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Access to the eukaryotic genome is key to the regulation of such DNA processes as transcription, replication and repair. About three quarters of the human genome is stored and shielded within arrays of nucleosomes, the fundamental structural units of chromatin, each of which contains about 150 bp of DNA and an octameric histone protein composed of two each of H2A, H2B, H3 and H4. Structural fluctuation of nucleosomes provides regulatory proteins with transient access to the internal DNA; however, nucleosome unwrapping, sliding and unstacking must occur to obtain full access of the DNA. In this review, we focus on the unwrapping of mononucleosomes and internucleosomal interactions to discuss our current understanding of the dynamics of the process.

An overlapping dinucleosome (OLDN) is a structure composed of one hexasome and one octasome and appears to be formed through nucleosome collision promoted by nucleosome remodeling factor(s). In the present study, the solution structure of the OLDN was investigated through integration of small-angle X-ray and neutron scattering (SAXS and SANS, respectively), computer modeling, and molecular dynamics simulations. Starting from the crystal structure, we generated a conformational ensemble based on normal mode analysis, and searched for the conformations that well reproduced the SAXS and SANS scattering curves. We found that inclusion of histone tails, which are not observed in the crystal structure, greatly improved model quality. The obtained structural models suggest that OLDNs adopt a variety of conformations stabilized by histone tails situated at the interface between the hexasome and octasome, simultaneously binding to both the hexasomal and octasomal DNA. In addition, our models define a possible direction for the conformational changes or dynamics, which may provide important information that furthers our understanding of the role of chromatin dynamics in gene regulation.

Eukaryotic genome is packaged in a nucleus in the form of chromatin. The fundamental structural unit of the chromatin is the protein-DNA complex, nucleosome, where DNA of about 150 bp is wrapped around a histone core almost twice. In cellular processes such as gene expression, DNA repair and duplication, the nucleosomal DNA has to be unwrapped. Histone proteins have their variants, indicating there are a variety of constitutions of nucleosomes. These different constitutions are observed in different cellular processes. To investigate differences among nucleosomes, we calculated free energy profiles for unwrapping the outer superhelical turn of CENP-A nucleosome and compared them with those of the canonical nucleosome. The free energy profiles for CENP-A nucleosome suggest that CENP-A nucleosome is most stable when 16 to 22 bps are unwrapped in total whereas the canonical nucleosome is most stable when it is fully wrapped. This indicates that the flexible conformation of CENP-A nucleosome is ready to provide binding sites for the structural integrity of the centromere.

Although genome-editing enzymes such as TALEN and CRISPR/Cas9 are being widely used, they have an essential limitation in that their relatively high molecular weight makes them unsuitable for capsulation. To develop a novel genome-editing enzyme with a smaller molecular weight, we focused on the engrailed homeodomain (EHD). We designed and constructed proteins composed of two EHDs connected by a linker to increase sequence specificity. In bacterial one-hybrid assays and EMSA analyses, the created proteins exhibited good affinity for DNA sequences consisting of two tandemly aligned EHD target sequences. However, they also bound to individual EHD targets. To avoid binding to single target sites, we introduced amino acid mutations to reduce the protein-DNA affinity of each EHD monomer and successfully created a small protein with high specificity for tandem EHD target sequences.

量研では、新しい学術領域「量子生命科学」の確立を目指した活動を、国内外の研究者と共に精力的に進めている。量子生命科学とは、最先端の量子技術と量子科学の知見を総合的に利用し、従来不可能であった極微の空間・時間スケールあるいは超高感度での生体内部の観測や生体分子の計測、生命機能のモデリングなどを実現することにより、量子論・量子力学を基盤とした視点から生命全般の根本原理を明らかにすることを目標とし、その原理の産業応用も視野に入れた分野である。本稿では、平成 31 年 4 月に量研内に発足した「量子生命科学領域」におけるアプローチを紹介する。

自己集合化するタンパク質動態について、計算科学的な立場から、分子シミュレーションとバイオインフォマティクス解析でどこまで理解が進み、今後どのようなことが問題になるかについて発表する。, 第6回蛋白質工学研究会ワークショップ

A small DNA binding protein to target desired sequences have the potential to become a scaffold of molecular tools such as genome-editing enzymes. We previously showed two engrailed homeodomains (EHDs) connected with a linker recognizes a target sequence twice as long as a single EHD in cells only when arginine 53 in each EHD in the tandem protein is mutated to alanine ((EHD[R53A])2). In this study, we determined the crystal structure of the (EHD[R53A])2-DNA complex. Most importantly, it shows the base-specific interactions necessary for the affinity and/or specificity of the wild-type EHD are preserved in (EHD[R53A])2. The mechanism of the specific recognition will be discussed based on the structure and cellular assays., 第58回日本生物物理学会年会

Hydration water dynamics were measured by quasi-elastic neutron scattering with H2O/D2O contrast for two DNA dodecamers, 5’CGCGAATTCGCG’3 and 5’CGCGTTAACGCG’3, which have been computationally shown to be structurally rigid and flexible, respectively. The dynamical transitions of the hydration water as well as DNA were observed for both sequences at approximately 240 K. Above the transition temperature, the mean square displacements of the hydration water for the rigid sequence were smaller than those for the flexible one. Furthermore, the relaxation time of the hydration water was longer in the rigid DNA than in the flexible DNA. We suggest that hydration water dynamics on the picosecond timescale are associated with sequence-dependent deformability of DNA.

