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Structural transition of the nucleosome during chromatin remodeling and transcription
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ScienceDirect Structural transition of the nucleosome during chromatin remodeling and transcription Wataru Kobayashi1,2 and Hitoshi Kurumizaka1,2 In eukaryotes, the nucleosome is the basic unit of chromatin. Since the genomic DNA is tightly wrapped around the histone octamer in the nucleosome, its function is severely restricted in chromatin. To overcome the nucleosome barrier, the nucleosome structure must dynamically change during genomic DNA functions. Recent structural studies revealed that chromatin remodelers and RNA polymerase II drastically alter the nucleosome structures during the chromatin remodeling and transcription processes. These results provide important information for understanding how genes are properly regulated in eukaryotes. Here, we review the recent structural studies of nucleosome versatility and dynamics during chromatin remodeling and transcription by RNA polymerase II. Addresses 1 Laboratory of Chromatin Structure and Function, Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan 2 Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan Corresponding author: Kurumizaka, Hitoshi ([email protected])
Current Opinion in Structural Biology 2019, 59:107–114 This review comes from a themed issue on Protein nucleic acid interactions Edited by Fred Allain and Martin Jinek
https://doi.org/10.1016/j.sbi.2019.07.011 0959-440X/ã 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4.0/).
Introduction Eukaryotes store genomic DNA in the form of chromatin, in which the nucleosome is the basic repeating unit [1]. In the nucleosome, 150 base pairs (bp) of DNA are tightly bound to histones H2A, H2B, H3, and H4 (Figure 1a) [2]. Histones are basic proteins that form specific heterodimers, H2A–H2B and H3–H4 [1,2,3]. The H3–H4 heterodimers are further associated and form a dimer of heterodimers, called the H3–H4 tetramer [1,2,3]. To form the complete nucleosome, called the ‘octasome’, two H2A–H2B heterodimers associate with an H3–H4 tetramer, and the negatively charged backbone phosphates of each DNA strand bind to the positively charged surface of the histone www.sciencedirect.com
octamer at every 10 bases [2,3]. Consequently, the DNA is lefthandedly wrapped 1.65 turns around the histone octamer in the nucleosome. The DNA locations in the nucleosome are referred to as ‘superhelical locations (SHLs)’ [2,3]. Each SHL location is defined as a site where the major-groove faces toward the histone surface, and the SHLs are numbered, as SHL 1, 2 . . . 7 and SHL +1, +2 . . . +7, relative to the nucleosomal dyad axis position (SHL 0) with 10 base-pair periodicity (Figure 1b). Nucleosome formation plays an important role in genomic DNA compaction into chromatin. However, the histone– DNA interaction profoundly restricts the accessibility of other DNA binding proteins to genomic DNA, and negatively regulates genomic DNA functions, such as replication, repair, recombination, and transcription [4–6]. To ensure the genomic DNA functions in chromatin, the nucleosome structure must be versatile and dynamic. The nucleosome has intrinsic dynamic properties, but its dynamics is also regulated by chromatin remodeling factors, which are pivotal factors for genomic DNA functions [7–9]. The structural versatility and dynamic behavior of the nucleosome during and after nucleosome remodeling are popular research topics. In addition, the dynamic structural transition of the nucleosome during the transcription elongation process has been reported. Here, we review the current structural studies of nucleosome versatility and dynamics.
Structures of nucleosome complexes with chromatin remodeling factors ATP-dependent chromatin remodelers alter the structure and/or positioning of nucleosomes, and allow regulatory proteins to access their target DNA sites in chromatin. Chromatin remodeling factors use the energy of ATP hydrolysis, and promote the translocation of the nucleosomal DNA by distorting or disrupting the histone–DNA contacts in the nucleosome. Consequently, chromatin remodelers catalyze histone octamer sliding along DNA, histone octamer disassembly, and/or histone variant exchange [7–9]. Recent technological advances in cryo-electron microscopy (cryo-EM) have revealed how these chromatin remodelers change the histone–DNA contacts in the nucleosome.
Swi2/Snf2
Swi2/Snf2 is the ATPase motor subunit of the SWI/SNF complex, which functions in transcription activation and Current Opinion in Structural Biology 2019, 59:107–114
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Figure 1
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Overview of the nucleosome structure. (a) High-resolution structure of the nucleosome core particle (PDB ID: 1KX5 [54]). Histones and DNA are shown in surface and cartoon representations, respectively. H2A, H2B, H3, H4, and DNA are colored magenta, yellow, blue, green, and grey, respectively. (b) The superhelical locations (SHLs) of the nucleosome. The nucleosomal DNA is lefthandedly wrapped around the histone octamer surface. The nucleosomal dyad (center) position is numbered as SHL 0. Each SHL location is numbered as SHL 1 to 7 and SHL +1 to +7 from SHL 0, with 10 base-pair periodicity.
