Molecular Mechanism for the Role of the H2A and H2B Histone Tails

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Molecular Mechanism for the Role of the H2A and H2B Histone Tails in Nucleosome Repositioning Kaushik Chakraborty, Myungshim Kang, and Sharon M. Loverde J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b07881 • Publication Date (Web): 27 Nov 2018 Downloaded from http://pubs.acs.org on November 28, 2018

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The Journal of Physical Chemistry

Molecular Mechanism for the Role of the H2A and H2B Histone Tails in Nucleosome Repositioning Kaushik Chakraborty1, Myungshim Kang1, Sharon M. Loverde1,2,3,4* 1

Department of Chemistry, College of Staten Island, The City University of New York, 2800 Victory Boulevard, Staten Island, New York, 10314, United States. 2

Ph.D. Program in Chemistry, The Graduate Center of the City University of New York, New York, NY, 10016

3

Ph.D. Program in Biochemistry, The Graduate Center of the City University of New York, New York, NY, 10016 Ph.D. Program in Physics, The Graduate Center of the City University of New York, New York, NY, 10016

4

E-mail: [email protected], Phone: 718-982-4075, Fax: 718-982-3910

ABSTRACT: The nucleosome core particle (NCP) is the basic packaging unit of DNA. Recently reported structures of

the NCP suggest that the histone octamer undergoes conformational changes during the process of DNA translocation around the histone octamer. Herein, we demonstrate with long-time all-atomistic molecular dynamics simulations that the histone tails play a critical role in this nucleosome repositioning. We simulate the NCP at high salt concentrations an order of magnitude higher than physiological conditions to screen the electrostatic interactions. We find that the positively charged H2B tail collapses and complexes with the minor groove of nucleosomal DNA. Upon collapse of the tail, counterions are released. This promotes the formation of a ~ 10 bp loop of nucleosomal DNA. The complexation of the tail increases the local flexibility of the DNA, as characterized by local force constants. Using normal mode analysis, we identify a ‘wave-like motion’ of nucleosomal DNA. We perform umbrella sampling to characterize two possible pathways of the initial stages of unwrapping, symmetric and asymmetric. These results suggest that regulation of the histone tail interactions with nucleosomal DNA may play a critical role in nucleosomal dynamics by acting as a switch to determine the initial pathway of unwrapping.

NCP determines the strength of these electrostatic interactions. Indeed, the unwrapping rate of the nucleosome core particle is increased in the presence of additional salt11. Although the nucleosome is stable in physiological conditions, it undergoes largescale structural re-arrangements during different cell cycles. DNA binding proteins such as transcription factors12, 13, chromatin remodelers14, and polymerases15, 16 need to bind nucleosomal DNA. Furthermore, the sequence of nucleosomal DNA may determine the strength of histone-DNA interactions and may encode organization of the nucleosome at larger length-scales17. However, in order for transcription to occur, the nucleosomal DNA needs to unwrap from the central histone core. Modification of the histone core can allow for easier access to nucleosomal DNA through modifications in the structure and dynamics of the NCP. Furthermore, mutations in chromatin components, such as the NCP, can lead to a range of human diseases13.

INTRODUCTION The nucleosome core particle (NCP) is the basic building block of chromatin, which is a compact, yet dynamic structure that packages DNA1-4. The NCP consists of a positively charged histone octameric core, surrounded by negatively charged nucleosomal DNA which is wrapped ~ 1.7 times around the core5-7. The central core consists of a highly ordered helical globular region composed by two copies of each of the four main histone core proteins (H2A, H2B, H3, and H4). Surrounding the core are the disordered, flexible, and positively charged histone tails that often play a critical role in many biological processes involving the nucleosome3 and contain many important epigenetic marker sites8, 9. Fundamentally, the electrostatic interactions between the negatively charged DNA and the positively charged histone proteins stabilizes the NCP10. The ionic environment around the

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Multiple theories exist concerning the translocation of nucleosomal DNA along the central histone core at shorter time-scales (10-100’s of milliseconds), as well as the pathway for unwrapping of nucleosomal DNA at much longer time-scales (100’s of milliseconds-seconds). For instance, the twist/diffusion18 as well as the bulge/loop19 propagation models both suggest defects or distortion in the local DNA structure during the sliding motion of DNA along the histone core. This sliding motion is inherently difficult to study both experimentally and computationally. However, recent time-resolved (TR) small-angle X-ray scattering (SAXS)20 and Förster resonance energy transfer (FRET)21, 22 experiments have shown that there exist multiple pathways (symmetric and asymmetric) for large-scale unwrapping. Intermediate states form after 100-200 milliseconds, while the timescale for the complete unwrapping is 1-2 seconds. However, the molecular mechanism triggering unwrapping, as well as how the shorter time-scale motion of nucleosomal DNA is correlated to the largescale unwrapping is largely unknown.

According to their prediction, nucleosome repositioning may happen via formation of two types of loops with different sizes (i) a small 10 base pair loop and (ii) a large loop with variable length. Li et al.33 also studied translation of the DNA loop around the nucleosome using Brownian dynamics simulation. Alternatively, based on twist diffusion, Mohammad-Rafiee et al.34 estimated nucleosome mobility using a deterministic model. Lequieu and co-workers found, using a coarse-grained model of the nucleosome core particle, based on DNA sequence, nucleosome repositioning happens either by loop propagation or twist diffusion or by both31. In addition, a number of both atomistic and coarse grained simulations of the nucleosome core particle have been performed near equilibrium9, 35-45. Biasing methods such as steered molecular dynamics (SMD) and umbrella sampling (US) also provide significant details about nucleosome unwrapping pathways46-48. Multiscale models offer a connection between molecular structure and mesoscale chromatin organization49.

Various experiments suggest that breathing22, 23 and sliding24 are two important motions of the nucleosomal DNA at short timescales. While breathing is related with the transient opening and closing of the DNA end regions, movement of the histone core along the DNA without disrupting the overall structure is known as sliding. Both these modes make the nucleosomal DNA accessible to DNA binding proteins transiently. Specifically, sliding becomes crucial in the presence of chromatin remodeler19. The “twist diffusion”18, 25 and “ loop/bulge propagation” models26-28 are two different models that allow for nucleosomal DNA sliding. According to the twist diffusion model, the exchange of a base pair between the linker DNA and the wrapped DNA around the protein core creates a twist defect within the nucleosomal DNA. This twist defect then diffuses (so-called twist diffusion) all the way over the nucleosome to other end and shifts the histone octamer by one base pair. The discovery of a large number of nucleosome core particle structures with a gain or loss of one base pair support this model5, 29, 30. Analogous to the twist diffusion model, the bulge propagation model suggests a loop/bulge formation along the nucleosome due to a transient shift of base pairs from the linker DNA and then the loop or bulge propagates through one dimensional diffusion19 until it meets the other end (exit/entry region) of the nucleosome with a complete translocation of DNA14, 18, 19. For internal translocation of the loop, the nucleosome undergoes a “wave-like motion” propagating toward the dyad19. The major difference between these two models is the number of the base pairs that translocate during nucleosome sliding. While twist diffusion allows the movement of only one base pair, bulge/loop propagation model involves translocation of up to 10 base pairs14, 17, 27. This makes the bulge propagation model energetically more expensive31 than the twist diffusion model due to the disruption of more histone-DNA contacts. Very recently, as revealed by cryoelectron microscopy, Liu and co-workers reported a wave-like motion of the nucleosome along with the bulging near SHL (super helix location) ±2 regions due to its binding with Snf2 chromatin remodeler32. Bilokapic et al7. also observed the presence of loops near SHL±5 and SHL±2 in the absence of any chromatin remodeler. (See Figure 1 (a)) which includes labeled super helix locations.)

