Multiscale physical effects of CpG methylation on DNA mechanics, nucleosome wrapping, and chromatin condensates

DNA methylation at CpG dinucleotides is a central epigenetic modification linked to transcriptional repression and heterochromatin, yet how it contributes to chromatin compaction beyond recruiting methyl-binding proteins remains unclear. Here, we integrate single-molecule force spectroscopy, FRAP, and nanorheology to show that CpG methylation alters chromatin organization across scales. CpG methylation modestly stiffens and extends DNA, while weakening both outer- and inner- turn nucleosome DNA wrapping. Despite these local destabilizing effects and similar thresholds for salt-induced phase separation, methylated chromatin condensates exhibit reduced internal mobility and increased viscoelasticity, indicating denser internal organization. Finally, CpG methylation cooperates with H3K9me3 to promote HP1α-mediated chromatin condensation, despite not altering HP1α affinity for naked DNA. These findings indicate that HP1α does not sense CpG methylation through direct recognition of the DNA modification itself, but rather through methylation-dependent changes in chromatin architecture and material properties. Together, our results support a multiscale physical model in which CpG methylation contributes to heterochromatin organization by tuning DNA rigidity, nucleosome wrapping energetics, and condensate material properties.