repression [10]. The cryo-EM structure of the nucleosome complexed with the Saccharomyces cerevisiae Swi2/Snf2 segment containing the ATPase core domain has been reported [11]. In this Swi2/Snf2–nucleosome complex, the structure was solved in the absence of ATP, and Swi2/Snf2 mainly contacts the nucleosomal DNA at SHL +2 (or 2) through its primary DNAbinding domain (Figure 2a). The histone H4 N-terminal tail of the nucleosome also binds to the DNA-binding domain of Swi2/Snf2 (Figure 2a). Mutational analyses of the H4 N-terminal tail and Swi2/Snf2 revealed that the interaction between H4 and Swi2/Snf2 facilitates its chromatin remodeling activity [11]. In addition to the SHL +2 (or 2) site, Swi2/Snf2 also contacts the adjacent DNA gyre at SHL 6 (or +6) through its secondary DNA-binding domain (Figure 2a). As a result, the nucleosomal DNA is locally distorted at the SHL +2 (or 2) site (Figure 2b). This local DNA distortion may stimulate further nucleosome remodeling driven by energy from ATP hydrolysis.
[17,18]. The structure was determined in the presence of a non-hydrolyzable ATP analog, ADP/BeF3. Similar to the Swi2/Snf2–nucleosome complex, Chd1 binds to the nucleosomal DNA at SHL +2 (or 2) in the Chd1–nucleosome complex, and the H4 N-terminal tail of the nucleosome also binds to the ATPase domain of Chd1 (Figure 2c). The SHL +2 (or 2) and H4 N-terminal tail binding mechanisms may be common between Swi2/Snf2 and Chd1.
Chd1
Interestingly, in the Chd1–nucleosome complex, a region containing about 20 base-pairs of nucleosomal DNA (from SHL 5 (or +5) to SHL 7 (or +7)) is peeled off from the histone surface (Figure 2c). This may be induced by the SANT and SLIDE DNA-binding domains, which apparently bind to the linker DNA cooperatively and peel a 20 base-pair stretch of the nucleosomal DNA in an ATPdependent manner [17,18]. This DNA-peeling process may induce the DNA binding of the double chromodomain to the SHL +1 (or 1) site with a swinging motion (Figure 2c). Eventually, the DNA binding of the double chromodomain could bring the ATPase domain closer to the nucleosomal DNA [17,18].
Chd1 reportedly regulates transcription processes [12–14]. Many transcription elongation factors have been reported as Chd1-interacting factors [12,13]. Chd1 functions as a monomeric remodeler, and contains the SANT and SLIDE DNA-binding domains, the double chromodomain, and the ATPase domain [15,16]. The cryo-EM structure of the S. cerevisiae Chd1–nucleosome complex has been reported
In the Chd1–nucleosome complex, the nucleosomal DNA translocates one base pair, as compared with the Swi2/Snf2–nucleosome complex. These Swi2/Snf2–nucleosome and Chd1–nucleosome structures may represent the pre-translocated and posttranslocated nucleosome structures, respectively.
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Structural dynamics of the nucleosome Kobayashi and Kurumizaka 109
Figure 2
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The structures of the nucleosomes in the complexes with chromatin remodeling factors. (a) The structure of the Swi2/Snf2–nucleosome complex (PDB ID: 5X0Y). Swi2/Snf2 (orange) binds to the nucleosomal DNA at SHL +2 (or 2) and SHL 6 (or +6) through its primary DNA-binding domain and secondary DNA-binding domain (white and black dashed circles, respectively). (b) Superimposition of the nucleosomal DNA bound by Swi2/ Snf2 (orange, PDB ID: 5X0Y) and the nucleosome containing a 145 base-pair Widom 601 sequence (blue, PDB ID: 2NZD [55]). The black dashed www.sciencedirect.com
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The SWR1 and INO80 complexes
The SWR1 and INO80 complexes are multi-subunit structures. The SWR1 and INO80 complexes both function in transcriptional regulation, DNA replication, and DNA repair [19,20]. The SWR1 complex is a chromatin remodeler that promotes nucleosomal H2A exchange with the histone variant H2A.Z [21,22]. In contrast, the INO80 complex catalyzes the reverse exchange reaction, by replacing the nucleosomal H2A.Z with H2A [23,24]. The cryo-EM structure of the S. cerevisiae SWR1 complex has been determined [25]. The SWR1 complex is composed of 14 subunits, and is bound to the nucleosome (Figure 2d). In the SWR1–nucleosome complex, the ATPase motor subunit, Swr1, binds to the nucleosomal DNA at SHL +2 (or 2), and a local DNA bulge is observed at the site (Figure 2d and e). This bulge formation may be induced by a 1 bp DNA translocation by the SWR1 complex. In this step, the histone core structure is also distorted [25]. The SWR1 complex subunits, Arp6 and Swc6, bind to the nucleosomal DNA at SHL +6 (or 6). The Arp6 and Swc6 subunits also interact with the C-terminal tail and the acidic patch of H2A in the nucleosome (Figure 2d). As a result, about 10 base pairs of the nucleosomal DNA (from SHL +6 (or 6) to +7 (or 7)) are unwrapped from the histone octamer surface (Figure 2d). These conformational changes require ATP binding, but not ATP hydrolysis [25]. However, a non-hydrolyzable ATP analog does not support the histone exchange, suggesting that both ATP binding and hydrolysis are required for histone exchange by the SWR1 complex. The nucleosome structure complexed with the INO80 complex has been determined by cryo-EM [26,27]. The ATPase motor subunit of the INO80 complex binds to the nucleosomal DNA at SHL +6 (or 6), and the accessory Arp5 and Ies6 subunits bind to SHL +2 (or 2) (Figure 2f). These nucleosome-binding locations of the ATPase motor and accessory proteins are opposite to those of the SWR1–nucleosome complex (Figure 2d). In the INO80–nucleosome complex, the nucleosomal DNA is detached from the aN helix of H3 and the loop2 of H2A, and consequently, the H2A–H2B dimer is partially exposed to the solvent (Figure 2f). The ATPase domain binds to the detached DNA region, and cooperatively translocates the nucleosomal DNA with the Arp5
and Ies6 subunits bound to the SHL +2 (or 2) and SHL +3 (or 3) sites, in a counter grip motion (Figure 2f). Thus, a DNA loop might be created between the ATPase motor and the Arp5–Ies6 subunits, resulting in large translocations with 10–20 step sizes [28], as well as histone exchange [23,24].
