Two are better than one: HPoxBS - hairpin oxidative bisulfite sequencing.

Pascal Giehr,Konstantin Lepikhov,Jörn Walter,Verena Wolf,Stefan Wallner,Charalampos Kyriakopoulos

doi:10.1093/nar/gky422

Pascal Giehr, Konstantin Lepikhov + Show 4 more

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https://doi.org/10.1093/nar/gky422

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Abstract

The controlled and stepwise oxidation of 5mC to 5hmC, 5fC and 5caC by Tet enzymes is influencing the chemical and biological properties of cytosine. Besides direct effects on gene regulation, oxidised forms influence the dynamics of demethylation and re-methylation processes. So far, no combined methods exist which allow to precisely determine the strand specific localisation of cytosine modifications along with their CpG symmetric distribution. Here we describe a comprehensive protocol combining conventional hairpin bisulfite with oxidative bisulfite sequencing (HPoxBS) to determine the strand specific distribution of 5mC and 5hmC at base resolution. We apply this method to analyse the contribution of local oxidative effects on DNA demethylation in mouse ES cells. Our method includes the HPoxBS workflow and subsequent data analysis using our developed software tools. Besides a precise estimation and display of strand specific 5mC and 5hmC levels at base resolution we apply the data to predict region specific activities of Dnmt and Tet enzymes. Our experimental and computational workflow provides a precise double strand display of 5mC and 5hmC modifications at single base resolution. Based on our data we predict region specific Tet and Dnmt enzyme efficiencies shaping the distinct locus levels and patterns of 5hmC and 5mC.

Highlights

In mammals, DNA methylation is restricted to the C5 position of cytosine and is predominantly found in a CpG context [1,2,3]
The optimal restriction enzyme (RE) used for hairpin bisulfite with oxidative bisulfite sequencing (HPoxBS) should fulfill the following conditions: The restriction site should be in close proximity to the CpGs of interest, as it provides the anchor for the hairpin linker ligation
Mouse embryonic stem cells (ESC) have a high genome wide methylation status when cultivated on serum/LIF, while loosing DNAmethylation in a replication dependent manner under 2i conditional medium [43,44,45,46]

Summary

Introduction

DNA methylation is restricted to the C5 position of cytosine and is predominantly found in a CpG context [1,2,3]. The protein complex modulates the preferred recognition of Dnmt for hemimethylated CpGs, such that Dnmt acts as a copying enzyme for existing methylation patterns from the old to the newly synthesised DNA strand, maintaining original methylation patterns across cell divisions [8,9]. This process is one of the key mechanisms of epigenetic inheritance

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