Single-molecule micromanipulation studies of methylated DNA

Abstract

Cytosine methylated at the 5-carbon position is the most widely studied reversible DNA modification. Prior findings indicate that methylation can alter mechanical properties. However, those findings were qualitative and sometimes contradictory, leaving many aspects unclear. By applying single-molecule magnetic force spectroscopy techniques allowing for direct manipulation and dynamic observation of DNA mechanics and mechanically driven strand separation, we investigated how CpG and non-CpG cytosine methylation affects DNA micromechanical properties. We quantitatively characterized DNA stiffness using persistence length measurements from force-extension curves in the nanoscale length regime and demonstrated that cytosine methylation results in increased DNA flexibility (i.e., decreased persistence length).In addition, we observed the preferential formation of plectonemes over unwound single-stranded "bubbles" of DNA, under physiologically relevant stretching forces and supercoiling densities. The stiffness and high structural stability of methylated DNA is likely to have significant consequences on the recruitment of proteins recognizing cytosine methylation and DNA packaging. Statement of SignificanceDespite countless structural and functional studies of DNA methylation, a key epigenetic mark in higher organisms, research towards the understanding of DNA intrinsic structural properties in the context of methylation layout representing different epigenetic landscapes is still in its initial stage. We utilize single molecule spectroscopy to analyze the effect of sparse symmetric and asymmetric 5-mC modification on the mechanical stability of long double-stranded DNA. Our findings establish that at physiologically relevant forces and supercoiling densities increased DNA flexibility of non-CpG methylated DNA translates to the high structural stability.

The Impact of the HydroxyMethylCytosine epigenetic signature on DNA structure and function

“…Our experimental and theoretical results can be compared with previous data from the literature. Thus, the impact of methylation reducing DNA flexibility found here agrees well with a myriad of experiments from our own group and others [ 12 , 40 , 41 , 43 – 45 ], but disagrees with the results in a recent study by Zaichuk and Marko [ 42 ] that point in the opposite direction, probably due to a different experimental set-up where no full control of the placement and level of methylation exists. The impact of hydroxymethylation is less clear, and our results disagree with previous claims by Ngo et al [ 41 ] derived from circularization experiments using short oligomers.…”

“…Two main possibilities emerge: i) a change in the DNA physical properties reverting to the unmethylated situation that leads to nucleosome repositioning; ii) a direct protein-mediated mechanism. However, there is still a controversy on the physical/conformational effect of mC and hmC on DNA flexibility and its ability to wrap around histones to form nucleosomes [12,[40][41][42][43][44][45]. Using a variety of experimental and Government AGAUR (SGR2017-134 to M.O.…”

Single molecule characterization of the binding kinetics of a transcription factor and its modulation by DNA sequence and methylation

“…While one possible mechanism is steric hindrance by the methyl group, the effect observed for a methylated CpG outside the core binding site, and the fact that methylation can stabilize as well as destabilize the bound complex, suggest that part of the effect is related to changes in the local structure of DNA. Although similar crystal structures were reported for methylated and unmethylated DNA ( 72 ), methylation was shown to affect the flexibility ( 73 , 74 ), hydration structure ( 75 ), and sugar pucker conformations ( 76 ) of DNA. Molecular dynamics simulations showed that methylation increases, locally, the propensity of DNA toward different values of roll and propeller twist, and that the position of the modification and the local sequence context has significant effects on the amount of structural variation observed ( 77 ).…”

Enhanced nucleosome assembly at CpG sites containing an extended 5-methylcytosine analogue

“…Indeed, CpG and non-CpG methylation affect DNA micromechanical properties, i.e. methylation adds stiffness to DNA ( 11 , 44 ) and therefore reduces the affinity of the DNA to assemble into nucleosomes ( 43 , 45 ). Additionally, it might affect the nucleosome positioning as it was shown for satellite 2 region in pericentric heterochromatin domain, whereas the nucleosome structures and thermal stability stays unaffected ( 46 ).…”