The structure of the overlapping dinucleosome In the process of the chromatin remodeling, a repositioned nucleosome may collide with a neighboring nucleosome and form unusual structures, such as the ‘altosome’ and ‘overlapping dinucleosome’ [29,30]. The formation of these unusual dinucleosome structures is enzymatically promoted, as consequence of the nucleosome remodeling by the SWI/SNF complex [29,30]. The overlapping dinucleosome is suggested to be composed of the hexasome, lacking one H2A–H2B dimer from the canonical nucleosome (octasome), and the octasome (Figure 3a). The crystal structure of the overlapping dinucleosome has been reported [31]. In the overlapping dinucleosome, the hexasome directly contacts the octasome without an obvious linker DNA segment. As a result, a 250 bp DNA segment is continuously wrapped in three turns around the histone core, containing three H2A–H2B dimers and four H3–H4 dimers (Figure 3b). The genome-wide analysis suggested that the overlapping dinucleosome is predominantly formed at the regions just downstream of transcription start sites (TSSs) [31]. This is consistent with the idea that the chromatin remodeling factor remodels the chromatin structure at the TSS to form the nucleosome free region (NFR) in actively transcribed genes, followed by the recruitment of the transcription machinery in the NFR. It is intriguing to study the relationship between overlapping dinucleosome formation and transcription activation. Further studies are awaited to reveal how chromatin remodelers promote overlapping dinucleosome formation at specific genomic loci.
The nucleosome structures in the complexes with transcribing RNA polymerase II The transcription of protein-coding and non-coding RNA genes is promoted by RNA polymerase II (RNAPII) [32,33]. Owing to the strong histone–DNA interactions, the nucleosome is inhibitory for the transcription process
(Figure 2 Legend Continued) circle represents the distorted DNA strand at SHL +2 (or 2). (c) The structure of the Chd1–nucleosome complex (PDB ID: 5O9G). Chd1 (orange, red, and brown) binds to the nucleosomal DNA at SHL +2 (or 2) and SHL+ 1 (or 1) through its ATPase domain and double chromodomain, respectively. The nucleosomal DNA (from SHL 5 (or +5) to SHL 7 (or +7)) is peeled off from the histone surface. The Chd1 ATPase domain, SANT SLIDE DNA-binding domain, and double chromodomain are colored orange, red, and brown, respectively. (d) The structure of the SWR1–nucleosome complex (PDB ID: 6GEJ). Swr1 (orange) and the Arp6–Swc6 (light blue) subunits bind to the nucleosomal DNA at SHL 2 (or +2) and SHL 6 (or +6), respectively. Other subunits are represented with transparency. The white dashed circle shows the interaction site with the acidic patch of H2A. (e) Superimposition of the nucleosome bound by the SWR1 complex (orange, PDB ID: 6GEJ) and the canonical yeast nucleosome (blue, PDB ID: 1ID3 [56]). The black dashed circle shows the bulged DNA strand at SHL 2 (or +2). (f) The structure of the INO80–nucleosome complex (PDB ID: 6FML). Ino80 (orange) and the Arp5–Isc6 (light blue) subunits bind to the nucleosomal DNA at SHL 6 (or +6) and SHL 2 (or +2), respectively. Other subunits are represented with transparency. Current Opinion in Structural Biology 2019, 59:107–114
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Figure 3
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Crystal structure of the overlapping dinucleosome. (a) A model for overlapping dinucleosome formation. In this model, the nucleosome repositioned by a chromatin remodeling factor collides with a neighboring nucleosome, forming the overlapping nucleosome concomitant with the release of an H2A–H2B dimer. (b) Overview of the overlapping dinucleosome (PDB ID: 5GSE). Histones and DNA are shown in surface representations. H2A, H2B, H3, H4, and DNA are colored magenta, yellow, blue, green, and grey, respectively. Figure 4
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Snapshots of the nucleosome structure transition during RNAPII passage. (a–d) The structures of RNAPII–nucleosome complexes paused at SHL 6, SHL 5, SHL 2, and SHL 1 (upper panel, PDB IDs: 6A5O, 6A5P, 6A5R, and 6A5T, respectively) are presented. The structural transitions of the nucleosomal DNA are shown in the bottom panel. RNAPII, H2A, H2B, H3, H4, and DNA are colored orange, magenta, yellow, blue, green, and grey, respectively. www.sciencedirect.com
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mediated by RNAPII [34–36]. In vivo and in vitro analyses suggested that, in the nucleosome, RNAPII preferentially pauses near the entry site and before the dyad (center) site of the nucleosomal DNA [37–42].
