Investigating the Influence of Arginine Dimethylation on Nucleosome

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Investigating the Influence of Arginine Dimethylation on Nucleosome Dynamics Using All-Atom Simulations and Kinetic Analysis Zhenhai Li, and Hidetoshi Kono J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b05067 • Publication Date (Web): 26 Sep 2018 Downloaded from http://pubs.acs.org on September 26, 2018

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The Journal of Physical Chemistry

Investigating the Influence of Arginine Dimethylation on Nucleosome Dynamics using All-atom Simulations and Kinetic Analysis Zhenhai Li and Hidetoshi Kono*

Molecular Modeling and Simulation (MMS) Group National Institutes for Quantum and Radiological Science and Technology (QST) 8-1-7, Umemidai, Kizugawa, Kyoto 619-0215 Japan tel: +81-774-71-3465 email: [email protected]

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ABSTRACT The dimethylation of Arg at the 42nd position (R42me2) of H3 histone, a posttranslational modification (PTM) in nucleosomes close to the DNA entry/exit region, showed controversial gene regulations. To address this discrepancy, we performed comprehensive all-atom replica-exchange molecular dynamics (REMD) 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 R42me2a nucleosome adopts a relatively more open conformation than that in the unmodified nucleosome; while in contrast, 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 dynamics change induced by a subtle modification.

INTRODUCTION Eukaryotic cells store the genomic DNA in the repeating basic units, nucleosomes, each of which consists of a histone octamer core and DNA wrapping around the core ~1.7 times. The core is formed by the globular domains of two copies each of H3, H4, H2A, H2B histone proteins1. The stability and dynamics of a nucleosome are regulated by the interaction between histone proteins and DNA, which sterically obstacles the access to the genes stored in the nucleosomal DNA and impedes both DNA transcription and

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replication1-3. Different mechanisms have been found to alter DNA-histone core interactions: replacement of canonical histones to their variants and post-translational modifications (PTM) in the histone core. The histone proteins are found to be heavily decorated with PTMs, including acetylation, methylation, phosphorylation, ubiquitinylation, etc. These PTMs either directly affect the histone–DNA interaction or provide a scaffold to recruit regulatory proteins, and eventually promote or repress gene expression and replication. The downstream events of the PTMs are determined by the types and locations of the modifications4, 5. Recently some PTMs in histone globular domains were identified, such as PTMs on H3(Y41), H3(R42), and H3(K56), H4(K77), and H4(K79) at the DNA–histone interface, and H4(K91) and H4(K92) at the histone– histone interface4, 5, though numerous studies have been focused on PTMs in the intrinsically unstructured histone tails6, 7. Among these amino acids, H3(R42), which is located at the entry/exit region of a nucleosome, is conserved across species8 and was found to be dimethylated in mammalian cells9, 10. Casadio et al. showed that the asymmetric dimethylation of R42 (R42me2a) in vitro promotes gene transcription9. Later Yaseen et al. found the H3(R42) in host cell nucleus can be dimethylated by a mycobacteria-secreted protein, Rv1988, which in turn represses the host gene transcription, and eventually impedes the host immune response10. The arginine can be methylated twice symmetrically or asymmetrically, and owing to the possible rotation around central carbon-nitrogen bond in the guanidinium group, two symmetric dimethylarginine stereoisomers, anti-syn and anti-anti symmetric dimethylarginine, exist (Figure 1a) 11. However since it is difficult to examine the atomic arrangement of dimethylated R42 experimentally and numerous MD simulations have 3



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been performed to study the PTM in nucleosomes showing the advantage of MD method in this field12-16, we carried out a series of Replica-Exchange Molecular Dynamics (REMD) simulations on a group of nucleosome variants with and without modifications of H3(R42), including the unmodified, R42me2a, two stereoisomers of R42me2s (indicated by anti-anti R42me2s and anti-syn R42me2s), and a mutant, R42A (Supporting Information,Table S1). The REMD simulations clearly showed the R42me2a and R42A nucleosome adopted a relatively opened conformation compared to the unmodified nucleosome. By adopting the methods of protein folding kinetics analysis with REMD, we obtained the opening/closing rates of the entry/exit DNA. The kinetics shows the asymmetric methylations and R42A mutation promoted the opening rate and impeded the closing rate of the nucleosome, which was not observed in the symmetrically methylated nucleosome. These findings highlight the distinct effect of R42me2a and R42me2s modifications on nucleosome structure and dynamics, which gives an explanation for the distinct gene activities of previous studies. Furthermore, this study unravels the delicate mechanism of nucleosome structure regulation by dimethylation of H3(R42) and provides an understanding of the different effects from the different dimethylation constitutional isomers on the atomic level.

