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Title: Free energy profiles for unwrapping the outer superhelical turn of nucleosomal DNA}
Article:
Abstract:
The eukaryotic genome is packaged into a nucleus in the form of chromatin. The fundamental structural unit of chromatin is a protein-DNA complex, the nucleosome, where 146 or 147 base pairs of DNA wrap 1. 75 times around a histone core. To function in cellular processes, however, nucleosomal DNA must be unwrapped. Although this unwrapping has been experimentally investigated, details of the process at an atomic level are not yet well understood. Here, we used molecular dynamics simulation with an enhanced sampling method to calculate the free energy profiles for unwrapping the outer superhelical turn of nucleosomal DNA. A free energy change of about 11. 5 kcal/mol for the unwrapping agrees well with values obtained in single molecule experiments. This simulation revealed a variety of conformational states, indicating there are many potential paths to outer superhelicdal turn unwrapping, but the dominant path is likely asymmetric. At one end of the DNA, the first five bps unwrap, after which a second five bps unwrap at the same end with no increase in free energy. The unwrapping then starts at the other end of the DNA, where 10 bps are unwrapped. During further unwrapping of 15 bps, the unwrapping advances at one of the ends, after which the other end of the DNA unwraps to complete the unwrapping of the outer superhelical turn. These results provide insight into the construction, disruption, and repositioning of nucleosomes, which are continuously ongoing during cellular processes.
Introduction:
The nucleosome is the fundamental structural unit of chromatin and is composed of histone proteins and DNA. Its crystal structure revealed that within the nucleosome, 146 or 147 bps of DNA wrap 1. 75 times around an octameric histone core[1]. However, for proper gene function, such as transcription, duplication and repair, this nucleosomal DNA must be unwrapped, at least partially. Single-molecule experiments showed that the energetics of protein-DNA interactions depend to varying degrees on the protein-DNA contact positions. FRET experiments[2–6] showed that in the absence of salt, the outer superhelical turn of DNA( hereinafter referred to as “outer DNA”) within the nucleosome repeats spontaneously and repeatedly unwrapping and rewrapping. Rewrapping is about 5 times faster than unwrrapping, which is within about 10 to 50 ms[7, 8]. Single-molecule experiments in which the two ends of the DNA in a nucleosomal array[2, 5] or individual nucleosome[6] are mechanically pulled in opposite directions demonstrated that the outer DNA is more easily unwrapped than the inner super-helical turn. Later, after performing a DNA unzipping experiment, Wang’s group reported the energetics of histone-DNA interactions within the nucleosome[4]. This single molecule experiment showed that distinct histone-DNA interactions occur at intervals of about five bps, indicating that the two phosphate backbones of double-stranded DNA interact independently with the histones. It is difficult, however, to determine at the atomic level details such as which interaction is lost first during unwrapping and then second and so on. All-atom molecular dynamics( MD) simulations have provided detailed information about the dynamics of the nucleosome[9–16]. Ettig et al. were the first to carry out all-atom MD simulations of the nucleosome to investigate unwrapping energetics[17] and Gribkova et al. calculated histone-DNA binding energies with a concept similar to this study[18]. Consistent with the unzipping experiment, Ettig et al. observed a 5-bp periodicity. However, because of the limited amount of time that could be computationally processed, a large force was imposed on the DNA during the unwrapping process, which caused unrealistic distortion of the DNA and histones. Coarse-grained MD is an attractive method because it enables processing time spans not achievable in all-atom simulations using current state-of-the-art computer systems. Kenzaki and Takada performed a coarse-grained MD simulation that captured residue-level structural details, and exhibited spontaneous unwrapping and rewrapping of the outer DNA[19]. In addition, their simulation showed that the unwrapping is mostly asymmetric, with one end unwrapping while the other end remains wrapped. Using a similar coarse-grained model, Lequieu et al. calculated the tension-dependent free energy surface and found that the contributions of histone tails to DNA stability differed between the H3/H4 and H2A/B tails[20]. Whereas H3/H4 contributes more to the stability of the outer DNA, H2A/B stabilizes the inner DNA. The results of these simulations are consistent with the experimental findings[2–6]. However, coarse-grained MD requires adjustable parameters for the target of interest and is still less reliable in dynamics than all-atom simulations with an explicit solvent model due to the nature of the simplification. Here, we carried out MD simulations with an enhanced sampling method to obtain conformational ensembles. We report the free energy profiles for unwrapping the outer-turn of nucleosomal DNA. The obtained profiles based on the ensembles show that unwrapping of the outer-turn costs about 11. 5 kcal/mol. Comparison of the numbers of unwrapped base pairs at the two ends of the DNA shows that the unwrapping occurs asymmetrically, though the nucleosome has a pseudo-symmetric structure and a symmetric DNA sequence.