進展のあった、OLDNの溶液構造 （Biophysical J. (2020)）、 ヌクレオソームの力応答 (in revision)、 HP1を介した構造 （ Biophysical Rev. (2020)）等について、 全原子モデル、クロスリンキング実験結果について、 報告し、今後の取り組みについて言及した。, 新学術領域クロマチンポテンシャル領域会議

Progress of theoretical and computational biophysics

Torsional stress has a significant impact on the structure and stability of the nucleosome. RNA polymerase imposes torsional stress on the DNA in chromatin and unwraps the DNA from the nucleosome to access the genetic information encoded in the DNA. Some chromatin Remodelers are thought to change nucleosome positioning by adding torsional stress on DNA. To understand how such torsional stress affects the stability of the nucleosome, we examined the unwraping of the outer superhelical turn of the nucleosomal DNA under positive or negative torsional stress with all-atom molecular dynamics simulations. The free energies for unwrapping the DNA indicate that positive stress that overtwists DNA facilitates asymmetric unwrapping of the DNA while negative stress that undertwists DNA does not. Under positive stress, DNA was straightened and rigid, which facilitated a large-scale asymmetric unwrapping of the DNA without a large extension of two DNA.In the structure, one end of the DNA was disconnected from H3 and H2A/H2B while the other end of the DNA stably remained wrapped. Under negative stress, the DNA became more bendable and flexible, which facilitated the binding of the unwrapped DNA to the octamer in a stable state. Our results suggest that the stability of individual nucleosomes in the chromatin can be changed by torsional stress, thereby DNA accessibility of regulatory proteins can be produced to maintain gene expression without large conformational changes even in heterochromatin., Biophysical Society Annual Meeting 2020

The dimethylation of Arg at the 42nd position (R42me2) of H3 histone, a post-translational modification (PTM) in nucleosomes close to the DNA entry/exit region, showed controversial gene regulations. To address this discrepancy, we performed comprehensive all-atom replicaexchange molecular dynamics simulations with and without a single PTM, either symmetric (R42me2s) or asymmetric (R42me2a) dimethylation. Together with a kinetics analysis, our simulations showed that DNA at the entry/exit region in the R42me2a nucleosome adopts a relatively more open conformation than that in the unmodified nucleosome,whereas R42me2s exhibits significantly weaker or even negligible effects on DNA dynamics and structures, which may provide clues of the discrepancy of gene regulation by R42me2. Our approach will be useful to study the mechanism of nucleosome dynamical change induced by a subtle modification.

Widely used genome-editing enzymes such as TALEN and CRISPR have a high molecular weight, which makes them difficult to be delivered to cells. To develop a novel genome-editing enzyme with a smaller molecular weight, we have focused on engrailed homeodomain protein (EHD). In our previous work, we have successfully created a novel DNA-binding protein by connecting two EHD domains with a linker (EHD2). The protein specifically recognizes the tandemly connected target site of the individual EHD’s target site in E.coli cells. The specific recognition for the tandem target sequence was achieved only when the conserved arginine 53 of the EHD was mutated to alanine in the tandem proteins ((EHD[R53A])2). The tandem proteins without that mutation mostly bound to sequences containing the target of the monomeric EHD. To reveal the molecular mechanisms for recognition of the tandem target site, we determined a crystal structure of ((EHD[R53A])2-DNA complex. We found that base-specific interactions of I47, K50 and N51 with DNA in the major groove observed in the wt were completely preserved in ((EHD[R53A])2. Together with the biological functional analysis, we conclude that ((EHD[R53A])2 realizes the precise recognition of the tandem target sites in cell by concurrently two individual EHD domains’ binding to the target sites., 3rd QST International Symposium

Nucleosome is the fundamental structural unit of chromatin and is composed of histone proteins and DNA. The individual core histones form the stable dimers of H2A/H2B and H3/H4. The H3/H4 dimers further assemble into a tetramer. In the nucleosome the H3/H4 tetramer and two H2A/H2B dimers form an octamer. The nucleosome undergoes structural changes such as DNA unwrapping and the eviction of the H2A/H2B dimer in cellular processes. To understand how the eviction of the H2A/H2B dimer occurs, an adaptively biased molecular dynamics (ABMD) simulation was carried out with a reaction coordinate, d, which is the distance between the two H2A/H2B dimers. In the process, the interaction between the two H2As was firstly lost at d = ~40 Å. The interaction between the (H2A/H2B)a dimer and (H3/H4)a dimer then started to break. The docking domain of the H2A strongly interacted with the (H3/H4) a dimer until the interaction was lost at d = ~50 Å. This indicates that the docking domain of the H2A plays an important role in stabilizing the nucleosome and blocking the eviction of the H2A/H2B dimer. In fact, the chromatin remodeling histone chaperone FACT is known to move the docking domain away from the nucleosome. Thus, the loss of interaction with this domain would significantly facilitate the eviction of the H2A/H2B dimer. In addition to the (H2A/H2B)a – (H3/H4)a interaction, the (H2A/H2B)a – (H3/H4)b interaction was also found to be strong. We will discuss the important regions of the nucleosome for the eviction in detail., 3rd QST International Symposium