Conflict of interest statement
To reveal the mechanism by which RNAPII transcribes on the nucleosomal DNA, cryo-EM studies of the nucleosome structures complexed with the transcribing RNAPII have been performed [43,44]. These studies provided snapshots of the nucleosome structure transitions during RNAPII passage. The structures revealed the RNAPII– nucleosome complexes paused at SHL 6, SHL 5, SHL 2, and SHL 1. In the SHL 6 state, the transcribing RNAPII pauses just in front of the nucleosome, and the nucleosomal DNA is entirely wrapped around the histone octamer (Figure 4a). RNAPII then advances by approximately 20 base pairs from the entry site of the nucleosome, and pauses at the SHL 5 site, which is a major RNAPII pausing site in cells [37,43]. In the SHL 5 complex, the H2A–H2B dimer–DNA interactions stall the transcription elongation by RNAPII, and the DNA end (20 base pairs) of the nucleosome is peeled from the histone surface (Figure 4b). Subsequently, RNAPII elongates the RNA with continuous peeling of the nucleosomal DNA, and pauses at the SHL 2 and SHL 1 sites (Figure 4c and d). Consequently, about 50–60 base pairs of DNA are detached from the histone surface. Interestingly, the H2A–H2B dimers remain in the nucleosome, although one of the H2A–H2B dimers loses its interaction with the DNA and is exposed to the solvent (Figure 4c and d). This may be supported by the direct interaction between RNAPII and an H2A–H2B dimer. In the SHL 1 state, ‘a foreign DNA’ is bound to the exposed H2A–H2B dimer region in trans. This state may represent an intermediate state for the histone transfer during the transcription elongation, such as a ‘template looping’ structure, as proposed by an atomic force microscopic study [45].
This work was supported in part by JSPS KAKENHI Grant Numbers JP18H05534 and JP17H01408 [to H.K.]. This work was also partly supported by JST CREST Grant Number JPMJCR16G1 and the Platform Project for Supporting Drug Discovery and Life Science Research (BINDS) from AMED, under Grant Number JP19am0101076 [to H.K.].
Concluding remarks Recent advances in high-resolution cryo-EM analysis have provided breakthroughs in the structural biology of nucleosome dynamics. In this review, we summarized how chromatin remodeling factors and RNAPII promote the dynamic structural transitions of the nucleosome. Chromatin remodelers translocate the nucleosomal DNA with two DNA binding domains. The nucleosomal DNA translocation mediated by chromatin remodelers may be coupled with the genomic DNA functions, such as transcription. In fact, RNAPII reportedly cooperates with transcription elongation factors, chromatin remodelers, and histone chaperones [44,46–51]. Structural studies of nucleosome transcription coupled with these factors are the next challenges. The chromatin remodeling activity may be required for RNAPII transcription activation and repression in higher ordered chromatin architectures. It is also intriguing to study how nucleosomes suppress transcription in inactive heterochromatin [52,53]. Current Opinion in Structural Biology 2019, 59:107–114
Nothing declared.
Acknowledgements
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38. Bondarenko VA, Steele LM, Ujva´ri A, Gaykalova DA, Kulaeva OI, Polikanov YS, Luse DS, Studitsky VM: Nucleosomes can form a polar barrier to transcript elongation by RNA polymerase II. Mol Cell 2006, 24:469-479. 39. Kulaeva OI, Gaykalova DA, Pestov NA, Golovastov VV, Vassylyev DG, Artsimovitch I, Studitsky VM: Mechanism of chromatin remodeling and recovery during passage of RNA polymerase II. Nat Struct Mol Biol 2009, 16:1272-1278. 40. Kulaeva OI, Hsieh FK, Chang HW, Luse DS, Studitsky VM: Mechanism of transcription through a nucleosome by RNA polymerase II. Biochim Biophys Acta 2013, 1829:76-83. 41. Weber CM, Ramachandran S, Henikoff S: Nucleosomes are context-specific, H2A.Z-modulated barriers to RNA polymerase. Mol Cell 2014, 53:819-830. 42. Ramachandran S, Ahmad K, Henikoff S: Transcription and remodeling produce asymmetrically unwrapped nucleosomal intermediates. Mol Cell 2017, 68:1038-1053. 43. Kujirai T, Ehara H, Fujino Y, Shirouzu M, Sekine SI, Kurumizaka H: Structural basis of the nucleosome transition during RNA polymerase II passage. Science 2018, 362:595-598. Snapshot structures of the RNAPII–nucleosome complexes during transcription elongation revealed that RNA polymerase II pauses at histone– DNA contact sites, and gradually unwraps the nucleosomal DNA. 44. Ehara H, Kujirai T, Fujino Y, Shirouzu M, Kurumizaka H, Sekine SI: Structural insight into nucleosome transcription by RNA polymerase II with elongation factors. Science 2019, 363:744-747. The cryo-EM structure of the RNAPII–nucleosome complex with elongation factors revealed that the transcription elongation factors Elf1 and Spt4/5 enhance the RNAPII processivity in the nucleosome. 45. Bintu L, Kopaczynska M, Hodges C, Lubkowska L, Kashlev M, Bustamante C: The elongation rate of RNA polymerase determines the fate of transcribed nucleosomes. Nat Struct Mol Biol 2011, 18:1394-1399. This paper reported the atomic force microscopy analysis of the RNAPII– nucleosome complexes. The authors proposed the template looping model during RNAPII passage through the nucleosome. 46. Orphanides G, LeRoy G, Chang CH, Luse DS, Reinberg D: FACT, a factor that facilitates transcript elongation through nucleosomes. Cell 1998, 92:105-116. 47. LeRoy G, Orphanides G, Lane WS, Reinberg D: Requirement of RSF and FACT for transcription of chromatin templates in vitro. Science 1998, 282:1900-1904. 48. Ehara H, Yokoyama T, Shigematsu H, Yokoyama S, Shirouzu M, Sekine SI: Structure of the complete elongation complex of RNA polymerase II with basal factors. Science 2017, 357:921-924. Current Opinion in Structural Biology 2019, 59:107–114