METHODS AND COMPUTATIONAL DETAILS Initial nucleosome structure for REMD and conventional MD simulations

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In conventional MD simulation, the entire nucleosome structure, with 10 bp extended at both ends was used. This structure was prepared as a hybrid of two nucleosome structures (PDB ID 1KX5 and PDB ID 1ZBB)1, 17, as described in Ref18, 19, which included eight histone proteins with all the tails and 167 bp of DNA. We assigned the base pair (bp) at the dyad axis as bp 0. The nucleosome structure in REMD is a part of this nucleosome structure, which includes two segments of DNA: inner (bp range: 29~+2) and entry/exit region to linker DNA (bp range: +54~+78); four segments of histones: H3 (amino acid range: 40~135), H4 (full-length), H2A (full-length), and H2B (amino acid range: 36~59) (Figure 1b). The conventional MD and REMD simulations were performed on the whole nucleosome and partial nucleosome structure with or without modifications, respectively (Supporting Information, Table S1). REMD simulation methods The REMD MD simulation was performed by GROMACS20. CHARMM2721, 22 force field combined with a PTM force field23 specifically designed for CHARMM27 were used to describe the interactions. The temperature and pressure control methods are the same as conventional MD simulation as described in Ref19. TIP3P model of CHARMM27 was used to describe water molecules. In brief, the nucleosomes were solvated in neutralized NaCl24 solution at 150 mM. Following that, minimizations of the systems were performed with both the steepest descent and conjugated gradient algorithms. Then, we generated 69 replicas by heating up the system to 69 different temperatures from 300 K to 372 K (Supporting Information, Table S2), which were selected to maintain a 20 % exchange probability25. Each replica was maintained at a selected 5



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temperature first in NVT and then in NPT ensemble with position restraints for heavy atoms of the nucleosome. Finally, we performed 20 to 50 ns replica-exchange production runs in NPT ensemble26, while keeping the restraint for C1’ atoms of both ends of the inner DNA and the +54 end of the entry/exit DNA, and Cα atoms of Cterminal of histone H3 and H2B segments and N-terminal of H2A segment to mimic the whole nucleosome structure.. All conducted REMD simulations are listed in Supporting Information, Table S1. The simulations were performed on the structure without or with one of the following modifications on H3 histone: R42me2a, anti-anti R42me2s, anti-syn R42me2s, and R42A (Supporting Information, Table S1). Conventional MD methods The same simulation package and force field were used as in REMD simulation. The MD procedure is as described in Ref19. In brief, the nucleosomes were solvated in neutralized NaCl solution at 150 mM. After minimization with both steepest descent and conjugated gradient algorithms, we heated up the system and then maintained the temperature at 353K first in NVT and then in NPT ensembles with the heavy atoms of the nucleosome restrained. Afterward, we performed 50-100 ns production runs in the NPT ensemble without any restraints. The temperature and pressure were controlled by V-rescale and Parrinello-Rahman respectively27, 28. The simulations were performed on this nucleosome structure or on this structure with both copies of H3 histone mutated or methylated as followings: R42me2a, R40me2a, or R42A. For each type of nucleosomes, the same procedure was repeated by 10 times with distinct initial assigned velocities to

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obtain independent results. All conventional simulations conducted are listed in Table S1. Opening and closing kinetics We calculated the kinetics of opening and closing rate based on the REMD simulations data. An angle, “DNA opening angle”, was introduced to quantify the nucleosome structural change and was defined by the nucleosome dyad axis and DNA end axis. The DNA end axis shown in Figures 2 and 3 was calculated from the 3rd to 10th base pairs from the end of the DNA. The first two base pairs were ignored to avoid the influence from the unwinding of the DNA end. The coordinates of the C1 atoms of these base pairs were collected and fitted to a straight line as the DNA axis. To calculate the kinetics of opening and closing rate, we used a method which has been developed to derive the protein folding kinetics from REMD simulations29, in which an RMSD threshold was selected as the indicator of protein folded or unfolded. Herein, we set a criterial angle as the indicator of a closed or open nucleosome DNA. Thus, we could group the DNA configurations into two groups by the criterial angle as shown in Figure 3a). A closed or open conformation was judged by whether the DNA opening angle is lower or greater than the criterial angle, respectively (Figure 3a). By applying this analysis to all 69 replicas, we calculated the opening angle of the nucleosome in each replica at a certain simulation time, and further classified it into either the closed or open group according to the criterial angle. Eventually, we can calculate the closed nucleosome fraction among 69 replicas with a certain criterial angle at a certain simulation time, C ( t ) . 7



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Theoretically, at the temperature in the mth replica, the DNA configurations are governed by the opening/closing kinetics which is given by:

dcm ( t ) = kclose om ( t ) − kopen cm ( t ) dt

(1)

where c m and om are closed and open nucleosome fractions of the mth replica, respectively. The sum of two fractions equals 1, om ( t ) + cm ( t ) = 1 . kclose and kopen are the closing and opening rate at the temperature of the mth replica. The transition rates are related to the activation energy E and the prefactor A as followed:

k = Ae− E / kBT

(2)

where kB is Boltzmann’s constant and T is temperature. By assuming the activation energies and prefactors, we can predict the closed nucleosome fraction change of all the replicas over time t:

Φ (t ) =

1 M

M

dcm (τ ) dτ + cm ( 0 ) 0 dt

∑∫ m =1

t

(3)

By minimizing the difference between the predicted and simulated closed nucleosome fraction in all the recorded steps (N) from 2 ns to 50 ns: χ 2 =

2 1 N  Φ ( ti ) − C ( ti )  ∑ N i =1

(thicker lines in Figure 3b), the activation energy E and prefactor A of DNA opening and closing were derived. The DNA opening and closing rates at 300 K were then obtained by substituting T with 300 K in Eq. 2. Considering the linker DNA was

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modeled based on a di-nucleosome crystal structure, we discarded the first 2 ns of the REMD trajectories when analyzing the data. Note that the average time to complete an opening-closing cycle can be estimated by T = 1/kclose + 1/kopen. and the fluctuation frequency of closing and opening DNA is defined as 1/T.

RESULTS To evaluate the effect of a single amino acid modification of H3R42, a group of nucleosome variants, including the unmodified, R42me2a, anti-anti R42me2s, anti-syn R42me2s, and R42A nucleosomes (Figure 1a, as highlighted by the red circle in Figure 1b), were studied with Replica-Exchange Molecular Dynamics (REMD), which was widely used in biomolecular simulations to enhance the sampling30-32. To save the computational cost and obtain a sufficient conformational sampling on the site of interest, we minimized the simulation system as shown in Figure 1b. A part of a nucleosome, highlighted by the red box, including a segment of each histone protein and two pieces of DNA strands, was used in this study (see details in Materials and Methods). The DNA base pairs were labeled sequentially with numbers, where the base pair at the center of dyad was labeled as 0. Thus, in the simulated partial nucleosome, the inner loop DNA is from -29 to +2, while the entry/exit DNA is from +54 to +78 (Figure 1b inset). To focus on the effect of R42 modifications, the H3 N-terminal tail was removed before Y41 in the REMD simulations. REMD simulations of 69 replicas from

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300.0 K to 371.7 K (Supporting Information, Table S2) for 50 ns (with an exception of 20 ns for R42A) were performed, yielding a total of ~3.5 µs (1.4 µs for R42A) simulation for each of variants. The temperatures of the replicas were selected to maintain an exchange probability of 0.225 and listed in Supporting Information, Table S2. The trajectories at 300 K were used for further analysis.

Figure 1. Simulated nucleosome structures and R42 modifications. 10


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A. Modification implemented on R42 in histone H3 includes three dimethylation isomers, asymmetric dimethylarginine (R42me2a), anti-anti symmetric dimethylarginine (R42me2saa), and anti-syn symmetric dimethylarginine (R42me2sas), and a mutation to Alanine (R42A). B. Simulated nucleosome. DNA and histone proteins are labeled with different colors (blue: H3; green: H4; yellow: H2A; pink: H2B; purple: entry-exit DNA; orange: inner DNA). The DNA sequence is labeled with numbers as indicated in the inset on the top left. R42 residues on histone H3 is labeled with a stick model and highlighted by red circles (Figure was created by using VMD33). R42me2a but not R42me2s promotes the opening of the nucleosome at the entry/exit site As expected, the REMD simulations showed a clear difference in the spatial distribution of DNA at the entry/exit region between the unmodified and R42me2a nucleosomes. We show the highly probable DNA locations that were obtained in the simulation in Figure 2a and b where the occupancy of the last 5 DNA base pairs was greater than 5 %. DNA of the unmodified nucleosome fluctuated in a region closer to the nucleosome core (black mesh in Figure2b), while that of R42me2a nucleosome fluctuated in a region apart from nucleosome core, indicating the DNA in R42me2a nucleosome tends to open at the entry/exit region (red mesh in Figure 2b). To quantify the opening induced by R42me2a, we fitted the axis of DNA at the entry/exit region in each frame of the simulated trajectories and calculated the angle between the DNA axis and nucleosome dyad axis (see detail in Materials and Methods). The angle was used to represent the entry/exit DNA axis direction (blue arrow in Figure 2a). The angle which fell above or 11



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below the nucleosome dyad axis (black arrow in Figure 2a) was defined as positive or negative, respectively. In R42me2a and R42A nucleosome, angle distribution was clearly shifted toward the positive direction and the average angle increased by ~12°, which indicates these nucleosomes adopted a relatively open conformation (Figure 2c). Considering the 147 base pairs wrap on histone core by 1.67 turns, 12° opening corresponds to ~3 base pairs exposure at entry/exit region. In contrast, changes in DNA end spatial distributions, as well as in the DNA opening angle, were subtle for both R42me2s stereoisomers comparing to the unmodified nucleosome (Figure 2b and c). The energy landscape can be extracted from the distribution by ε i = −kBT ln ( fi ) + ln C (Figure 2d). fi and εi represent the fraction and the energy at any opening angle, respectively. Similarly, the R42me2a and R42A nucleosomes, but not R42me2s ones, induce shifts of the energy landscape to more widely opened conformation. These data suggest the increase in DNA accessibility by R42me2a and R42A, but not by either R42me2s. Carefully examining the DNA end distribution and the energy landscape, we found that compared to R42A, R42me2a nucleosome exhibited less population and higher energy in low angle regions corresponding to the closed conformation (Figure 2). This might be due to the steric bulk introduced by methylation, which prevents the entry/exit DNA from being closely attached to nucleosome core.