Discussion:
We carried out simulations of DNA unwrapping from nucleosomes to obtain the free energy profiles. Nucleosomes have a pseudo-symmetric structure, and we used a symmetric DNA sequence. Consequently, there are two sets of interactions, as nucleosomes contain two copies for each of histones and two DNA ends. For instance, H3a-end2 and H3b-end1 contacts are expected to be very similar to one another. In fact, the contact profile of H3a and DNA end2( Fig 4A) is very similar to that of H3b and DNA end1( S4A Fig). This was also true for interactions between other histones and DNA, and demonstrates that the sampling in this study was sufficient to analyze these data. We considered free energy for the outer DNA unwrapping in two stages. In each stage, we observed unusual DNA stretch at longer end-to-end distances in ABMD simulations and therefore excluding those regions before the umbrella samplings. After checking the DNA conformations obtained by the umbrella samplings, we estimated the free energy cost of unwrapping a total of 44 bps, which corresponds to nearly the entire outer DNA, to be about 11. 5 kcal/mol( 5. 5 kcal/mol in stage 1 and 6 kcal/mol in stage 2). The full unwrapping free energy has been estimated about 40 kcal/mol using an integrating model of a coarse-grain and fine-grain models[24] or a theoretical model[25]. The calculated cost of 11. 5 kcal/mol for unwrapping 44 bps in this study is about a quarter of the cost for the full unwrapping, indicating the obtained value is quite reasonable compared with the reported values. The corresponding free energy value obtained by experiments ranges about 5 to 12 kcal/mol[2, 6, 26, 27], which likely depends on interpretation of the experimental results. Because DNA unwrapping starts at one of the DNA ends( Fig 6), gaps in the free energy curves are well explained by the loss of interaction between the DNA and histones. However, there is a discrepancy between our model and a previous model[28]. Forties et al. proposed a quantitative theoretical model of DNA unwrapping and disassociation from the histone octamer[28]. By calibrating the model to a high-resolution nucleosome unzipping experiment[4], they successfully explained the differences in the rate of nucleosome disassembly caused by hMSH2-hMSH6 between two DNA sequences and between histone modification states. In their model, free energy increases stepwise as unwrapping advances, which is consistent with our findings. However, they found that the cost to unwrap 20 bps was about 1 kBT, which is 10 times smaller than our calculated value. Most likely, this discrepancy arises from the following differences. First, unwrapping and rewrapping rates are highly dependent on the salt concentration[3, 29, 30]. Second, in the simulation, we counted the number of unwrapped bps from both DNA ends. In the unzipping experiment, by contrast, the model building always occurred from one DNA end. Third, the definition of unwrapping is context dependent. They assumed that the number of broken base pairs equals the number of base pairs unwrapped from the nucleosome; however, it is quite possible that there is a gap in time between the unwrapping and the dissociation, which would lead to the observed small free energy for unwrapping 20 bps. In an alanine mutation experiment, Ferreira et al. showed that each residue within the H3 αN helix revealed different aspects of nucleosome dynamics[31]. We found that the measured effects on DNA wrapping( see S1 Table in the reference) are well correlated with the contact ratio between the protein and DNA( Fig 4A). For example, R49A caused the largest decrease in the FRET ratio between the two DNA ends, followed by R52 and R53. Mutation of V46, L48 or K56 had a moderate effect. This trend is in good agreement with the contact ratios observed in the present study and in our earlier MD study[12], which indicates these residues are directly involved in protein-DNA interaction to stabilize the nucleosome. Interestingly, alanine mutation of L48 or Q55, which have little or no direct contact with DNA, destabilized histone octamer formation[31]. Most likely, these mutations destabilize the H3 αN helix causing favorable protein-DNA interactions to be lost. Thus, these residues are well conserved among eukaryotes. During stage 2, we observed that T76 and R77 of H2A and Y39 and K43 of H2B gradually lose direct hydrogen bonds to the DNA. Consequently, the free energy curve showed a more gradual increase during the unwrapping process than during stage 1. However, most interactions with H2A and H2B were well maintained in stage 2 as shown in S3C and S3D Fig. Thus, the unwrapping process advances with a subtle increase in free energy. Interesting, the residues( R29, R32, R77 of H2A and Y37, K43 G50 and I51 of H2B) which are in contact with DNA as well as H2A or H2B in the form of the H2A-H2B heterodimer