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ScienceDirect Structural transition of the nucleosome during chromatin remodeling and transcription Wataru Kobayashi1,2 and Hitoshi Kurumizaka1,2 In eukaryotes, the nucleosome is the basic unit of chromatin. Since the genomic DNA is tightly wrapped around the histone octamer in the nucleosome, its function is severely restricted in chromatin. To overcome the nucleosome barrier, the nucleosome structure must dynamically change during genomic DNA functions. Recent structural studies revealed that chromatin remodelers and RNA polymerase II drastically alter the nucleosome structures during the chromatin remodeling and transcription processes. These results provide important information for understanding how genes are properly regulated in eukaryotes. Here, we review the recent structural studies of nucleosome versatility and dynamics during chromatin remodeling and transcription by RNA polymerase II. Addresses 1 Laboratory of Chromatin Structure and Function, Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan 2 Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan Corresponding author: Kurumizaka, Hitoshi ([email protected])
Current Opinion in Structural Biology 2019, 59:107–114 This review comes from a themed issue on Protein nucleic acid interactions Edited by Fred Allain and Martin Jinek
https://doi.org/10.1016/j.sbi.2019.07.011 0959-440X/ã 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4.0/).
Introduction Eukaryotes store genomic DNA in the form of chromatin, in which the nucleosome is the basic repeating unit [1]. In the nucleosome, 150 base pairs (bp) of DNA are tightly bound to histones H2A, H2B, H3, and H4 (Figure 1a) [2]. Histones are basic proteins that form specific heterodimers, H2A–H2B and H3–H4 [1,2,3]. The H3–H4 heterodimers are further associated and form a dimer of heterodimers, called the H3–H4 tetramer [1,2,3]. To form the complete nucleosome, called the ‘octasome’, two H2A–H2B heterodimers associate with an H3–H4 tetramer, and the negatively charged backbone phosphates of each DNA strand bind to the positively charged surface of the histone www.sciencedirect.com
octamer at every 10 bases [2,3]. Consequently, the DNA is lefthandedly wrapped 1.65 turns around the histone octamer in the nucleosome. The DNA locations in the nucleosome are referred to as ‘superhelical locations (SHLs)’ [2,3]. Each SHL location is defined as a site where the major-groove faces toward the histone surface, and the SHLs are numbered, as SHL 1, 2 . . . 7 and SHL +1, +2 . . . +7, relative to the nucleosomal dyad axis position (SHL 0) with 10 base-pair periodicity (Figure 1b). Nucleosome formation plays an important role in genomic DNA compaction into chromatin. However, the histone– DNA interaction profoundly restricts the accessibility of other DNA binding proteins to genomic DNA, and negatively regulates genomic DNA functions, such as replication, repair, recombination, and transcription [4–6]. To ensure the genomic DNA functions in chromatin, the nucleosome structure must be versatile and dynamic. The nucleosome has intrinsic dynamic properties, but its dynamics is also regulated by chromatin remodeling factors, which are pivotal factors for genomic DNA functions [7–9]. The structural versatility and dynamic behavior of the nucleosome during and after nucleosome remodeling are popular research topics. In addition, the dynamic structural transition of the nucleosome during the transcription elongation process has been reported. Here, we review the current structural studies of nucleosome versatility and dynamics.
Structures of nucleosome complexes with chromatin remodeling factors ATP-dependent chromatin remodelers alter the structure and/or positioning of nucleosomes, and allow regulatory proteins to access their target DNA sites in chromatin. Chromatin remodeling factors use the energy of ATP hydrolysis, and promote the translocation of the nucleosomal DNA by distorting or disrupting the histone–DNA contacts in the nucleosome. Consequently, chromatin remodelers catalyze histone octamer sliding along DNA, histone octamer disassembly, and/or histone variant exchange [7–9]. Recent technological advances in cryo-electron microscopy (cryo-EM) have revealed how these chromatin remodelers change the histone–DNA contacts in the nucleosome.