Herein, we perform a 5 μs long molecular dynamics (MD) simulation of the nucleosome core particle in the presence of high salt concentration (2.0 M NaCl). The high salt concentration (an order of magnitude higher than physiological conditions) screens the electrostatic attraction between the DNA and the histone core, promoting the unwrapping of the nucleosome core particle. We identify breathing and stretching as the two important modes of the nucleosome in the early stages of unwrapping. Stretching is associated with loop or bulge formation near the SHL-5 region of the nucleosomal DNA. Although the loop or bulge formation is energetically expensive, our results also suggest that condensation of the highly charged histone tail on DNA can initiate the bulge/loop formation by modifying the local electrostatic environment. Along with loop formation, we also observe a wavelike motion of the nucleosomal DNA due to the inward breathing of the DNA tail region (SHL-6) and the outward stretching of the SHL-5 region. While the inward breathing of SHL-6 is related to the stabilization effect of the H2A C-terminal tail, destabilization by the H2B N-terminal tail promotes the outward stretching of SHL-5 region of the nucleosomal DNA. Hence, the highly charged and flexible histone tails can act as a switch to maintain both the stability and the plasticity of nucleosome. Furthermore, free energy methods in molecular dynamics, we probe the connection between loop formation and the symmetric/asymmetric nature of the initial stages of the unwrapping of the nucleosome.

Significant efforts have been made to understand nucleosome sliding computationally. Kulic and co-workers28 examined the loop propagation model of nucleosome sliding computationally.

2. SIMULATION The fully flexible nucleosome core particle was simulated in the presence of 2.0 M NaCl salt. The starting configuration was taken from the crystal structure6 as reported in the Protein Data Bank (PDB ID: 1KX5). After adding hydrogen atoms, the nucleosome core particle was immersed in a rectangular box containing 95,172 water molecules. To avoid any unfavorable contacts with solvent, we removed all the water molecules within 2 Å from the nucleosome surface. Then, we added 3972 Na+1 and 3856 Cl-1 ions to achieve 2.0 M salt concentration. In total, the solvated system contained 318,444 atoms. For both the protein50, 51 and DNA52-54 we used the CHARMM36 force field, while for water we used the TIP3P55 model.


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To begin with, we minimized the system to avoid any initial stress. Minimization was done using the conjugate gradient energy minimization method56. Following minimization, the temperature of the system was increased to 310 K at 1 atm pressure within a short NPT run. Afterwards, we equilibrated the system for 50 ns duration under the same NPT ensemble conditions. The Langevin dynamics method with a friction constant of 1 ps-1 was used to control the temperature of the system while the pressure of the system was controlled by the Nosé-Hoover Langevin piston method57. The simulation was continued for another 5 μs under NPT conditions with a time step of 2 fs. Trajectories were stored after every 50 ps for subsequent analysis. The simulation was performed on the highly efficient Anton-2 supercomputer at the Pittsburgh Supercomputing Center58. While the opening and the closing of the nucleosomal DNA termini happen on the micro to millisecond time scales, the rate of unwrapping increases significantly at higher salt concentrations due to the highly charged nature of the complex. We performed a 5 μs MD simulation of the nucleosome core particle in 2.0 M NaCl solution. The Debye length (λB) of water at room temperature is around 7 Å. In the presence of 2 M NaCl salt it reduces to ~2 Å. As a result, the strength of the electrostatic interactions between the DNA and histone proteins reduces accordingly and the unwrapping of the nucleosome core particle happens at a much faster rate11. Additionally, to further speed up the unwrapping process, we also carried out the simulation at 310 K. Though the length of our simulation is significantly shorter than the actual opening and closing time scales of the nucleosomal DNA, it is almost five times longer than the previously reported longest trajectory22 of the nucleosome core particle. Umbrella Sampling To construct the free energy profile of the nucleosome unwrapping, we used umbrella sampling (US) techniques59 for efficient sampling. The reaction coordinate for the US is the radius of gyration (rg) of the DNA backbone heavy atoms. A harmonic poten1 tial, 2 𝑘𝑟 (𝑟𝑔 − 𝑟𝑔0 )2 , was employed during simulation. Here, the −1 −2

𝑟𝑔0

force constant 𝑘𝑟 = 2 𝑘𝑐𝑎𝑙𝑚𝑜𝑙 Å and is the reference radius of gyration of the DNA. 13 individual reference points were chosen from 43.5 to 50 Å with an increment of 0.5 Å. For each window we performed 20 ns simulation with a time step of 2 fs. Two sets of US sampling were performed with different starting structures (initial crystal structure and the final configuration after 5 μs equilibrium simulation). For each case, 260 ns trajectory in total was generated. Thus, a total 520 ns US simulation was performed using NAMD 2.1156. Finally, to obtain the potential of mean force (PMF) as a function of rg we used the weighted histogram analysis method (WHAM)60, 61.

we search for a minima by varying λ2 and keeping the λ1 constant as obtain from the first step. The search is continued until the entire set of λ1 and λ2 are covered. The minimum path ε is constructed by connecting these points with minimum free energy. 3.2 Elastic Properties of Nucleosomal DNA To study the elastic properties of nucleosomal DNA, we compute the elastic matrix (F) of each base pair using the equation65 𝐹 = 𝑘𝐵 𝑇𝑉 −1 where V is the 6 x 6 covariance matrix of the DNA deformation variables (roll, tilt, twist, rise, shift and slide). Diagonal elements of the matrix are proportional to force constants (K) of the six helical parameters. Roll, tilt, twist, rise, shift and slide are calculated using the Curves+ algorithm66. Later, the Cholesky Decomposition67 method is employed to get the inverse of the covariance matrix at different time frames. 3.3 Localized Dissociation of DNA Bases To compute the relative dissociation constant of each base-pair, we follow the approach as proposed by Ghaemi and co-workers68. They have determined the relative dissociation constant of wild type and mutated protein-RNA complexes based on fluctuation theory69. As the electrostatic interaction is the major binding force in any protein-nucleic acid complex, Ghaemi and co-workers found that a change in the electrostatic environment due to mutation is directly related to the atomic fluctuation of the complex, as well as to its dissociation70. Like mutation, unwrapping of the nucleosome also modifies the electrostatic interactions between the DNA and the histone proteins. The change in the electrostatic environment of individual bases due to unwrapping is directly proportional to their dissociation rates. According to this method, the electrostatic force, Fi, on i-th atom due to interaction with j-th atom can be represent as ∆𝐹𝑗 (𝑖) ≈ −𝑅𝑇〈𝛿𝑟𝑖 . 𝛿𝑟𝑗 〉−1 ∆𝑟𝑖