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Figure 2. R42me2a but not R42me2s enhances nucleosome opening at the entry/exit site. A. A superposition of the DNA end distributions of all the nucleosomes at 300 K in REMD simulations. The colored meshes highlight the location where linker DNA occupancy is > 5%. B. The comparisons of DNA end distribution between the unmodified and each modified nucleosome. The color code for (A and B) is as followed: Black, Unmodified; red, R42me2a; magenta, R42A; blue, R42me2saa; green, 13



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R42me2sas. C. Changes in linker DNA orientation caused by modifications. The linker DNA orientation is represented by an angle between the DNA axis and the nucleosome dyad axis as shown in (A). The angle which falls below the dyad axis (as the representative angle in (A)) is defined as a negative angle. Left and right panels in (C) are the distribution and average of the angles, respectively. The error bars in the right panel represents the standard errors of the angle. The simulation trajectories at 300 K were divided into 69 pieces corresponding to the 69 replicas. The standard errors were calculated among the 69 replicas. The numbers indicate the P-values, which were calculated by Student’s T-test. D. The energy landscape profile of the nucleosome linker DNA. The modification-induced kinetics changes To investigate the DNA opening/closing kinetics, we adopted a data analysis technique, which has been developed to derive the protein folding kinetics from REMD simulations29. We first determined a nucleosome conformation state by comparing the DNA opening angle with a criterial angle (Figure 3a), and further, we can calculate the closed nucleosome fraction among 69 replicas with this criterial angle (see details in Methods). In Figure 3b, we show trajectories of the fraction of the closed conformation on different nucleosomes. We set first the criterial angle to be −42° which was lower than the DNA opening angle of the initial nucleosome structure. So, most of the nucleosomes were in an open conformation at the beginning. The closed fraction trajectories first rapidly increased from ~0 and then gradually approached plateaus (thinner lines in Figure 3b). The unmodified and R42me2a nucleosome had respectively 14


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the highest and lowest fraction of closed conformations after reaching the plateaus (Figure 3b). The fluctuation dynamics of the entry/exit DNA are hidden in the trajectories. We adopted a method which has been developed to derive the protein folding kinetics from REMD simulations29 and succeeded in extracting the opening/closing rates ( k open , k close ) and the equilibrium constant ( K = k open / k close ) by fitting the trajectories to the kinetics master equation (see details in Methods). The unmodified nucleosome clearly had the lowest opening and highest closing rates (black bars in Figure 3c left & middle), while R42me2a nucleosome had the highest opening and lowest closing rates (red bars in Figure 3c left & middle). The kinetics of symmetrically dimethylated nucleosome were in between of the unmodified and R42me2a nucleosomes (blue and green bars in Figure 3c left & middle). The equilibrium constant of R42me2a is 4 times larger than that of the unmodified nucleosome (black and red bars in Figure 3c right), but those of symmetrically dimethylated nucleosomes were less than twice of the unmodified one (blue and green bars in Figure 3c right). R42A exhibited a fast opening and a slow-closing rate as R42me2a (pink bars in Figure 3c left & middle), and the equilibrium constant of R42A was more than three times of the unmodified nucleosome but lower than R42me2a (pink bar in Figure 3c right). This suggests R42me2a nucleosome has the highest opening tendency.

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Figure 3. DNA opening and closing kinetics. A. A criterial angle is an angle between the selected criterial and the dyad axes, which defines the nucleosome DNA opening state. The nucleosomal DNA is defined as open when the DNA axis is above the criterial axis, and vice versa. B. Trajectories of the closed fraction of all the replicas (thinner lines) of the nucleosomal DNA with a criterial angle of −42°. Different nucleosomes and the fitted fraction trajectories (thicker lines) are shown in different colors. C. The computed opening rates (left), closing rates 16