Swi2/Snf2
Swi2/Snf2 is the ATPase motor subunit of the SWI/SNF complex, which functions in transcription activation and Current Opinion in Structural Biology 2019, 59:107–114
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Figure 1
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(b) H2A
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Overview of the nucleosome structure. (a) High-resolution structure of the nucleosome core particle (PDB ID: 1KX5 [54]). Histones and DNA are shown in surface and cartoon representations, respectively. H2A, H2B, H3, H4, and DNA are colored magenta, yellow, blue, green, and grey, respectively. (b) The superhelical locations (SHLs) of the nucleosome. The nucleosomal DNA is lefthandedly wrapped around the histone octamer surface. The nucleosomal dyad (center) position is numbered as SHL 0. Each SHL location is numbered as SHL 1 to 7 and SHL +1 to +7 from SHL 0, with 10 base-pair periodicity.
repression [10]. The cryo-EM structure of the nucleosome complexed with the Saccharomyces cerevisiae Swi2/Snf2 segment containing the ATPase core domain has been reported [11]. In this Swi2/Snf2–nucleosome complex, the structure was solved in the absence of ATP, and Swi2/Snf2 mainly contacts the nucleosomal DNA at SHL +2 (or 2) through its primary DNAbinding domain (Figure 2a). The histone H4 N-terminal tail of the nucleosome also binds to the DNA-binding domain of Swi2/Snf2 (Figure 2a). Mutational analyses of the H4 N-terminal tail and Swi2/Snf2 revealed that the interaction between H4 and Swi2/Snf2 facilitates its chromatin remodeling activity [11]. In addition to the SHL +2 (or 2) site, Swi2/Snf2 also contacts the adjacent DNA gyre at SHL 6 (or +6) through its secondary DNA-binding domain (Figure 2a). As a result, the nucleosomal DNA is locally distorted at the SHL +2 (or 2) site (Figure 2b). This local DNA distortion may stimulate further nucleosome remodeling driven by energy from ATP hydrolysis.
[17,18]. The structure was determined in the presence of a non-hydrolyzable ATP analog, ADP/BeF3. Similar to the Swi2/Snf2–nucleosome complex, Chd1 binds to the nucleosomal DNA at SHL +2 (or 2) in the Chd1–nucleosome complex, and the H4 N-terminal tail of the nucleosome also binds to the ATPase domain of Chd1 (Figure 2c). The SHL +2 (or 2) and H4 N-terminal tail binding mechanisms may be common between Swi2/Snf2 and Chd1.
Chd1
Interestingly, in the Chd1–nucleosome complex, a region containing about 20 base-pairs of nucleosomal DNA (from SHL 5 (or +5) to SHL 7 (or +7)) is peeled off from the histone surface (Figure 2c). This may be induced by the SANT and SLIDE DNA-binding domains, which apparently bind to the linker DNA cooperatively and peel a 20 base-pair stretch of the nucleosomal DNA in an ATPdependent manner [17,18]. This DNA-peeling process may induce the DNA binding of the double chromodomain to the SHL +1 (or 1) site with a swinging motion (Figure 2c). Eventually, the DNA binding of the double chromodomain could bring the ATPase domain closer to the nucleosomal DNA [17,18].
Chd1 reportedly regulates transcription processes [12–14]. Many transcription elongation factors have been reported as Chd1-interacting factors [12,13]. Chd1 functions as a monomeric remodeler, and contains the SANT and SLIDE DNA-binding domains, the double chromodomain, and the ATPase domain [15,16]. The cryo-EM structure of the S. cerevisiae Chd1–nucleosome complex has been reported
In the Chd1–nucleosome complex, the nucleosomal DNA translocates one base pair, as compared with the Swi2/Snf2–nucleosome complex. These Swi2/Snf2–nucleosome and Chd1–nucleosome structures may represent the pre-translocated and posttranslocated nucleosome structures, respectively.