(1)

where 𝛿𝑟𝑖 is the displacement vector of i-th atom and 〈𝛿𝑟𝑖 . 𝛿𝑟𝑗 〉−1 is the inverse of the covariance matrix. Here, i-th

3. METHODS 3.1 Property Based Potential of Mean Force The 2D potential of mean force (PMF) or free energy landscape can be defined as62, 63 𝐺(𝜆1, 𝜆2) = −𝑘𝐵 𝑇[𝑙𝑛𝑃(𝜆1, 𝜆2) − 𝑙𝑛𝑃𝑚𝑎𝑥 ] where λ1 and λ2 are reaction coordinates and P(λ1,λ2) is the probability distribution. Pmax denotes maximum probability. To construct the minimum free energy path64, first we search for a minima by varying λ1 and keeping λ2 constant. In the next step,



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Figure 1. (a) The crystal structure of nucleosome core particle (PDB ID: 1KX5) contains 147 DNA base pairs wrapped around two copies of each of the four histone proteins H2A, H2B, H3, and H4. Different regions of nucleosomal DNA are shown in different colors. The histone proteins are in blue. (b) The nucleosome core particle after 5 μs simulation. Here the backbone of the nucleosomal DNA is shown in transparent blue and the histone core in grey. Loop formation near SHL (super helix location)-5 is highlighted in red. (c) Top and (d) bottom view of the initial (black) and final (red) conformations of nucleosomal DNA. Here each dot represents a base-pair. Bulging near SHL-5 is clearly evident. X and Y are in Å. The backbone of the DNA in the reported CryoEM structure from Bilokapic et al (reference 7) is shown in blue. (e) The average distance (R) of each base pair from the center of mass of the whole DNA of its initial crystal structure over last 1 μs (red). Corresponding data for the initial configuration (black) is also given for comparison. Difference in R between the simulated and the initial configurations (∆R) are incorporated in the inset. Both inward (SHL-6) and outward (SHL-5) movements at the same time suggest a wave-like motion of nucleosomal DNA. (f) Variation of DNA loop size, Lp, near SHL-5 as a function of time. Any base pair near the SHL-5 region with ∆R greater than 2 Å is considered a part of the loop. Lp is the number of base pairs within the loop. The red and blue regions indicate loop formation and loop propagation respectively. atom is referred to as an “observation point”. As the nucleosome unwraps, the free energy change of i-th atom due to the change in electrostatic energy with j can be written as70 ∆𝐺𝑗 (𝑖) ≈ 𝑅𝑇(〈𝛿𝑟𝑖 . 𝛿𝑟𝑗 〉−1 ∆𝑟𝑖 ). ∆𝑟𝑗

(2)

where ∆𝑟𝑖 is the displacement vector of i-th atom from the initial crystal structure. Thus, ∆𝐺𝑗 (𝑖) is the change in the free energy of the i-th atom due to j-th atom during unwrapping. The total free energy change of i-th atom for all the atoms is −1 ∆𝐺(𝑖) ≈ 𝑅𝑇 ∑𝑁 𝑗 (〈𝛿𝑟𝑖 . 𝛿𝑟𝑗 〉 ∆𝑟𝑖 ). ∆𝑟𝑗

(3)

Then, the dissociation constant (𝐾𝑑 ) can be written as −1 ln 𝐾𝑑 = ∑𝑁 𝑗 ((〈𝛿𝑟𝑖 . 𝛿𝑟𝑗 〉 ∆𝑟𝑖 ). ∆𝑟𝑗 )

To estimate the free energy of each base pair, we consider two phosphorous (P) atoms of the tagged base pair as the observation points. The average of the two free energies of these P atoms is taken as a free energy of the particular base pair. Free energies are computed from equation (3). Now, to calculate the free energy of individual base pairs, we assume that binding free energy of the base pairs are independent of each other. This allows us to construct the covariance matrix in equation (3) by considering the displacement vectors of the tagged P atoms and Cα atoms of the histone proteins. To get the average positions of both the histones and the DNA bases in the covariance matrix, the entire 5 μs trajectory has been taken into account. Then, from the free energies of the individual base pairs, we compute their dissociation constants.

(4) 4. RESULTS & DISCUSSION


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4.1 Formation of Loop on the Histone Surface Our 5 μs long simulation reveals a bulge/loop formation near the SHL-5 region of the nucleosome after ~ 2 μs in the presence of 2.0 M salt. The bulge/loop remains stable and starts to grow over the next 3 μs. The structure of the loop is highlighted after 5 μs in Figure 1(b). Next, in Figure 1 (c) and (d) we present the superimposed conformation of the nucleosomal DNA after 5 μs as compared with the initial crystal structure. Each dot represents the center of mass of a base-pair. Indeed, there are two loops near the SHL-5 and SHL-5 regions, both with a different size and shape. Furtheremore, the loop size near SHL-5 shows a good agreement between the simulation and the structures reported by Bilokapic et al7, the position of the loop is shifted 5 base-pairs. The difference in DNA sequences between these experiments and our simulation may be associated with such a small shift in the

Next, we calculate the distance (R) of each base-pair of the simulated configurations with respect to the center of mass of the whole DNA heavy atoms of the initial crystal structure. The average distance of each base pair over the last 1 μs trajectory is depicted in Figure 1 (e) as compared with the initial configuration. As the distances are measured with respect to the crystal structure, any difference in R between the simulated and the initial configurations (∆R) suggests a displacement of the DNA bases from the starting structure. ∆R of each base pair is given in the inset. A loop of approximately ten base pairs bends outwards with a maximum displacement of nearly 6 Å near base pair 21 is highlighted in the inset of Figure 1 (e). Similar displacements (~ 5 Å ) of the DNA backbone due to the loop formation in the presence of chromatin remodeler snf2 is also reported by Liu and coworkers32 using cryo-EM. Furthermore, while SHL-5 forms a loop, the SHL-6 region also moves nearly 5 Å, but more towards

Figure 2 . Structural rearrangements of α-helices within the histone core. Crystal and simulated structures are shown in green and red respectively. Cryo-EM structure of the histone core from reference 7 for the distorted Class-3 nucleosome is also presented in blue. loop position. The loop near SHL±5 also agrees well with the crystal structure reported by Ong and co-workers30 with an extreme kinking around SHL±5 and SHL±2 regions of the NCP.

