Sensitive Sequence Dependency of energy.
Our physics-based method is used with a template from a high-resolution crystal structure (25) to predict the nucleosome formation energy, En ? El (where En is the energy of the particular sequence on DNA that is bent to fit the nucleosome and El is the energy of the same sequence on ideally straight B-DNA, termed “linear DNA”). El is used as reference energy to eliminate the dependence on trivial effects such as the number of hydrogen bonds made between the two strands. Fig. 2 compares our predicted energy with the in vitro experimental occupancy for sequence positions from 187,000 to 207,000 in yeast chromosome 14 (26, 27); it shows clear negative correlation between the two data sets: The in vitro nucleosome occupancy is higher at the sequence positions where the nucleosome formation energy is lower. Position-dependent correlations (Fig. 2A) show that the correlation is generally uniform along the sequence although there are regions with high correlation (195,000–199,000) and others with low correlation (187,000–191,000). Fig. 2B depicts the in vitro experimental nucleosome occupancy and computed nucleosome formation energies. The overall correlation between the experimental and modeled data are ?0.612. Fig. S2 shows the ab initio nucleosome occupancy profiles obtained when energies are converted to probabilities of occupancy using the Boltzmann formula (SI Materials and Methods).
Nucleosome formation energy and the in vitro occupancy profile for sequence positions from 187,000 to 207,000 with single-position increments in yeast chromosome 14. (A) The position-dependent negative correlation of the in vitro profile and nucleosome formation energy is shown using windows of 2,000 (violet) and 4,000 (brown) bp. The nucleosome formation energy is the difference between the energy of DNA bent as if on a nucleosome and a linear B-DNA (one type of right-handed DNA conformation) structure with the same sequence, i.e., (En ? El). Calculations were performed using the AMBER99-bsc0 force field, an implicit electrostatic solvent description, and PDB 1kx5 and linear B-DNA templates. (B) The nucleosome formation energy (cyan) and experimental profile (red) plotted along the sequence 
. The overall correlation of the nucleosome formation energy and in vitro profile is ?0.613.
The effect off DNA Methylation.
Although methylation does not occur in yeast, we aimed to study its enhanced physical effect. Therefore, we methylated all C bases of our studied sequence (used in Fig. 2) at the 5 position (5Me-C). At first sight, the energy values of nucleosome formation (EnMe ? ElMe) (Fig. 3A) look very much like the corresponding energy values for normal DNA (En ? El). Closer examination shows that whenever the nucleosome formation energy of normal DNA is particularly large or small, the energy of 5Me-C DNA is less extreme. Thus, methylation moderates the sequence dependence of the nucleosome formation energy. Quantitatively, this moderating effect is reflected by the smaller SD of the formation energies for the methylated sequence compared with those of the normal sequence (43.0 and 52.1 kcal/mol, respectively). These observations are further supported by Fig. 3B showing how the effect of methylation on the nucleosome formation energy, ?EMe defined as ?EMe = (EnMe ? ElMe) ? (En ? El), is negatively correlated with (En ? El) with a correlation coefficient of ?0.584. Fig. 3 C and D plots the methylation energies for both linear and nucleosomal DNA and indicates that nucleosome methylation (EnMe ? En) and nucleosome formation energy (En ? El) are strongly anticorrelated [correlation coefficient (CC) = ?0.739], whereas the methylation energy change on the linear form (ElMe ? El) has only weak anticorrelation with (En ? El) (CC = ?0.196). From this we infer that the effect of methylation on the nucleosome formation energy arises from methylation of the nucleosomal form and not the linear form. Additional correlation plots are presented in Fig. S3.