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Structural dynamics of the nucleosome Kobayashi and Kurumizaka 109
Figure 2
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The structures of the nucleosomes in the complexes with chromatin remodeling factors. (a) The structure of the Swi2/Snf2–nucleosome complex (PDB ID: 5X0Y). Swi2/Snf2 (orange) binds to the nucleosomal DNA at SHL +2 (or 2) and SHL 6 (or +6) through its primary DNA-binding domain and secondary DNA-binding domain (white and black dashed circles, respectively). (b) Superimposition of the nucleosomal DNA bound by Swi2/ Snf2 (orange, PDB ID: 5X0Y) and the nucleosome containing a 145 base-pair Widom 601 sequence (blue, PDB ID: 2NZD [55]). The black dashed www.sciencedirect.com
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The SWR1 and INO80 complexes
The SWR1 and INO80 complexes are multi-subunit structures. The SWR1 and INO80 complexes both function in transcriptional regulation, DNA replication, and DNA repair [19,20]. The SWR1 complex is a chromatin remodeler that promotes nucleosomal H2A exchange with the histone variant H2A.Z [21,22]. In contrast, the INO80 complex catalyzes the reverse exchange reaction, by replacing the nucleosomal H2A.Z with H2A [23,24]. The cryo-EM structure of the S. cerevisiae SWR1 complex has been determined [25]. The SWR1 complex is composed of 14 subunits, and is bound to the nucleosome (Figure 2d). In the SWR1–nucleosome complex, the ATPase motor subunit, Swr1, binds to the nucleosomal DNA at SHL +2 (or 2), and a local DNA bulge is observed at the site (Figure 2d and e). This bulge formation may be induced by a 1 bp DNA translocation by the SWR1 complex. In this step, the histone core structure is also distorted [25]. The SWR1 complex subunits, Arp6 and Swc6, bind to the nucleosomal DNA at SHL +6 (or 6). The Arp6 and Swc6 subunits also interact with the C-terminal tail and the acidic patch of H2A in the nucleosome (Figure 2d). As a result, about 10 base pairs of the nucleosomal DNA (from SHL +6 (or 6) to +7 (or 7)) are unwrapped from the histone octamer surface (Figure 2d). These conformational changes require ATP binding, but not ATP hydrolysis [25]. However, a non-hydrolyzable ATP analog does not support the histone exchange, suggesting that both ATP binding and hydrolysis are required for histone exchange by the SWR1 complex. The nucleosome structure complexed with the INO80 complex has been determined by cryo-EM [26,27]. The ATPase motor subunit of the INO80 complex binds to the nucleosomal DNA at SHL +6 (or 6), and the accessory Arp5 and Ies6 subunits bind to SHL +2 (or 2) (Figure 2f). These nucleosome-binding locations of the ATPase motor and accessory proteins are opposite to those of the SWR1–nucleosome complex (Figure 2d). In the INO80–nucleosome complex, the nucleosomal DNA is detached from the aN helix of H3 and the loop2 of H2A, and consequently, the H2A–H2B dimer is partially exposed to the solvent (Figure 2f). The ATPase domain binds to the detached DNA region, and cooperatively translocates the nucleosomal DNA with the Arp5
and Ies6 subunits bound to the SHL +2 (or 2) and SHL +3 (or 3) sites, in a counter grip motion (Figure 2f). Thus, a DNA loop might be created between the ATPase motor and the Arp5–Ies6 subunits, resulting in large translocations with 10–20 step sizes [28], as well as histone exchange [23,24].
The structure of the overlapping dinucleosome In the process of the chromatin remodeling, a repositioned nucleosome may collide with a neighboring nucleosome and form unusual structures, such as the ‘altosome’ and ‘overlapping dinucleosome’ [29,30]. The formation of these unusual dinucleosome structures is enzymatically promoted, as consequence of the nucleosome remodeling by the SWI/SNF complex [29,30]. The overlapping dinucleosome is suggested to be composed of the hexasome, lacking one H2A–H2B dimer from the canonical nucleosome (octasome), and the octasome (Figure 3a). The crystal structure of the overlapping dinucleosome has been reported [31]. In the overlapping dinucleosome, the hexasome directly contacts the octasome without an obvious linker DNA segment. As a result, a 250 bp DNA segment is continuously wrapped in three turns around the histone core, containing three H2A–H2B dimers and four H3–H4 dimers (Figure 3b). The genome-wide analysis suggested that the overlapping dinucleosome is predominantly formed at the regions just downstream of transcription start sites (TSSs) [31]. This is consistent with the idea that the chromatin remodeling factor remodels the chromatin structure at the TSS to form the nucleosome free region (NFR) in actively transcribed genes, followed by the recruitment of the transcription machinery in the NFR. It is intriguing to study the relationship between overlapping dinucleosome formation and transcription activation. Further studies are awaited to reveal how chromatin remodelers promote overlapping dinucleosome formation at specific genomic loci.