the histone protein core. Such inward and outward displacements of SHL-6 and SHL-5 respectively at the same time suggest a wave-like motion19 of nucleosomal DNA. According to the wave-bulging model19, along with the formation of a loop on the histone surface, the wave-like motion of DNA is an integral part



of nucleosome sliding. Although the size of the loop agrees reasonably well with experiments, it increases throughout the simulation trajectory. Hence, the loop size may increase further at longer timescales (10’s -100’s of microseconds). To further examine the effect of the loop formation on histone-DNA contacts, we also calculate the number of contacts between the histone core and the base-pairs present near the loop region of the DNA, as shown in SI-2. Here contacts between the histone tails and the DNA are not considered. As expected, the dissociation of histoneDNA contacts upon loop formation is evident. Following, we further compute the number of base-pairs involved in loop formation (Lp). Any base pair near the SHL-5 region with a ∆R greater than 2 Å is considered part of the loop. The time evolution of Lp (Figure 1(f)) reveals that the loop propagates through a series of events. To begin with, a 5 base-pair loop first forms near ~2 μs and remains stable for next 1 μs. After 3 μs, as the loop starts to propagate on the histone surface, the Lp increases from 5 base-pairs to 10 base-pairs. For next 1.5 μs, Lp shows minor fluctuations. Near the end of the simulation (~ 4.5 μs), Lp

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again displays a slight increase. Hence, like nucleosome unwrapping48, 71, the formation of a loop on the histone surface also happens in a step-wise manner. In addition, different time scales suggest different free energy barriers for these individual steps. The barrier associated with these combined steps may determine the overall nucleosome sliding rate. Very recently Lequieu and coworkers31 also explored sequence dependent nucleosome sliding computationally. The study provided significant insight into the energetics of the loop formation. They found that, based on sequence, the energy barrier of DNA loop formation can vary from 2 to 20 kBT. This raises an important question. Though it is freeenergetically expensive, why (and how) the loop actually forms? Our long, unbiased, and atomistic simulation provides a rare opportunity to explore the molecular mechanism driving loop formation compared to recent experimental as well as simulation studies.

Figure 3. (a) Superimposed structure and the fluctuating conformations of the first three modes of nucleosomal DNA obtained from NMA. Initial and final configuration of the corresponding modes are shown in red and blue respectively. (b) Square-fluctuations (mobility) of DNA backbone heavy atoms for the first three modes of nucleosomal DNA. The corresponding dynamics of DNA is also included in Figure 3(c) (from red (rigid) to blue (flexible)). SHL -6, SHL 6, and SHL -5 (dark blue regions) are the most flexible regions for mode1, mode2, and mode3 respectively.


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The Journal of Physical Chemistry loop on the histone surface. Later we will discuss more about the possible role of the histone proteins for loop formation near SHL5.

4.2 Structural Rearrangement of Histone Core Along with the DNA, we also notice structural rearrangements of α-helices within the histone core, as shown in Figure 2. The Cryo-EM structure of the histone core for distorted Class-3 nucleosome is also given for comparison7. A nice agreement between the experiment and the simulation is evident from the superimposed structures. Previously, Bilokapic et al7 suggested that such minor structural rearrangements of the histone core can di-

4.3 Normal Mode Analysis of Nucleosomal DNA So far it is clear that SHL-6 and SHL-5 are the most flexible regions of nucleosomal DNA. While the displacement of SHL-6 is associated with the breathing motion of the complex, outward

Figure 4. (a) Wave-like motion of nucleosomal DNA. The stabilization effect of the H2A C-terminal tail is responsible for the inward breathing motion of SHL-6, whereas the destabilization by the H2B N-terminal tail due to condensation promotes the outward stretching motion of SHL-5. A combination of these two motions creates a wave-like motion within the nucleosome. (b) Electrostatic map of the nucleosome core particle after 5 μs. Here, the positively charged regions are shown in blue and the negatively charged regions are shown in red. At higher salt concentrations, the H2B N-terminal tail collapses on the minor groove of DNA and creates a positively charged region near SHL-5 region. This destabilizes the DNA and creates a bulge. (c) Two dimensional representation of the free energy landscape of the bulging of nucleosomal DNA. Rg (radius of gyration of H2B histone tail) and LP (number of base pairs involve in loop formation) are considered as the reaction coordinates. The minimum around Lp 5 associates with formation of the loop and the loop propagation is responsible for the minima near 8 and 10. (d) The free energy profiles (G) along the minimum energy pathways for loop formation as constructed from the free energy landscape. Here ε is a generalized reaction co-ordinate. rectly lead to the formation of a loop on the histone surface. For instance, while the movement of α-1 and α-2 helices of H3 and α-1 helix of H4 (see Figure -2(a) and (b)) is associated with the formation of a loop near SHL-2 region, tilting of α-1 helices of H2A and H2B histone proteins (see Figure -2(c) and (d)) towards the DNA is responsible for the outward bending of the SHL-5 region. With tilting, the α-1 helices of H2A and H2B push the SHL-5 region of the nucleosomal DNA outward and create a

bending of SHL-5 is related with the formation of loop. Hence, these are important low frequency modes of nucleosome in the early stage of unwrapping. Normal mode analysis (NMA) has been successfully used to study the slow dynamics or low frequency modes of both protein and DNA72, 73. To study the low frequency global modes, we consider the very last configuration of the nucleosomal DNA after 5 μs simulation. VMD74 and Prody75 packages are used for normal mode analysis. The first



three modes are shown in Figure 3(a). The two slowest global modes of the nucleosome involve large scale in-plane and out-ofplane motions of the DNA tails regions. They are the in-plane and out-of-plane breathing dynamics of the DNA tails. The third mode is the lateral stretching of nucleosomal DNA along the histone core. Stretching has been reported for multiple different DNA sequences76. Unlike breathing, in the case of stretching, the DNA tail regions show very little fluctuation. Ramaswamy and co-workers73 also explored the low frequency modes of the nucleosome for a number of crystal structures. Unlike us, the stretching mode was absent in their study due to the wrapped configuration of the nucleosome in the crystal structure. In Figure 3(b), we show the square-fluctuations (mobility) of DNA backbone atoms for the three modes. The corresponding dynamics of DNA is also included in Figure 3(c) colored from red (rigid) to blue (flexible). The higher mobility of the DNA tail regions in the case of both the first and second mode again suggest that these two modes are the breathing motions of nucleosomal DNA. A close comparison between these two modes further reveals that in in-plane breathing Tail-1 (SHL-6) has a higher mobility whereas Tail-2 (SHL 6) is the most dynamic region for out-ofplane breathing. Different mobility of the two DNA tails for the two modes agrees well with our previous77 findings that the breathing motions of the DNA tails are both asymmetric and anticorrelated with each other. The mobility plot also reveals that SHL-5 is the most dynamic region for stretching. Similar outward stretching of SHL-5 is also reported experimentally7 for the nucleosome in its distorted conformation. This particular stretching mode is absent for the initial crystal structure, as shown in SI-3. Hence, while breathing is associated with the closing and the opening of the nucleosome exit/entry regions, lateral stretching is responsible for loop formation. A combination of breathing and stretching of SHL-6 and SHL-5 respectively creates a wave-like motion of DNA on the histone surface. 4.4 Wave-like Motion of the Nucleosomal DNA A number of studies have already proposed a wave/bulging model19 on the octamer surface for the nucleosome sliding. According to this model, the torsional strain generated within the DNA due to binding with the remodeler creates a wave-like motion on the histone surface. However, very little is known about the structural basis of the wave-like motion of the nucleosome. In Figure 4 (a), we outline molecular interactions governing the wave-like motion of the DNA. During simulation, while the SHL6 region of the DNA moves toward the histone core, the neighboring SHL-5 region shows an outward displacement with the formation of a loop (also see Figure 1(e)). Both the inward and outward displacements of the DNA at the same time is characteristic of wave-like motion. In addition, breathing is related with the inward movement of SHL-6 and stretching is responsible for the outward bending of SHL-5. To explore the wave-like motion of the DNA over time in SI-4 we show the super-imposed conformation of the nucleosomal DNA at different time periods. As time progresses the SHL-6 region moves toward the histone core and the SHL-5 region moves away from it. The combination of both inward and outward motions of the SHL-6 and SHL-5 regions respectively creates a wave-like motion on the histone surface. Due to strong interactions with the H2A tail, the SHL-6 region moves toward the histone protein core. While the C-terminal tail of the H2A histone stabilizes the SHL-6 region, the long N-terminal tail of H2B condenses into the SHL-5 minor groove

