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

Abstract

We present a comprehensive, experimental and theoretical study of the impact of 5-hydroxymethylation of DNA cytosine. Using molecular dynamics, biophysical experiments and NMR spectroscopy, we found that Ten-Eleven translocation (TET) dioxygenases generate an epigenetic variant with structural and physical properties similar to those of 5-methylcytosine. Experiments and simulations demonstrate that 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC) generally lead to stiffer DNA than normal cytosine, with poorer circularization efficiencies and lower ability to form nucleosomes. In particular, we can rule out the hypothesis that hydroxymethylation reverts to unmodified cytosine physical properties, as hmC is even more rigid than mC. Thus, we do not expect dramatic changes in the chromatin structure induced by differences in physical properties between d(mCpG) and d(hmCpG). Conversely, our simulations suggest that methylated-DNA binding domains (MBDs), associated with repression activities, are sensitive to the substitution d(mCpG) ➔ d(hmCpG), while MBD3 which has a dual activation/repression activity is not sensitive to the d(mCpG) d(hmCpG) change. Overall, while gene activity changes due to cytosine methylation are the result of the combination of stiffness-related chromatin reorganization and MBD binding, those associated to 5-hydroxylation of methylcytosine could be explained by a change in the balance of repression/activation pathways related to differential MBD binding.

5-Hydroxymethylcytosine: the many faces of the sixth base of mammalian DNA

“…In general, hmC is considered to reverse the impacts of methylation on DNA physical properties. Numerous studies have shown that while mC increases the duplex melting temperature, hmC brings it back closer to the one observed with C. 68,126,127,129…”

“…In general, hmC is considered to reverse the impacts of methylation on DNA physical properties. Numerous studies have shown that while mC increases the duplex melting temperature, hmC brings it back closer to the one observed with C. 68,126,127,129 A single-molecule mechanical force study showed that the mechanical stability of DNA (resistance to zipper-mode strand separation) increases with the number of hmC bases. 137 Different approaches have been taken to evaluate the effects of epigenetic DNA modifications on the DNA flexibility with anticipation of finding a direct mechanistic connection to nucleosome binding and gene expression regulation.…”

“…However, DNA circularisation with either ligation or FRET, nucleosome assembly or disassembly, and different molecular dynamics simulations have led to contrasting conclusions. 129,136 Finally, there can be differences between effects of solitary hmC modifications and their cooperative impact in larger clusters. 129,131 Among non-canonical DNA structures, G-quadruplexes (G4) have emerged as a significant player in regulation of gene expression, replication and other genome metabolic processes.…”

“…However, DNA circularisation with either ligation or FRET, nucleosome assembly or disassembly, and different molecular dynamics simulations have led to contrasting conclusions. 129,136 Finally, there can be differences between effects of solitary hmC modifications and their cooperative impact in larger clusters. 129,131…”

Studies of the Mechanism of Nucleosome Dynamics: A Review on Multifactorial Regulation from Computational and Experimental Cases

“…A series of molecular dynamics simulation research studies and experiments have studied the effect of methylation on the physical properties and dynamic features of DNA. Battistini et al performed molecular dynamics simulations, biophysical experiments, and NMR spectroscopy to study the impact of 5-hydroxymethylation on DNA cytosine and demonstrated that the modified cytosines (5mC or 5hmC) made the DNA stiffer than the normal cytosine [ 76 ]. Further studies performed by Hognon et al also explained how different levels of methylation in CpG sites affect the behavior of DNA [ 77 ].…”