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(middle), and equilibrium constants (right) with a criterial angle at −42°. The color code in (B, C) is the same as in Figure 2c. The transition kinetics at different criterial angles In principle, the criterial angle selection affects the fraction of closed nucleosome and also the transition rates, thus different criterial angles from −56° to −12° were tested. A gradual decrease in opening rates with criterial angle increasing from −56° to −40° was followed by a fast decrease for all the nucleosomes (Figure 4a). In contrast, the closing rates increased rapidly from −56° to −40° and then kept slowly increasing afterward (Figure 4b). Intriguingly, although the opening and closing rate non-uniformly changed with criterial angle changing, the equilibrium constants linearly decreased in the semilog plot (Figure 4c). In the entire range of criterial angle, the R42me2a nucleosome had the highest opening and the lowest closing rates; the unmodified nucleosome, on the other hand, had the lowest opening and the highest closing rates. Similarly, the R42me2a and unmodified nucleosomes had the highest and the lowest equilibrium constants. The kinetics and the equilibrium constants of the R42me2s nucleosomes fell in between of the unmodified and R42me2a nucleosomes. However, in some criterial angles for R42A nucleosome, the trajectory of the fraction of the closed nucleosome reached equilibrium too rapid (within 2 ns). Therefore, we did not obtain enough data points before the equilibrium to compute reliable opening and closing rate values (Supporting Information, Figure S1). However, the equilibrium constant of R42A nucleosome was obtained since it depends

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only on the fraction of closed nucleosome after the system reaching the equilibrium. Obviously, R42A and R42me2a both had very close equilibrium constants (Figure 4c). We further calculated the equilibrium angle of each of the nucleosomes where the equilibrium constant equals 1 (open black bars in Figure 4d). The equilibrium angles are indistinguishable from the average opening angle obtained from the opening angle distribution (right panel of Figure 2c and stripped bars in Figure 4d). The opening (or closing) rate at the equilibrium angle is mathematically equivalent to double of the fluctuation frequency of the entry/exit DNA (red open bars in Figure 4d). Clearly, the fluctuation frequencies of different nucleosomes are comparable, suggesting the modifications of R42 in H3 do not affect the fluctuation frequency of entry/exit DNA but only induce the equilibrium angle shifted to a more open conformation.

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Figure 4. DNA opening and closing kinetics variation with criterial angle changing. A. Opening rates. B. Closing rates. C. Equilibrium. The color code in A to C is the same as in Figure 2c. D. The comparison of calculated equilibrium angle and the average opening angle and the fluctuation frequency of the entry-exit DNA.

Y41 and R42 H-bonds greatly reduce in R42me2a but not in R42me2s

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To investigate why R42 asymmetric, but not symmetric, methylation influences the entry/exit DNA opening, we examined the H-bond formation between each H3 amino acid and the entry/exit DNA in all the nucleosome variants. Intriguingly, not only H-bond of R42 but also that of Y41 to the entry/exit DNA was affected by R42me2a and R42A (Figure 5a). More specifically, R42me2a and R42A greatly reduced H-bond formation for both Y41 and R42 with the entry/exit DNA (Figure 5a). R42me2sas but not R42me2saa reduced H-bond formation between R42 and the entry/exit DNA, while both R42me2s stereoisomers had limited effects on H-bond formation between Y41 and the entry/exit DNA. The cumulative moving averages of H-bonds (the average from the beginning to the current time) of the entry/exit DNA to R42 or Y41 reached plateaus at ~25 ns, indicating the simulation is sufficiently long (Supporting Information, Figure S2). R42A induced more reduction of H-bond compared to R42me2a. As a result, the DNA end distribution became broader with a smaller peak than R42me2a, indicating less conformational restraints from R42A (Figure 2b and c). To check if these changes were observed due to the truncated nucleosome system (Figure 1b), we further carried out the conventional simulations for the entire nucleosome. The result showed that H-bond counts of R42–entry/exit DNA and Y41–entry/exit DNA both dropped in the R42me2a nucleosome (Supporting Information, Figure S3a); the H-bond count of R42–inner DNA dropped in the R42me2a nucleosome (Supporting Information, Figure S3b); total Hbond counts of R42–DNA and Y41–DNA both dropped in the R42me2a nucleosome (Supporting Information, Figure S3c); and accordingly the co-occurrence frequency of R42 and Y41 to form H-bond with both the inner and entry/exit DNA significantly

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dropped in the R42me2a nucleosome. These results corroborated the observations in the REMD simulations.

Figure 5. The average count of R42/Y41–entry/exit DNA H-bond and the entry/exit–inner DNA distance.

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A. Average H-bond count between the entry/exit DNA and Y41 (left) or R42 (right) in the nucleosomes with and without modifications. The error bars represent the standard errors. The numbers in the figure indicate the P-values. The standard errors and the statistical test are obtained in the same way as in Figure 2. B. H-bond frequency of each donor hydrogen atom in the unmodified and modified R42. The H-bond frequency is denoted by colors from white to red. The hydrogen atoms are highlighted with large beads. C. A close-up view of the nucleosome entry/exit region showing the most frequent H-bond configuration in the unmodified nucleosome. The DNA strands are represented by purple (entry/exit DNA) and orange (inner DNA) tubes, respectively. The histone H3 is represented by a cyan ribbon. The R42/Y41 amino acids and the DNA bases forming H-bond with R42/Y41 are highlighted by the stick model. D. Distance distributions between the inner DNA acceptor 5O1P and entry/exit DNA acceptor 68O3’. The different colored curves indicate the distance distributions of different nucleosomes as labeled. The brown area indicates such distribution when in the unmodified nucleosome both 68O3’–Y41 and 5O1P–R42 form H-bond simultaneously. The gray area indicates the distribution of Y41–R42 length defined in C. The left and right edges of lighter areas indicate the 10% and 90% percentile of the distance distributions, while those edges of the darker areas indicate the 25% and 75% percentile of the distributions. Only the trajectories at 300 K were used in to analyze the H-bond occupancies and distances in this figure. The sidechain of R42 resides in between the inner and the entry/exit DNA. The guanidinium group capping arginine has 5 potential donor atoms (Figure 1b and 5b), thus R42 has high opportunity to form H-bond with the acceptors, the oxygens, of both 22