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around 2 μs (see SI -5(a)). Incidentally, the loop near SHL-5 region also forms at the same time. This suggests a close connection between the H2B N-terminal tail condensation and the formation of the loop on the histone surface. Due to their condensation into the minor groove of DNA, the long and highly charged histones tails can destabilize the complex by modifying the electrostatic environment (with increased salt concentration). To study the electrostatic environment, in Figure 4 (b) we present the electrostatic map of the nucleosome core particle as calculated using APBS78. Red and blue represent highly negatively and positively charged regions respectively. Positively charged LYS and ARG residues of the H2A histone are also shown in green. The blue color near the loop region of DNA (SHL-5) indicates a higher population of positively charged residues of H2B histone proteins condensing in the minor groove. It suggests that at ~2 μs, a large number of LYS and ARG residues of the H2B tail are deeply inserted into the DNA minor groove and make extensive contacts with both the DNA bases and the backbone phosphate groups near SHL-5. The presence of such large number of LYS and ARG residues inside the highly confined minor groove of DNA may create overcharging and can destabilize the complex through formation of a loop. Two distinct electrostatic environments with low and high charge near the DNA loop due to the H2B N-terminal are also evident from the distribution of charge (see SI-5(b)). To directly correlate the condensation of the H2B tail and bulging of nucleosomal DNA, we further construct a two-dimensional potential of mean force (2D PMF) using radius of gyration of the H2B histone tail (Rg) and loop size (Lp) as reaction coordinates. The free energy landscape, shown in Figure 4(c), is characterized by four minima. Relatively high Rg (~ 11.5 Å) and low LP (~ 0) suggest that the first minimum corresponds to the native conformation of the nucleosome without any localized deformation of DNA as well as with no restructuring of H2B tail. In the next step, the N-terminal tail of H2B histone starts to collapse (Rg ~ 9.5 Å) with a small bulging (Lp ~ 5) on the histone surface. Finally, as the restructuring of the histone tail destabilizes the nucleosomal DNA and the loop starts to propagate on the histone surface, it creates two additional minima. The minimum around Lp ~ 5 is associated with the formation of the loop. Loop propagation is responsible for the minima near Lp 8 and 10. To understand the molecular mechanism of loop formation from the free energy surface, we apply a simple approach, as describe in the methods section, to calculate the minimum energy path. It reveals the most probable transition of the nucleosome from the fully wrapped conformation to its distorted state. Lequieu et. al. 31 used the string method to construct minimum free energy path from the free energy profile of nucleosomal repositioning around the histone core. The minimum free energy pathway of loop formation, as shown in Figure 4(d), shows four minima. In addition, the minimum free energy path suggests that the energy barriers for these steps are of different magnitude from each other and varies between 0.5-1.0 kcal mol-1. Previously, Lequieu and co-workers31 found that, based on sequence, the energy barrier of DNA loop formation varies from 2 to 20 kBT. The magnitude of the energy barriers we are finding here are much lower. Most likely, this is due to the different salt concentrations that are used in these two studies.


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Figure 5. (a) Restructuring of the H2B tail can facilitate nucleosome sliding through formation of a loop near the SHL-5 region. The H2B facilitated nucleosome sliding involves two major structural rearrangements of the particular histone tail: tail collapse and formation of a stable secondary structure (coil to helix transition). Conformations of the H2B tail before and after 2 μs are shown. Structural rearrangements of the H2B tail destabilize the complex, forming a loop near SHL-5. Apart from the unbiased 5 s simulation, a 50 ns targeted MD (TMD) simulation starting from the initial crystal structure considering radius of gyration of the H2B tail as a reaction co-ordinate also revels loop formation near SHL-5, as shown in SI-8. Thus, the collapse/restructuring of the histone core tails directly leads to a bulge/loop formation. A number of experimental studies also report that H2B N-terminal tail is crucial for nucleosome polymorphism7, 79. Hence, the interactions between DNA and histone tails are dynamic in nature. For example, formation of salt bridges between the DNA phosphate groups and positively charged residues of Cterminal tails of the H2A histone77 stabilize the closed conformation of the nucleosome with an inward breathing motion of the DNA tails. At the same time, condensation of the long H2B Nterminal tail into the DNA minor groove, destabilizes the complex with an outward stretching of the DNA. The combination of both the stabilizing and the destabilizing effects of the H2A and the H2B tails respectively, creates a wave-like motion on the histone octamer surface. In other words, a combination of stabilization and destabilization effects of H2A and H2B tails respectively make the bulge formation free-energetically less expensive. Thus, the highly charged and flexible histone tails can act as a switch to maintain both the stability and the plasticity of the nucleosome. 4.5 H2B Facilitated Nucleosome Sliding

Even in the bound state, the disordered H2B histone tail has at least two different states: collapsed and extended. While the extended conformation of the H2B tail stabilizes the complex, the primary effect of the collapsed state is the formation of the loop/bulge. By creating a loop/bulge, the H2B tail can also promote nucleosome sliding. Previously, Hemiche et. al.80 studied the role of disordered histone tails for the sliding of NCP experimentally. They found that during sliding the H2B histone plays a crucial role. In fact, deletion of the H2B N-terminal tail promotes the nucleosome sliding rate, in the absence of any chromatin remodeler. As, the H2B tails make extensive histone-DNA contacts near the gyres (SHL 5) of the DNA superhelix, modulation the histone-DNA contacts near the gyres promote nucleosome sliding due to the removal of H2B tails. Furthermore, Swi/Snf a chromatin remodeling complex also targets the H2B N-terminal region81 and deletion of the H2A/H2B N-tail promotes nucleosome traversal by RNA Polymerase II82. Hence, the H2B N-terminus is an important modulator for chromatin structure and function. On top of this, our data further reveals that even in the bound state, restructuring of H2B the tail can facilitate nucleosome sliding through formation of a loop near the SHL-5 region. The H2B facilitated nucleosome sliding involves two major structural rearrangements of the particular histone tail: tail collapse and formation of a stable secondary structure (coil to helix transition), as shown schematically in Figure 5. Both happen at ~ 2 μs, suggesting a close connection between the two intermediate states.