The nucleosome structures in the complexes with transcribing RNA polymerase II The transcription of protein-coding and non-coding RNA genes is promoted by RNA polymerase II (RNAPII) [32,33]. Owing to the strong histone–DNA interactions, the nucleosome is inhibitory for the transcription process
(Figure 2 Legend Continued) circle represents the distorted DNA strand at SHL +2 (or 2). (c) The structure of the Chd1–nucleosome complex (PDB ID: 5O9G). Chd1 (orange, red, and brown) binds to the nucleosomal DNA at SHL +2 (or 2) and SHL+ 1 (or 1) through its ATPase domain and double chromodomain, respectively. The nucleosomal DNA (from SHL 5 (or +5) to SHL 7 (or +7)) is peeled off from the histone surface. The Chd1 ATPase domain, SANT SLIDE DNA-binding domain, and double chromodomain are colored orange, red, and brown, respectively. (d) The structure of the SWR1–nucleosome complex (PDB ID: 6GEJ). Swr1 (orange) and the Arp6–Swc6 (light blue) subunits bind to the nucleosomal DNA at SHL 2 (or +2) and SHL 6 (or +6), respectively. Other subunits are represented with transparency. The white dashed circle shows the interaction site with the acidic patch of H2A. (e) Superimposition of the nucleosome bound by the SWR1 complex (orange, PDB ID: 6GEJ) and the canonical yeast nucleosome (blue, PDB ID: 1ID3 [56]). The black dashed circle shows the bulged DNA strand at SHL 2 (or +2). (f) The structure of the INO80–nucleosome complex (PDB ID: 6FML). Ino80 (orange) and the Arp5–Isc6 (light blue) subunits bind to the nucleosomal DNA at SHL 6 (or +6) and SHL 2 (or +2), respectively. Other subunits are represented with transparency. Current Opinion in Structural Biology 2019, 59:107–114
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Structural dynamics of the nucleosome Kobayashi and Kurumizaka 111
Figure 3
(a) H2A-H2B dimer
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Chromatin remodeling Chromatin remodeler Overlapping dinucleosome Hexasome
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Crystal structure of the overlapping dinucleosome. (a) A model for overlapping dinucleosome formation. In this model, the nucleosome repositioned by a chromatin remodeling factor collides with a neighboring nucleosome, forming the overlapping nucleosome concomitant with the release of an H2A–H2B dimer. (b) Overview of the overlapping dinucleosome (PDB ID: 5GSE). Histones and DNA are shown in surface representations. H2A, H2B, H3, H4, and DNA are colored magenta, yellow, blue, green, and grey, respectively. Figure 4
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Snapshots of the nucleosome structure transition during RNAPII passage. (a–d) The structures of RNAPII–nucleosome complexes paused at SHL 6, SHL 5, SHL 2, and SHL 1 (upper panel, PDB IDs: 6A5O, 6A5P, 6A5R, and 6A5T, respectively) are presented. The structural transitions of the nucleosomal DNA are shown in the bottom panel. RNAPII, H2A, H2B, H3, H4, and DNA are colored orange, magenta, yellow, blue, green, and grey, respectively. www.sciencedirect.com
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mediated by RNAPII [34–36]. In vivo and in vitro analyses suggested that, in the nucleosome, RNAPII preferentially pauses near the entry site and before the dyad (center) site of the nucleosomal DNA [37–42].
Conflict of interest statement
To reveal the mechanism by which RNAPII transcribes on the nucleosomal DNA, cryo-EM studies of the nucleosome structures complexed with the transcribing RNAPII have been performed [43,44]. These studies provided snapshots of the nucleosome structure transitions during RNAPII passage. The structures revealed the RNAPII– nucleosome complexes paused at SHL 6, SHL 5, SHL 2, and SHL 1. In the SHL 6 state, the transcribing RNAPII pauses just in front of the nucleosome, and the nucleosomal DNA is entirely wrapped around the histone octamer (Figure 4a). RNAPII then advances by approximately 20 base pairs from the entry site of the nucleosome, and pauses at the SHL 5 site, which is a major RNAPII pausing site in cells [37,43]. In the SHL 5 complex, the H2A–H2B dimer–DNA interactions stall the transcription elongation by RNAPII, and the DNA end (20 base pairs) of the nucleosome is peeled from the histone surface (Figure 4b). Subsequently, RNAPII elongates the RNA with continuous peeling of the nucleosomal DNA, and pauses at the SHL 2 and SHL 1 sites (Figure 4c and d). Consequently, about 50–60 base pairs of DNA are detached from the histone surface. Interestingly, the H2A–H2B dimers remain in the nucleosome, although one of the H2A–H2B dimers loses its interaction with the DNA and is exposed to the solvent (Figure 4c and d). This may be supported by the direct interaction between RNAPII and an H2A–H2B dimer. In the SHL 1 state, ‘a foreign DNA’ is bound to the exposed H2A–H2B dimer region in trans. This state may represent an intermediate state for the histone transfer during the transcription elongation, such as a ‘template looping’ structure, as proposed by an atomic force microscopic study [45].
This work was supported in part by JSPS KAKENHI Grant Numbers JP18H05534 and JP17H01408 [to H.K.]. This work was also partly supported by JST CREST Grant Number JPMJCR16G1 and the Platform Project for Supporting Drug Discovery and Life Science Research (BINDS) from AMED, under Grant Number JP19am0101076 [to H.K.].
Concluding remarks Recent advances in high-resolution cryo-EM analysis have provided breakthroughs in the structural biology of nucleosome dynamics. In this review, we summarized how chromatin remodeling factors and RNAPII promote the dynamic structural transitions of the nucleosome. Chromatin remodelers translocate the nucleosomal DNA with two DNA binding domains. The nucleosomal DNA translocation mediated by chromatin remodelers may be coupled with the genomic DNA functions, such as transcription. In fact, RNAPII reportedly cooperates with transcription elongation factors, chromatin remodelers, and histone chaperones [44,46–51]. Structural studies of nucleosome transcription coupled with these factors are the next challenges. The chromatin remodeling activity may be required for RNAPII transcription activation and repression in higher ordered chromatin architectures. It is also intriguing to study how nucleosomes suppress transcription in inactive heterochromatin [52,53]. Current Opinion in Structural Biology 2019, 59:107–114
Nothing declared.
Acknowledgements
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