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entry/exit and inner DNA. A careful survey was done over all the hydrogen bond donors in R42. Since we were interested in how R42 restrains the entry/exit DNA, the frequency for each hydrogen atom (highlighted with larger beads in Figure 5b) to form H-bond with the entry/exit DNA was analyzed and shown with the scaled color in Figure 5b and in Supporting Information, Figure S4a. We adopted the atom name nomenclature used in CHARMM force field. H21 and HE atoms in the guanidinium group and HN atom in the backbone of R42 dominated the H-bond formation in the unmodified nucleosome (Figure 5b and Supporting Information, Figure S4a). As H21 was replaced by a methyl-group in R42me2a and anti-syn R42me2s, the H-bond with the donor H21 was abolished. In addition, since the same methyl-group in R42me2a and anti-syn R42me2s partially blocks HE atom, the frequency of H-bond formation with the donor HE was impaired. However the modifications did not affect the backbone donor, thus the H-bond was maintained between the backbone donor HN, and the acceptor in the entry/exit DNA (Figure 5b and Supporting Information, Figure S4a). Eventually, the numbers of H-bond with the entry/exit DNA in R42me2a, R42me2sas and R42A reduced to a half or even less of that in the unmodified nucleosome while anti-anti R42me2s kept the number of H-bond unchanged (Figure 5a). Y41 has one donor atom HH in its sidechain, which formed H-bond with the oxygens of the entry/exit DNA backbone. The most frequent H-bond acceptor for Y41 was an O3’ atom in +68th DNA base pair (shortened as +68.O3’). It formed ~0.4 H-bond with Y41 on average in the unmodified and both R42me2s nucleosomes (Figure 5a and c), comparable with other donors in R42 (Figure 5b and Supporting Information, Figure S4a). This value reduced to less than 0.2 in R42me2a and R42A nucleosomes on 23



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average. Intriguingly, unlike R42, the asymmetric methylations of R40 had undetectable effects on the H-bond formation of Y41 (Supporting Information, Figure S3a), suggesting the reduction of H-bond of Y41 is affected with R42 modification. We found that the distance between −5.O1P and +68.O3’ was critical for Y41 to form Hbond with the entry/exit. R42 sidechain formed H-bond with oxygen O1P of the −5th DNA base pair (−5.O1P) in the inner region (Supporting Information, Figure S4b). The average number of this H-bond slightly decreased in the modified nucleosomes compared to the unmodified nucleosome, suggesting the distance from R42 to the inner DNA was almost unchanged. On the other hand, Y41 sidechain tended to form H-bond with +68.O3’ in the entry/exit region (Supporting Information, Figure S4c). Since the extension between the HH atom of Y41 and guanidinium of R42 are restrained by the serial of the covalent bonds in the amino acids (Figure 5c and d, the area highlighted by gray color), it is considered that H-bond to the DNA on either end affects the H-bond on the other end. If the distance between the two acceptors −5.O1P and +68.O3’ were too long, Y41 and R42 would not be able to form H-bonds simultaneously. By analyzing the unmodified nucleosome simulation trajectories, we obtained the distribution of this distance with H-bonds formed on both ends simultaneously (Figure 5d, the area highlighted by brown color). Clearly, R42me2a and R42A induced right-shifts of the distance distributions, dramatically lost the coverage of the distance distribution with Hbonds on both ends in the unmodified nucleosome (Figure 5d, red and magenta curves). As a result, the H-bond count between Y41 and the entry/exit DNA dropped (Figure 5a). In contrast, the distance distribution of −5.O1P – +68.O3’ was not significantly affected 24