In the following section, we discuss these two steps in more detail. 4.5.1 Collapse of the H2B Tail and the Role of Salt To begin with, the H2B N-terminal tail is long (~ 30 amino acids), highly charged, and lacks a stable secondary structure. Interestingly, a recent experiment83 further suggests that the different residues of H2B tail possess heterogeneous binding affinities with the DNA, depending on their location. So it is possible that based on binding, different parts of the H2B tail can show heterogeneous behavior. In fact, a wide range of Rg for three different regions of H2B tail (region-1 (1 to 10), region-2 (11 to 20) and region-3 (21 to 30)), as shown in SI 8(a), reveal heterogeneous flexibility within the histone tail. A closer comparison among the three regions demonstrates that region-1 is the most flexible part of H2B tail due to its weak binding with the nucleosomal DNA. This agrees well with the experimentally reported low binding affinities of the terminal region (region-1) of H2B tail83. While region-1 adopts a multiple of conformations, the strongly bound region-2 and region-3 have two distinct states with high and low Rg . In fact, both the regions collapse at ~ 2μs. Along with the weak binding of the H2B terminal region, Wand and co-workers83 also identified cooperative interactions of region-2 and region-3 with the DNA. The near simultaneous collapse of region2 and region-3 may be due to their cooperative interactions with the DNA. Hence, in agreement with experiments83, our study

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within 5 Å from the heavy atoms of the SHL-5 region of nucleosomal DNA. As the H2A tail condenses on the DNA minor groove, the number of both Na+ and Cl-1 ions near the SHL-5 region start to decrease. Complexation of the histone tail with the DNA releases condensed counterions into the system and thus favors the loop formation process entropically. In addition to the entropic role of the salt, we also find a direct molecular role in helix formation. In SI 8(b), we show the average number of bound Na+ (PNa) ions for each H2B histone tail residue. A higher population of Na+ ions (> 1 Na+ ion) is found on average around the negatively charged ASP22. Next, to explore whether the interactions of ASP22 with the Na+ ion is important for the restructuring of H2B tail, in SI 8(c), we depict the number of Na+ ions bound with ASP22 (NNa) during the 5 μs trajectory. A shift of NNa from 1 to 2 after 2 μs indicates a correlation between the binding of the Na+ ion with ASP and the collapse of the H2B tail. Furthermore, the conformations of the H2B tail before (red) and after 2 μs (blue), as shown in SI 8 (e), demonstrate a spatial rearrangement of the histone tail due to binding of Na+ ion with ASP22. The positions of ASP22 before and after 2 μs are also highlighted. In the presence of Na+ ions, which minimizes the repulsion between the DNA backbone and negatively charged ASP, the H2B histone tail moves toward the DNA (shown in black arrow in SI-8 (e)) and collapses into the minor groove.

Figure 6. (a) Variation of rise force constant over the nucleosome (from red (low force constant) to blue (high force constant)). The red color suggests that SHL -6, SHL -5, and SHL 2 are most flexible regions of the nucleosomal DNA. (b) Variation of rise, shift, and slide force constants as a function of time for five base pairs near the loop regions (base pair 21 to 25). shows that region-2 and region-3 of the H2B tail mainly interact with the DNA. Next, we characterize the role of salt during the collapse of the tail. Fundamentally, the nucleosome core particle is a polyelectrolyte complex. The salt concentration in solution is well known to modulate the stability of these highly charged complexes. Indeed, upon formation of a highly charged complex, counterions are released in solution. Here, we examine if the same effect is true. SI-6 represents the variation of numbers of Na+ and Cl-1

4.5.2 Coil to Helix Transition of the H2B Tail Upon collapsing, residue number 16-23 of H2B form a stable helical structure. The time evolution of the secondary structure of the H2B tail (SI-9(a)) reveals helix formation after 2 μs that remains stable for the rest of the 5 μs simulation. Both experimental and computational studies have suggested helical conformations of the H3 and H4 tails with nucleosome assays35, 84. Similarly, residue number 10–21 of the H2B tail were predicted to form a α-helix85 conformation experimentally83, 86, 87. In agreement with


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experiments, we also find that residues 16-23 in the H2B tail have a very high helical propensity, as shown in SI 9(b). Hence, as the tail collapses, residues 16-23 in the H2B tail fold to form a compact helical structure. The helix formation can also modulate protein-protein and protein-DNA contacts within the NCP. In SI-10 we show variations of both protein-protein (SI 10 (a)) and protein-DNA (SI 10 (b)) contacts (distance between two heavy atoms ≤ 4 Å) of the H2B tail over the 5 μs trajectory. A slight increase in a number of contacts between the protein residues with the formation of helix occurs after 2 μs. Most importantly, after helix formation, the contacts between the DNA and non-helical region of the tail increase at ~4 μs, which coincides with the increased loop size of the DNA (see Figure 1(f)). Increasing contacts are associated with the formation of nearly 21 % new contacts between the DNA and LYS and ARG residues from the non-helical part (especially from α3 region) of H2B tail. In addition, the average contact numbers, as shown in Table-1, display significantly more contacts between the last six LYS and ARG residues from residue 27 to 31 of the H2B tail and the nucleosomal DNA. The presence of six positively charged residues next to each other with a large number of contacts with the DNA may play two different roles in regulating NCP stability and dynamics. Being positively charged, these LYS and ARG residues can increase stability of the complex by creating more contacts with the DNA. At the same time, being neighbors, these covalently linked (close proximity) LYS and ARG residues can create an excess positive charge (overcharging) near DNA, destabilizing the nucleosome. A delicate balance between the two opposite effects may play a key role to modulate both the stability and plasticity of the NCP. Incidentally, the above mentioned LYS and ARG residues are a part of the histone H2B repression (HBR) domain88 (residue 2431). The HBR domain is known to play a crucial role in gene expression89, chromatin assembly7, and DNA damage and repair90. Considering the relevance of electrostatic interactions between the DNA and the histone proteins for the stabilization of the NCP, our study shows how the HBR domain may control nucleosome dynamics and DNA accessibility at the molecular level. A 2D PMF, as shown in SI-9(c), considering the protein-protein contacts of the helical region and histone-DNA contacts of the non-helical region of the H2B tail, depicts three clear minima. The first minimum corresponds to a lower number of contacts both between the helical protein residues and also between histone and DNA. Then, due to helix formation, a neighboring minimum appears with more protein-protein contacts. Finally, the helix formation modulates the contacts between the DNA and nonhelical regions of the H2B tail and creates a minimum with more histone-DNA contacts. This suggests that the coil-to-helix transition of the H2B tail has a close connection with the formation of a loop near SHL-5 region of the DNA. 4.6 Molecular Level Interaction between DNA and the H2B Histone Tail Being negatively charged, DNA mainly interacts with the LYS and ARG residues of histone proteins. In the H2B tail, there are 10 LYS (K5, K11, K12, K15, K16, K20, K23, K24, K27, and K28) and 2 ARG residues (R29 and R30). While the position of

