis (Fig. 1A). We observed substantial pausing of RNAPII near the entry [SHL(−5)] and before the dyad [SHL(−1)] of the nucleosome (Fig. 1A). These major pausing sites correspond well to the in vivo pausing sites within gene-body nucleosomes (7). In addition, we detected minor but clear RNA bands corresponding to the paused RNAPII at SHL(−6) and SHL(−2) (Fig. 1A).

Fig. 1 Cryo-EM structures of the transcribing RNAPII-nucleosome complexes.

(A) Elongated RNAs in the purified transcribing RNAPII-nucleosome complexes were analyzed by denaturing gel electrophoresis. After the transcription reaction, the complexes were fractionated by the GraFix method (GraFix lane), desalted on a gel filtration column (gel-filtrated lane), and concentrated (concentrated lane). (B to E) Cryo-EM densities of the RNAPII-nucleosome complexes paused at SHL(−6), SHL(−5), SHL(−2), and SHL(−1) with fitted structural models. The density for the Rpb4/Rpb7 stalk of RNAPII is weak, because of its inherent flexibility (31, 32). Focused classification clearly reveals the stalk density (fig. S11). RNAPII, DNA, H2A, H2B, H3, and H4 are colored gray, orange, magenta, pink, light blue, and blue, respectively. The arrows indicate the SHL(0) positions. Arrowheads represent transitions between the complexes.

We then collected cryo-EM images of the transcription reaction mixtures, and extensive three-dimensional classifications delineated seven distinct states of the RNAPII-nucleosome complexes, which belong to the complexes paused around SHL(−6), SHL(−5), SHL(−2), and SHL(−1) (Fig. 1, B to E; figs. S2 to S6; tables S1 and S2; and movie S1). These structures revealed the nucleosome transition during the RNAPII passage, whereas no major conformational change was observed in the RNAPII (see materials and methods). In these complexes, RNAPII is stalled at the major contact sites between the DNA and the core histones (Fig. 2). These sites are either the H2A-H2B contact site [SHL(−5)] or the H3-H4 contact sites [SHL(−6), SHL(−2), and SHL(−1)] (Fig. 2). These strong histone-DNA interactions should be the major cause of the RNAPII pausing and thus explain the previous observations that the histone mutations that weaken histone-DNA interactions relieve the RNAPII pausing (12). The nucleosome orientations relative to RNAPII are similar among these structures (fig. S7, A to E), in which the curved nucleosomal DNA fits on the RNAPII surface, interacting with the clamp head domain of the Rpb1 subunit (fig. S7F). These RNAPII-DNA interactions seem to stabilize the RNAPII stalled at each SHL.

Fig. 2 Structural transition of the nucleosome.

Improved density maps after focused refinement (see materials and methods) for the SHL(−6) (A), SHL(−5) (B), SHL(−2) (C), and SHL(−1) (D) complexes are shown with the atomic models. Top panels indicate overall nucleosome structures. These views are the same orientations as those in Fig. 1, B to E (top panels). Close-up views of the regions enclosed by the dashed squares in the top panels are presented in the bottom panels. The arginine (R) and lysine (K) residues that potentially contact the DNA at the RNAPII pausing sites are highlighted.

In the RNAPII-nucleosome complexes stalled around SHL(−6) and SHL(−5), the RNAPII is located near the entrance of the nucleosome. In the SHL(−6) complex, the nucleosomal DNA is entirely wrapped around the histone octamer (Figs. 1B and 2A). In the SHL(−5) complex, the RNAPII has advanced by ~20 bp and the DNA end of the nucleosome is torn away from the N-terminal part of histone H3 (Figs. 1C and 2B). The H3 N-terminal region contains many modification sites, which may affect the DNA affinity for the histone surface (1315). A similar 20-bp detachment reportedly occurs with nucleosome remodelers, such as CHD1 and INO80 (1618). Therefore, the histone modifications and the nucleosome remodelers may modulate the nucleosome accessibility of RNAPII at the nucleosomal entry site.