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by either of R42me2s stereoisomers (Figure 5d, blue and green curves), and the changes in the average number of H-bond between Y41 and the entry/exit DNA were negligible compared to the unmodified nucleosome (Figure 5a). To investigate whether Y41 and R42 are the critical amino acids for stabilizing the nucleosome structure in the H3 histone, we performed an additional REMD on the same partial unmodified nucleosome model, but with full-length H3 histone, and examined the H-bond between the amino acids from V35 to E56 and the entry/exit or inner DNA strands (Supporting Information, Figure S5a and b). Again, R42 amino acid had a higher chance to form H-bonds with both DNA than any other amino acids (Supporting Information, Figure S5 and Supporting Information). R42me2a alters the H2A C-terminal tail distribution to induce DNA dissociation We previously showed that the H2A C-terminal tail can regulate nucleosome structure by interacting with DNA at different locations by MD simulations19. The study showed that the more distal DNA H2A C-terminal tail binds to, the more closed conformation the nucleosome adopts. Herein we examined the H2A C-terminal tail distributions in all the nucleosome variants and displayed the distributions together with DNA end distributions (Figure 6a). Except for R42me2a, in all the nucleosome variants, the spatial distributions of the DNA end and H2A C-terminal tail shared a small overlap, indicating potential interactions between these two partners. For clarity, we recalculated the H2A C-terminal distribution after aligned the simulated trajectories using 5 base pairs at the end of DNA (Figure 6b). Clearly, compared with the unmodified nucleosome and the other variants, R42me2a nucleosome exhibited a spatial distribution of H2A C-terminal 25



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tail less close to the DNA end, indicating the linker DNA would receive less restraint from H2A C-terminal tail. This suggests the interaction between DNA and H2A Cterminal tail is weakened by R42me2a, but not by the other modifications. This is possible because R42me2a-induced dissociation of the DNA end from nucleosome core enlarges the DNA–H2A C-terminal tail distance, impairing the interaction between the H2A C-terminal tail and DNA. The loss of this interaction, in turn, further amplifies the destabilization of the nucleosome structure, which realizes a positive feedback mechanism in destabilizing nucleosome structure.

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Figure 6. Spatial distributions of H2A C-terminal tails obtained by REMD. A. H2A C-terminal tail (yellow) and the DNA end (magenta) distributions in different nucleosome variants. B. H2A C-terminal distribution relative to the DNA end in different nucleosome variants. The H2A C-terminal distribution is recalculated after aligning the 27



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trajectories to the DNA end. The meshes highlight the location where the spatial occupancy of the H2A C-terminal tail is > 5%.

DISCUSSION The methylations in histone have different effects depending on the locations and the isomer types4, 5, 11. Furthermore, it has been found that the symmetric and asymmetric methylations of some arginine residues exhibit opposite effects in gene regulation. H3R2me2a abrogates H3K4 trimethylation, as a result, represses the euchromatic gene activity34-36. Oppositely, H3R2me2s mediated by PRMT5 or PRMT7 excludes binding of RBBP7, a repressor, but enhances binding of WDR5, a common component of the coactivator, and eventually induces transcriptional activation37, 38. H4R3me2a facilitates transcriptional activation39, 40. In contrast, H4R3me2s recruits DNA methyltransferase and results in gene silencing41, 42. H3R8me2a is recognized by Spindlin1 to activate Wnt target gene43, 44. Whereas H3R8me2s catalyzed by PRMT5 suppresses the expression of suppressor of tumorigenicity 7 and nonmetastatic 2345, 46. These reports demonstrate that gene regulation is under delicate control of PTMs but the effect is contextdependent. The dimethylation of H3(R42) showed distinct gene regulations in previous studies9, 10. Yaseen et al. reasoned the discrepancy between these two studies might be due to either or both of the following two reasons: 1. different experimental condition, as in vitro transcription assay vs. in vivo reporter gene assay, 2. distinct constitutional isomers of

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the arginine methylation, asymmetric (R42me2a) vs. symmetric (R42me2s) dimethylation of H3(R42)10. Herein, we performed atomic simulations to study the opposite gene regulation activities of dimethylation H3(R42) and the results support Yaseen’s second hypothesis10. The small structural change by asymmetric dimethylation in the arginine residue produces a clear kinetics change, which directly promotes the opening tendency as the equilibrium constant shifts to a relatively open conformation of the nucleosome accordingly, while R42me2s has limited effects on nucleosome structure and dynamics (Figures 2~4). The average opening angle of R42me2a nucleosome was about 12° larger than that in the unmodified nucleosome in the simulation, corresponding to ~3 base pairs exposure at the entry/exit region, which may contribute to the positive effects on gene transcription9, whereas changes in the angle of R42me2s nucleosome was indistinguishable from the unmodified one (Figure 2). However, we cannot exclude the possibility that R42me2a may also recruit the regulatory proteins and induce gene activation. R42me2s, which impedes gene transcription10, does not directly affect nucleosome structure in our study. It might imply that R42me2s works as a maker for other regulatory proteins to bind to the nucleosome and repress the genes expressions. A detailed, structural analysis with the 3DNA package47 shows that the modifications did not distort the DNA conformation, i.e. B-form was maintained in all systems (Supporting Information, Figures S6-S8). We did not observe any changes in the contents of histone H3 secondary structures analyzed by DSSP package48 either (Supporting Information, Figure S9). Further analysis suggested that opening angle changes were due to the loss of hydrogen bond (H-bond) between nucleosome core and DNA. Comparing to the 29