LYS5 is closest to region-1, region-2 (or the central region) contains LYS11 to LYS20. The remaining LYS23 to LYS28 and ARG29 and ARG30 are in region-3. Now, to study the interactions of the DNA with LYS and ARG in different regions of the H2B tail, we have computed the time-dependent solvent accessible surface area (SASA) of the residues mentioned above. Detailed results can be found in SI-11. Briefly, the large surface area suggests a lack of or very little interaction between the DNA and LYS5. Variation of SASA further suggests that although the LYS residues from region-2 and region-3 interact strongly with the DNA, the nature of their interactions vary widely. LYS residues from region-2 interact mainly with the DNA backbone without going into the minor groove. By making contacts with the phosphate groups of the DNA backbone, these relatively mobile LYS residues stabilize the nucleosome. Incidentally, LYS 11, LYS 12, LYS 15 and LYS 1689, 91, 92 are important epigenetic markers of the H2B tail. In contrast, LYS23, LYS24, LYS27, LYS28, ARG29 and ARG30 move deeply into the SHL-5 minor groove after 2 μs. These above mentioned positively charged residues create a highly charged as well as crowded environment around the DNA. This can lead to loop formation near SHL-5 due to the overcharging of DNA. Hence, our data suggests that the interactions of H2B tail with the DNA are highly complex. Even within the same histone tail, the interaction with the nucleosomal DNA can vary from no interaction to stabilization to destabilization,

Figure 7. Relative dissociation constant, 𝐾𝑑𝑟 , of each base pair of the nucleosomal DNA. Variation of 𝐾𝑑𝑟 over the nucleosome is also incorporated in the inset in a colored fashion. The base pair with 𝐾𝑑𝑟 equal to 1 has the highest dissociation constant. The blue color near SHL 5 and SHL-5 are associated with the higher localized dissociations of these regions of nucleosome. which may act like an ideal molecular switch to control both the stability and plasticity of NCP. 4.7 Elastic Properties of Nucleosomal DNA The ability of highly mobile histone tails to locally deform the nucleosome is expected to be associated with the local elastic properties of DNA. Here, we characterize the elastic properties of the nucleosomal DNA at the base pair level. Average diagonal force constants of the six elastic parameters of DNA (roll, tilt, twist, rise, shift and slide) are shown in SI -13 for each base pair. In addition, Figure 6 (a) reveals the nucleosomal DNA backbone illustrating the local variation of rise force constant (from red (low K) to blue (high K)) averaged over 5 μs. Irrespective of base



pairs, the force constants for translational parameters (rise, shift, and slide) are significantly higher compared to rotational parameters (roll, tilt, and twist). This agrees well with previous simulation studies93, 94. A comparison between the different regions further suggest that SHL-6, SHL-5, and SHL-2 are the three most flexible regions of the nucleosome with very low force constants (red regions in Figure 6 (a)). NMA also reveals that SHL-6 and SHL-5 are the most dynamic regions for breathing and lateral stretching of nucleosome respectively. Ong and co-workers30 have reported a crystal structure of the NCP with an extreme kinking around both SHL±5 and SHL±2 regions. Very recently, Liu et al. also noticed loop formation near SHL±2 regions of NCP

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Skorupa et al96 found that twist oscillations in circular DNA are directly related to its intrinsic fluctuations and not due to proteinDNA interactions. In SI-14, we show the variation of twist force constant for base pairs from 21 to 25 over time. The sudden decrease in the twist force constant after 2 μs suggests that the histone-DNA interactions have profound effect on DNA elasticity and are not due to intrinsic fluctuations in this case. 4.8 Localized Dissociation of DNA Bases Bulging of nucleosomal DNA is also closely related with the higher dissociation rate of the DNA bases. Here, we characterize relative values of the dissociation constant of the DNA at base

Figure 8. Free energy profile as a function of radius of gyration, Rg, for the unwrapping of the nucleosome. (b) Average number of bound base pairs with the histone protein core as a function of Rg. (c) Symmetrical and (d) Asymmetrical pathways of Nucleosome unwrapping. Two different sets of simulations have been carried out so far with different starting configurations of the nucleosome core particle: the final configuration after 5 μs simulation (S2) and the initial crystal structure (S1). in the presence of chromatin remodeler snf2 using cryo-EM 32. Although, it is well known that the elastic properties of DNA depends on its sequence93, 95, all these studies suggest that perhaps SHL±5 and SHL±2 are the most easily deformable sites of nucleosome due to the proximity with the histone tails, as well as low force constants of base pairs in these regions. Next, in Figure 6 (b), we show the variation of force constants as a function of time (only rise, shift, and slide force constants are shown due to their higher values) for five base pairs from 21 to 25. These five base pairs are primarily involved in loop formation around SHL-5. As the histone tail collapses at ~2 μs, the force constants for the base pairs in SHL-5 region also suddenly decrease, making the region more flexible through formation of a loop or bulge. Recently

pair level. As relative values are more important here than the actual dissociation constant, in Figure 7, we have shown the relative dissociation constant (𝐾𝑑𝑟 ) with respect to 𝐾𝑚 [𝐾𝑑𝑟 = 𝐾𝑑 ] . 𝐾𝑚 is the dissociation constant of the base pair having the 𝐾 𝑚

highest dissociation rate. Hence, a base pair with 𝐾𝑑𝑟 equal to 1 has the highest dissociation constant. 𝐾𝑑𝑟 of each base pair has been calculated over 2000 snapshots from the last 2 μs of the trajectory. The average values are shown in the Figure 7. The dissociation constants of DNA bases are also included in the inset from red (low dissociation) to blue (high dissociation)). A wide range of 𝐾𝑑𝑟 values again indicates the heterogeneous binding affinity of DNA bases. Notably, DNA base pairs near the central


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dyad region have low dissociation constants. Hall and co-workers97 were able to determine a histone-DNA interaction map at a nearly single base-pair resolution along the DNA sequence of nucleosome from single molecule experiments. They found that the central dyad region and ± 40 from the dyad are the three most strongly binding sites of the nucleosome. Thus, our results agree qualitatively well. Furthermore, the alternating red/light red or white indicates relatively strong and weak binding sites of nucleosomal DNA respectively. This agrees well with the step-wise release of nucleosome under tension71. As much higher force is essential to unwrap the strongly bound regions of the nucleosome, under tension it unwraps in a stepwise manner. Most notably, the base pairs near the loop or bulge regions (blue regions in the inset) have a nearly 5 times higher dissociation constant. Around SHL-5 the values are even higher. Hence, condensation of H2B tail onto the SHL-5 region destabilizes the DNA and enhances the dissociation rate of the DNA bases locally. 4.9 Unwrapping of Nucleosomal DNA Thus, by altering the electrostatic environment (increasing the salt concentration to 2.0 M), condensation of the long and highly charged H2B histone tail enhances the dissociation rate of nucleosomal DNA locally. Such localized deformation of the complex may play a critical role at least in the early stage of its unwrapping. For instance, depending on the binding strength, the dissociation of DNA starts either from extreme terminal region or from the central dyad region. Weak binding makes the exit/entry regions of the nucleosomal DNA a potential unwrapping site97. On the contrary, loop or bulge formation near the dyad region can also facilitate the dissociation of the nucleosome, but it initiates from the central region18, 19.

