Effects of cytosine modifications on DNA flexibility and nucleosome mechanical stability.

Thuy T M Ngo,Aleksei Aksimentiev,Taekjip Ha,Qing Dai,Qiucen Zhang,Chuan He,Jejoong Yoo

doi:10.1038/ncomms10813

Abstract

Cytosine can undergo modifications, forming 5-methylcytosine (5-mC) and its oxidized products 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). Despite their importance as epigenetic markers and as central players in cellular processes, it is not well understood how these modifications influence physical properties of DNA and chromatin. Here we report a comprehensive survey of the effect of cytosine modifications on DNA flexibility. We find that even a single copy of 5-fC increases DNA flexibility markedly. 5-mC reduces and 5-hmC enhances flexibility, and 5-caC does not have a measurable effect. Molecular dynamics simulations show that these modifications promote or dampen structural fluctuations, likely through competing effects of base polarity and steric hindrance, without changing the average structure. The increase in DNA flexibility increases the mechanical stability of the nucleosome and vice versa, suggesting a gene regulation mechanism where cytosine modifications change the accessibility of nucleosomal DNA through their effects on DNA flexibility.

Highlights

Cytosine can undergo modifications, forming 5-methylcytosine (5-mC) and its oxidized products 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC)
We report a comprehensive survey of the effect of cytosine modifications on DNA flexibility using a single-molecule cyclization assay: 5-fC remarkably increases DNA flexibility while 5-mC reduces and 5-hmC enhances flexibility, and 5-caC does not have a measurable effect
We used a single-molecule DNA cyclization assay[38] to quantify the effect of cytosine modification on DNA flexibility. In this assay (Fig. 1a), DNA molecules terminated with two complementary 50 overhangs of 10 nucleotides each were immobilized on a polyethylene glycol (PEG)-coated quartz slide using the biotin–neutravidin interaction

Summary

Introduction

Cytosine can undergo modifications, forming 5-methylcytosine (5-mC) and its oxidized products 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). Despite their importance as epigenetic markers and as central players in cellular processes, it is not well understood how these modifications influence physical properties of DNA and chromatin. CpG methylation reduces DNA flexibility affecting the binding of some proteins to their cognitive sequences such as the EcoRI restriction site or the cAMP DNA responsive element. Little is known about the effects of 5-hmC, 5-fC and 5-caC modifications on DNA flexibility. Cytosine modifications may affect gene regulation by changing nucleosome stability and unwrapping. How cytosine modifications including 5-mC and its oxidation products (5-hmC, 5-fC and 5-caC) affect nucleosome unwrapping under tension is not known

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Conclusion