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unmodified nucleosome, R42me2a and R42A dramatically reduced the H-bond formation between R42 and the entry/exit DNA, but surprisingly barely did R42me2saa. The R42me2sas resulted in H-bond loss between R42 and the entry/exit DNA, but the loss was less pronounced than R42me2a. One of the primary H-bond donors, H21, was replaced by a methyl group in R42me2sas, but not in R42me2saa. Thus, the R42me2sas exhibits a significant drop in H-bond formation rate. However, the R42me2sas and R42me2saa are stereo-chemically interchangeable at room temperature by rotating the corresponding C–N bond49 and the lost H-bond could be recovered by the rotation. Surprisingly, very frequent C–N bond rotation was observed in R42me2saa, but not at all in R42me2sas simulation (Supporting Information, Figure S10), indicating that the transition between two isomers was allowed in R42me2saa but not in R42me2sas simulation. This may suggest that the initial structure of R42me2sas was trapped in a sub-stable conformation and the simulation time was not enough for the system. Nevertheless, we only observed limited effects on nucleosome conformation in R42me2sas and R42me2saa. This indicates, besides forming one Hbond, the steric effect of two methyl group on the same side in R42me2a also play an important role. Intriguingly, the H-bond between Y41 and the entry/exit DNA significantly decreased in R42me2a and R42A nucleosomes but was not affected in both R42me2s ones. Additional conventional Molecular Dynamics (MD) simulations (Supporting Information, Table S1) confirmed reductions in H-bond counts of R42–entry/exit DNA and Y41–entry/exit DNA in R42me2a nucleosome were not due to the truncated system using in the REMD simulations.

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Clear Y41–DNA H-bond reductions were observed in R42me2a and R42A nucleosomes (Figure 5a). Two possible reasons can be considered; 1) the loss of R42–entry/exit DNA H-bond releases the constraint on the entry/exit DNA, allowing DNA to freely fluctuate, leading to the entry/exit and inner DNA distance increasing and Y41–DNA H-bond decreasing (releasing effect); 2) the replacement of hydrogen atoms to methyl groups induces the increase of van der Waals radius, so the bulky groups push the entry/exit DNA strands outward (steric effect). These two causes are not mutually exclusive. In the conventional MD simulation using the entire nucleosome systems, the Y41– entry/exit DNA H-bond count dropped in R42me2a nucleosome but not in R42A (Supporting Information, Figure S3a). Nevertheless, the reduction of Y41–DNA H-bond can in turn further destabilize the nucleosome structure. Together with the decrease in H2A C-terminal interactions with the DNA, the decrease in Y41–DNA H-bond formation may be the second positive feedback, which facilitates destabilization of the nucleosome structure and induces a clear DNA dissociation from the histone core in R42me2a nucleosome.

CONCLUSIONS We utilized REMD simulation to investigate a single amino acid modification effect on the nucleosome structure and dynamics. R42me2a and R42A nucleosomes exhibit the DNA opening tendency while R42me2saa and R42me2sas little showed such an effect. Our study shed light on the distinct dynamics of nucleosomal DNA in R42me2a and

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R42me2s. The REMD simulation approach with the kinetics analysis could be used in studying the influence of other PTM in histone globular domains. ASSOCIATED CONTENT Supporting Information Table S1, the summary of REMD and conventional MD simulations; Table S2, the list of temperatures used in REMD; Figure S1, the simulated histories of closed fraction (thinner lines) of the nucleosome DNA with a criterial angle at −30°; Figure S2, the cumulative moving average of H-bond count; Figure S3, the average count of H-bond between DNA and individual amino acid along histone H3 from V35 to E50 in conventional MD simulation; Figure S4, the H-bond count of atoms; Figure S5, the analysis of H-bond between DNA and amino acids from VAL35 to LYS56 in histone H3; Figure S6, the analysis of DNA conformation; Figure S7, the distribution of Zp parameters; Figure S8, the distribution of slide parameters; Figure S9, the histone H3 secondary structure; Figure S10, the trajectory of C-N χ6 angle in R42me2saa and R42me2sas. This material is available free of charge via the Internet at http://pubs.acs.org.

CONFLICTS OF INTEREST There are no conflicts to declare. ACKNOWLEDGMENTS

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We appreciate the extensive discussion with our lab members, Drs. Hisashi Ishida, Atsushi Matsumoto, Tomoko Sunami, Shun Sakuraba, and Di Luo. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan as “Priority Issue on Post-K computer” [Project ID: hp170255, hp180191] and Grants-in-Aid for Scientific Research from MEXT [JP25116003, JP26330339, and 18H05534] to H.K.

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TOC figure

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Figure 1 85x157mm (300 x 300 DPI)


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Figure 2 85x176mm (300 x 300 DPI)



Figure 3 177x161mm (300 x 300 DPI)


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Figure 4 177x146mm (300 x 300 DPI)



Figure 5 177x177mm (300 x 300 DPI)


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Figure 6 177x193mm (300 x 300 DPI)


Investigating the Influence of Arginine Dimethylation on Nucleosome

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