Figure 1.
Mutation sites and thermodynamic cycle.
(A) Example of two extreme cases of MeC positioning (spheres) along the nucleosomal DNA with respect to the histone core (in grey) used in this work: (top) the CpG step minor groove faces the histones; (bottom) the CpG major groove faces the histones. The methyl carbon is colored in green. The images on the right show a lateral view of the DNA and the protein as seen from the solvent. (B) Diagram of the thermodynamic cycle used to extract the free energy variation (ΔΔGb (kJ/mol)) in nucleosome-DNA stability due to methylation of CpG steps. The calculations of the unbound reference state for the single mutations were performed on shorter DNA chains, using the nearest 3 neighbors of the CpG steps in the nucleosome sequence, and 4 bases to cap the duplex termini (5′-CGAT and TACG-3′). As the histone proteins are not affected by the cytosine methylation in the unbound state, they were not included in the calculations related to such state. In case of multiple methylations, large fragments of different length were used (further details in SI material). The free energy difference associated with the removal of the methyl group is calculated using discrete thermodynamic integration (DTI). The methyl group is shown as a green sphere.
Table 1.
List of the molecular dynamics simulations performed in this work.
Figure 2.
Differential binding free energy (ΔΔG bind. (kJ/mol)) of nucleosomal DNA.
(A) Differential binding free energy values for single and multiple methylated CpG steps with respect to un-methylated sequences; methylations at major groove positions (blue) are better tolerated than at minor groove positions (light orange). Multiple methylations show a cumulative effect on the differential binding energy, following the trend of single methylations (methylation in major groove in dark blue, mixed major-minor groove in green, and minor groove in dark red). The exact location of each mutation is listed in Table S1 in Text S1. (B) Correlation between the variation in free energy (ΔΔG (kJ/mol)) and the variation in elastic energy (ΔΔE (kJ/mol)) for single (black dots) or multiple (red squares) methylated CpG steps in the nucleosomal DNA.
Figure 3.
Methylated and non-methylated DNA elastic deformation energies.
(A) Distribution of deformation energies for 147 bp-long random DNA sequences with CpG steps positioned every 10 base steps (one helical turn) in minor (red and dark red) and major (light and dark blue) grooves respectively. The energy values were rescaled by the slope of a best-fit straight line of figure 2, which is 0.23, to map the elastic deformation energies to ΔΔG. Methylation of the DNA causes a higher energetic cost for nucleosome formation, especially when the MeCpG steps are positioned in the minor groove facing the histones (dark red). (B) Correlation between the additional energetic cost due to methylation (〈EMeth〉-〈E〉, kJ/mol) to form a nucleosome and the phasing of CpG steps respect to the histone (the zero is the reference phase position in which the CpG minor groove directly faces the histones). The cartoons illustrate two extreme positions of the methyl groups with respect to the histone core, which translate into a change in the rotational phase: the position of the methyl group, pointing to the solvent in 0 (red dot) and to the histones in 5 (blue dot).
Figure 4.
Examples of predicted impact of methylation on gene activity.
(A) Predicted impact of methylation on the accessibility of transcription factors. The black line corresponds to the experimental population of nucleosomes (data from Deniz et al. [45]) in a portion of the yeast XV chromosome (close to the putative transcription start site of a gene, located at base pair 201879). The blue line represents the theoretical nucleosome probability, predicted from the elastic deformation energy for un-methylated DNA, and the red line shows the resulting nucleosome probability after CpG methylation. A vertical box highlights the binding position of the ABF1 transcription factor. (B) Example of the impact of methylation in nucleosome phasing. The blue line corresponds to the probability to wrap a nucleosome in a region of yeast chromosome VIII, where we have experimentally detected a stable nucleosome (Deniz et al. [45]) next to the PHD1 recognition box. The red line illustrates the nucleosome probability profile found when the sequence is methylated. We have depicted the associated change in translational positioning in the cartoons embedded in the figure: minor groove facing the histones in green, major groove facing the histones in red, and the PHD1 recognition box as yellow balls.