increases, but in the early stage of unwrapping here, the two extreme end regions (exit/entry) of nucleosomal DNA remain attached to the histone core. In contrast, for the canonical structure of the NCP (S1), unwrapping initiates from the exit and entry regions of the nucleosomal DNA (Figure 8 (d)), without the formation of any loop/bulge. While for S2 both the end regions of the nucleosomal DNA unwrap simultaneously, for S1 only one end moves away from the histone core. Previously, Zhang and co-workers48 also explored unwrapping of the same crystal structure of nucleosome using US. Consistent with our finding, they also reported that unwrapping happens asymmetrically from the end regions of nucleosomal DNA without any loop formation. In contrast, recently, using time resolved (TR) SAXS and FRET techniques it was found that salt-induced disassembly of the NCP can undertake multiple pathways of unwrapping—symmetric and asymmetric20, 98. The asymmetric pathway allows for the formation of an intermediate 'teardrop' shape. To explore nucleosome unwrapping pathways in more detail, we also compute angle (A) between the nucleosome exit and entry regions. Variation of A over the US trajectories for S1 and S2 are shown in SI- 15. There is a much larger variation of A for the S2 pathway than that for S1. Hence, dependent on the starting configuration of the nucleosome, we find that the initial stages of unwrapping can occur both symmetrically and asymmetrically. To understand whether bulging is essential for symmetric unwrapping or not, further free energy calculations including variable starting structures of the NCP from the different stages of bulging are essential. Furthermore, many hidden reaction coordinates may exist and must be chosen carefully to sample the free energy surface.

To further explore the pathway nucleosome unwrapping, we have employed US considering the radius of gyration of the DNA (Rg) as a reaction coordinate. Two different sets of simulations have been carried out so far with different starting configurations of the nucleosome core particle: the initial crystal structure (S1) and the final configuration after 5 μs simulation with the presence of loops near SHL±5 (S2). The detailed PMFs (potential of mean force) obtained from S1 and S2 simulations are shown in Figure 8 (a). To begin with, the free energy cost for the S2 simulation is relatively lower than S1. The first minimum in the PMF for S2, indicated by the green arrow corresponds to a Rg of DNA around ~ 45 Å. With further unwrapping a neighboring minimum appears at ~ 46.5 Å, again indicated by green arrow. The second minimum is absent for the S1 simulation. In addition to the PMFs, the average number of bound DNA bases as a function of Rg is also depicted in Figure 8 (b). A step-wise dissociation of DNA bases with increasing Rg both for S1 and S2 is evident. The step-wise unwinding of nucleosomal DNA is consistent with other simulation and experimental studies48, 71. Furthermore, a sudden drop of ~ 5 bound DNA bases around 45 Å and 46.5 Å, coincide well with the presence of two minimum, as mentioned above. The corresponding snapshots of these two minima along with initial and final configurations for S2 and S1 simulation are shown in Figure 8 (b) and (c) respectively.

5. CONCLUSIONS

Two very very different unwrapping pathways of S1 and S2 are observed, one asymmetric and one symmetric. For S2, the unwrapping starts from the SHL ±5 regions (Figure 8(c)). As the loops gradually diffuse towards the end of the DNA, the loop size

Supporting Information. Supplementary figures concerning structural features of nucleosomal DNA, salt concentration in SHL-5 region, targeted molecular dynamics simulations, second-

Herein, we perform 5μs long MD simulations of the nucleosome core particle in 2.0 M NaCl solution. After 5μs we found that condensation of the H2B N-terminal tail into the minor groove of DNA promotes the formation of a 10bp loop of nucleosomal DNA. Upon condensation, counterions are released in solution. As the loop forms, this increases the flexibility of the DNA in this particular region. The structure of the loop agrees well with experimental observations7, 80. Along with bulging, we also identify a wave-like motion of the nucleosomal DNA due to the inward breathing of the DNA tail region (SHL-6) and outward stretching of the SHL-5. While inward breathing of SHL-6 is related with the stabilization effect of the H2A C-terminal tail, the destabilization by the H2B N-terminal tail is due to the outward stretching of SHL-5 region of the nucleosomal DNA. Hence, the interactions between the DNA and the histone tails are highly dynamic in nature. In addition, the nature of this interaction may be governed by the salt concentration in solution. The combination of both the stabilizing and the destabilizing effects of the H2A and the H2B tails respectively, creates a wave-like motion on the histone octamer surface. In other words, a combination of stabilization and destabilization effects of H2A and H2B tails respectively makes the bulge formation free-energetically less expensive.



ary structure of H2B tail, protein-protein and protein-DNA contacts, as well as solvent accessible surface area surrounding charged amino acids in H2B tail. Acknowledgements. This research was supported, in part, by the NSF through TeraGrid resources under grant number TGCHE130099 and a grant of computer time from the City University of New York High Performance Computing Center under NSF Grants CNS-0855217, CNS-0958379 and ACI-1126113. S. M. L. acknowledges start-up funding received from College of Staten Island and City University of New York. K.C. thanks to P. K. and Phu Tang for valuable suggestions during manuscript preparation. This research was also indirectly supported by grants from the NIH (R15EB020343-01A1) and by the NSF through grant 1506937. Anton 2 computer time was provided by the Pittsburgh Supercomputing Center (PSC) through Grant R01GM116961 from the National Institutes of Health. The Anton 2 machine at PSC was generously made available by D.E. Shaw Research.

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Table-1. The average contact numbers between the LYS and ARG residues of H2B N-terminal tail and the nucleosomal DNA. Corresponding data for the initial crystal structure is also given for comparison.

Location

Residue

Simulation

Crystal

Region-1

LYS5

0.27 (0.03)

0.00

LYS11

2.90 (0.18)

2.00

LYS12

4.38 (0.29)

2.00

LYS15

1.03 (0.58)

1.00

LYS16

2.98 (0.05)

3.00

LYS20

0.12 (0.01)

0.00

LYS23

0.10 (0.04)

0.00

LYS24

0.54 (0.03)

6.00

LYS27

4.75 (0.08)

2.00

LYS28

6.36 (0.10)

3.00

ARG29

4.23 (0.26)

4.00

ARG30

2.57 (0.34)

1.00

LYS31

6.42 (0.41)

3.00

Region-2

Region-3


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Molecular Mechanism for the Role of the H2A and H2B Histone Tails

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