Before the dyad, RNAPII is stalled at SHL(−2) and SHL(−1) of the nucleosome (Figs. 1, D and E, and 2, C and D). In the SHL(−2) and SHL(−1) complexes, ~50- and 60-bp DNA regions are peeled away from the histone surface by RNAPII, respectively. An H2A-H2B dimer is reportedly released from the nucleosome when a 40-bp stretch of the nucleosomal DNA is removed (19). However, the H2A-H2B dimer density is clearly observed in the SHL(−1) and SHL(−2) complexes (Fig. 3, A and B). Consistently, we observed no nucleosome particles lacking H2A-H2B during the classification processes. In the SHL(−2) complex, we observed a density connection between the Rpb2 lobe domain of RNAPII and the H2A-H2B dimer, and this interaction may retain the H2A-H2B dimer in the nucleosome (Fig. 3C).

Fig. 3 Interaction between an H2A-H2B dimer and RNAPII.

(A) A close-up view of an H2A-H2B dimer in the nucleosomes of the SHL(−1) complex. Cryo-EM density maps of the nucleosomes in the SHL(−1) complex, fitted with the structure, are shown. (B) The overall density map with the atomic model structure of the SHL(−2) complex. Rpb2 of RNAPII is colored pale blue. (C) The region enclosed by a dashed square in (B) is enlarged.

The majority of the complexes stalled at SHL(−1) contained a ~60-bp DNA segment of unknown origin (“foreign” DNA), which was bound to the DNA-peeled region of the nucleosome (fig. S8, A to C). We also observed foreign DNA association in the non–cross-linked sample (fig. S9), indicating that the DNA binding is not due to cross-linking. Further analyses of these complexes yielded three distinguishable classes with the foreign DNA: the SHL(−1), tilted SHL(−1), and SHL(−1)+1 complexes (fig. S8, A to C). In the SHL(−1)+1 complex, RNAPII proceeded by 1 bp (fig. S8C) and rotated by ~36° (fig. S8D and movie S2) compared with the positions in the SHL(−1) complex. The tilted SHL(−1) complex seems to be an intermediate between the SHL(−1) and SHL(−1)+1 complexes (fig. S8, B and D, and movie S2). In these complexes, RNAPII is stalled because of the DNA contacts with histones H3-H4. The acetylation of Lys64 in H3, which may reduce the H3-DNA contact at SHL(−1) (Fig. 2D), is reportedly accumulated at the transcription start sites of active genes (20). Therefore, SHL(−1) is probably another important site for transcriptional regulation coupled with histone modifications.

The foreign DNA is derived from a linker DNA region of other nucleosomes or disassembled nucleosomes. We observed the former particles in the raw cryo-EM images (fig. S10). This suggests that the DNA-peeled region (mainly H2A-H2B) has a propensity to associate with DNA to regenerate nucleosome-like structures. Therefore, the foreign DNA could serve as an intermediate for histone transfer to other DNA regions in cis or trans. It has been proposed that the histone is transferred from ahead of the transcribing RNA polymerase to behind it via a “template looping” intermediate (2124). If a region of the upstream DNA behind RNAPII interacted with the DNA-peeled part of H2A-H2B, like the foreign DNA observed here, then it would function as a histone transfer intermediate.

The present study unveiled the sequential transitions of nucleosomes during RNAPII passage (Fig. 4). The transcribing RNAPII first encounters the nucleosome [step 1, SHL(−6)] and begins its invasion with the peeling of a helical pitch of the DNA segment from the histone surface [step 2, SHL(−5)]. The RNAPII elongates the RNA with continuous peeling of the nucleosomal DNA and stalls at SHL(−2) of the nucleosome (step 3). Finally, RNAPII stalls at SHL(−1) (step 4), where the nucleosome interacts with the foreign DNA (step 5). At or beyond SHL(−1), transcription results in either histone transfer to the upstream DNA behind RNAPII or histone eviction from the DNA. We have revealed the structures of the RNAPII-nucleosome complexes formed in the presence of the transcription elongation factor TFIIS. However, in cells, several other elongation factors, including Spt4, Spt5, Spt6, PAF1C, and Elf1, interact with RNAPII (9, 2527) and function in proper transcription in chromatin (2830). Further studies are needed to understand the mechanism of chromatin transcription by RNAPII.