are well conserved among eukaryotes[32, 33] though H2A and H2B histones are the most sequence variable among all histones. It should be noted that all simulations in this study were carried out on nucleosomes whose histone tails were truncated because our preliminary calculations showed that positively charged histone tails interact with the negatively charged DNA backbone, and sampled conformations are highly dependent on the initial conformations. This treatment has a possibility of underestimating the unwrapping free energy of mono-nucleosome though the tails have more important role in inter-nucleosome interaction as discussed below. Histone tails are known to mediate interactions between nucleosomes, enabling them to fold into the chromatin solenoid[34], or they may mediate nucleosome assembly[35–38]. In particular, H4 tails probably play the most important role in inter-nucleosome interaction. All atom MD simulations by Langwoski’s group showed that H4K16 acetylation drastically reduces H4 tail-acidic patch of the stacked nucleosome and increases the tail conformational diversity[39]. Ishida & Kono computationally show that the diversity of nucleosome-nucleosome stacking depends on the way of H4 tail interactions[40]. These results indicate the impact of post-translational modification to chromatin conformation. At mono-nucleosome level, what is the role of tails? At least in terms of thermodynamic view, thermal stability of the entire nucleosome changes little with or without the tails at mono-nucleosome level. Reported theoretical[24] and experimental[41, 42] works show that the tails have little effect( at most 1. 1 kcal/mol) on thermodynamic stability of the entire nucleosome, but the tails can affect the partial unwrapping kinetics of the DNA. For instance, in a single molecule study, Brower-Toland et al. showed that tail truncation reduces the overall affinity of histones for outer DNA[43]. Interestingly, in SAXS and FRET experiments, Andersen et al. showed that truncation of the H3 or H4 tail has opposite effects. In their FRET studies, whereas H3 truncation induces dissociation of DNA, H4 truncation stabilizes the wrapped conformation to a greater degree than the intact nucleosome[44]. When Biswas et al. used all-atom MD simulations to examine nucleosome dynamics with and without the histone tails, they found that tail truncation destabilized the histone core[11]. Consistent with that finding, using an implicit solvent model, Erler et al. found that attractive interactions between positively charged Lys and Arg residues in the histone tails and negatively charged phosphate groups in the DNA stabilized the nucleosome structure[45]. In addition, a MD simulation carried out by one of the authors showed that the H3 N-terminal tail and H2A C-terminal tail both interact with linker DNA[13], and that the latter stabilizes the closed form of the linker DNA. Finally, biochemical[46] and MD[16, 47–49] studies showed that acetylation of the tails increases the α-helix content, and one of studies indicated that acetylation of the H3 tail induces DNA dissociation from the histone core[47], which is consistent with a single molecule FRET experiment[29, 50]. Here, we summarize the unwrapping outer DNA process. The calculated free energy profile suggests that about 11. 5 kcal/mol are required for unwrapping of the first 22 bps from both DNA ends, which is consistent with the previously estimated values. The number of unwrapping bp differences between two DNA ends clearly exhibits DNA dissociation process is stochastic and asymmetrically advances up to the first 10 bps unwrapping, then unwrapping of another 10 bps follows at the other end. This process occurs once more to complete the unwrapping of the outer superhelical turn.
The eukaryotic genome is compactly packaged into the cell nucleus . Within the nucleus , DNA forms protein-DNA complexes called nucleosomes . The nucleosome is the fundamental structural unit of chromatin . It is composed of a histone octamer around which DNA wraps about twice . During cellular processes such as DNA transcription , replication and repair , the DNA must be unwrapped from the structure . This unwrapping has been investigated experimentally , but details of the process at the atomic level are not yet well understood . We carried out molecular dynamics simulations using an enhanced sampling method to obtain free energy profiles for unwrapping the outer superhelical turn of the nucleosomal DNA . Our analysis shows that some energy cost is required to unwrap the first 5 bps at one end of the DNA , after which a second 5 bps unwrap with no increase in free energy . The unwrapping then proceeds at the other end of the DNA for up to 10 bps . In this way , DNA unwrapping occurs asymmetrically from one end . These findings provide insight into how DNA dynamics are related to gene regulation .