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Abstract Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that methylates histone H3 at Lysine 27. PRC2 is critical for epigenetic gene silencing, cellular differentiation and the formation of facultative heterochromatin. It can also promote or inhibit oncogenesis. Despite this importance, the molecular mechanisms by which PRC2 compacts chromatin are relatively understudied. Here, we visualized the binding of PRC2 to naked DNA in liquid at the single-molecule level using atomic force microscopy. Analysis of the resulting images showed PRC2, consisting of five subunits (EZH2, EED, SUZ12, AEBP2 and RBBP4), bound to a 2.5-kb DNA with an apparent dissociation constant ($K_{\rm{D}}^{{\rm{app}}}$) of 150 ± 12 nM. PRC2 did not show sequence-specific binding to a region of high GC content (76%) derived from a CpG island embedded in such a long DNA substrate. At higher concentrations, PRC2 compacted DNA by forming DNA loops typically anchored by two or more PRC2 molecules. Additionally, PRC2 binding led to a 3-fold increase in the local bending of DNA’s helical backbone without evidence of DNA wrapping around the protein. We suggest that the bending and looping of DNA by PRC2, independent of PRC2’s methylation activity, may contribute to heterochromatin formation and therefore epigenetic gene silencing. 

The forces that stabilize membrane proteins remain elusive to precise quantification. Particularly important, but poorly resolved, are the forces present during the initial unfolding of a membrane protein, where the most native set of interactions is present. A high‐precision, atomic force microscopy assay was developed to study the initial unfolding of bacteriorhodopsin. A rapid near‐equilibrium folding between the first three unfolding states was discovered, the two transitions corresponded to the unfolding of five and three amino acids, respectively, when using a cantilever optimized for 2 μs resolution. The third of these states was retinal‐stabilized and previously undetected, despite being the most mechanically stable state in the whole unfolding pathway, supporting 150 pN for more than 1 min. This ability to measure the dynamics of the initial unfolding of bacteriorhodopsin provides a platform for quantifying the energetics of membrane proteins under native‐like conditions.

 

The forces that stabilize membrane proteins remain elusive to precise quantification. Particularly important, but poorly resolved, are the forces present during the initial unfolding of a membrane protein, where the most native set of interactions is present. A high‐precision, atomic force microscopy assay was developed to study the initial unfolding of bacteriorhodopsin. A rapid near‐equilibrium folding between the first three unfolding states was discovered, the two transitions corresponded to the unfolding of five and three amino acids, respectively, when using a cantilever optimized for 2 μs resolution. The third of these states was retinal‐stabilized and previously undetected, despite being the most mechanically stable state in the whole unfolding pathway, supporting 150 pN for more than 1 min. This ability to measure the dynamics of the initial unfolding of bacteriorhodopsin provides a platform for quantifying the energetics of membrane proteins under native‐like conditions.