Fig. 4 Scheme of transcription elongation through a nucleosome by RNAPII.

RNAPII encounters a nucleosome [SHL(−6), step 1] and then proceeds to SHL(−5), which is a major pausing site for RNAPII (step 2). After passing through SHL(−5), the RNAPII pauses at SHL(−2) (step 3) and proceeds to SHL(−1) (step 4). In the SHL(−2) and SHL(−1) complexes, an H2A-H2B dimer is exposed. Then foreign DNA is bound to the DNA-peeled region of the nucleosome in the complex (step 5). Histones may be removed from the region ahead of the RNAPII, by transfer in cis or trans or by eviction. The red DNA cartoons indicate possible orientations of the upstream DNAs.

The nucleosome-dependent transcriptional pause is important for gene regulation. The first (+1) nucleosome is implicated in the promoter-proximal pausing of transcription, which regulates nucleosomal entry by RNAPII (7). The partially DNA-peeled nucleosome within the paused complexes should be the target of histone chaperones, nucleosome remodelers, and modification enzymes. Our present RNAPII-nucleosome complex structures at the major pause sites shed new light on such regulation mechanisms.

Supplementary Materials

www.sciencemag.org/content/362/6414/595/suppl/DC1

Materials and Methods

Figs. S1 to S11

Tables S1 and S2

References (3347)

Movies S1 and S2

References and Notes

Acknowledgments: We thank K. Katsura and T. Yokoyama (RIKEN) for their help with the RNAPII preparation and cryo-EM analyses and Y. Iikura (University of Tokyo) for her assistance. Funding: This work was supported in part by the RIKEN Dynamic Structural Biology project (to M.S., S.S., and H.K.); JSPS KAKENHI grants JP18H05534 (to H.K.), JP25116002 (to H.K.), and JP15H04344 (to S.S.); JST CREST grant JPMJCR16G1 (to H.K.); and the Platform Project for Supporting Drug Discovery and Life Science Research (BINDS) from AMED under grants JP18am0101076 (to H.K.) and JP18am0101082 (to M.S.). Author contributions: T.K. and Y.F. prepared the RNAPII-nucleosome complexes and performed biochemical analyses. H.E., T.K., M.S., and S.S. performed cryo-EM analyses. S.S. and H.K. conceived, designed, and supervised all of the work. T.K., H.E., S.S., and H.K. wrote the paper. All of the authors discussed the results and commented on the manuscript. Competing interests: The authors declare no competing interests. Data and materials availability: The cryo-EM reconstructions and atomic models of the RNAPII-nucleosome complexes have been deposited in the Electron Microscopy Data Bank and the Protein Data Bank (PDB) under the following accession codes: EMD-6981 and PDB ID 6A5O for the RNAPII complex stalled at SHL(−6) of the nucleosome; EMD-6982 and PDB ID 6A5P for the RNAPII complex stalled at SHL(−5) of the nucleosome; EMD-6983 and PDB ID 6A5P for the RNAPII complex stalled at SHL(−2) of the nucleosome; EMD-6984 and PDB ID 6A5T for the RNAPII complex stalled at SHL(−1) of the nucleosome; EMD-6980 and PDB ID 6A5L for the RNAPII complex stalled at SHL(−1) of the nucleosome, with foreign DNA; EMD-6985 and PDB ID 6A5U for the RNAPII complex stalled at SHL(−1) of the nucleosome, with foreign DNA, tilt conformation; and EMD-6986 and PDB ID 6A5V for the RNAPII complex stalled at SHL(−1)+1 of the nucleosome, with foreign DNA.

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