A genome-wide search for epigenetically [corrected] regulated genes in zebra finch using MethylCap-seq and RNA-seq.

Sandra Steyaert,Nina Sohnius-Wilhelmi,Sarah De Keulenaer,Jeroen Galle,Jolien Diddens,Carolina Frankl-Vilches,Wim Van Criekinge,Ellen De Meester,Antje Bakker,Annemie Van Der Linden,Wim Vanden Berghe,Tim De Meyer

doi:10.1038/srep20957

Sandra Steyaert, Nina Sohnius-Wilhelmi + Show 10 more

Open Access

https://doi.org/10.1038/srep20957

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Abstract

Learning and memory formation are known to require dynamic CpG (de)methylation and gene expression changes. Here, we aimed at establishing a genome-wide DNA methylation map of the zebra finch genome, a model organism in neuroscience, as well as identifying putatively epigenetically regulated genes. RNA- and MethylCap-seq experiments were performed on two zebra finch cell lines in presence or absence of 5-aza-2′-deoxycytidine induced demethylation. First, the MethylCap-seq methodology was validated in zebra finch by comparison with RRBS-generated data. To assess the influence of (variable) methylation on gene expression, RNA-seq experiments were performed as well. Comparison of RNA-seq and MethylCap-seq results showed that at least 357 of the 3,457 AZA-upregulated genes are putatively regulated by methylation in the promoter region, for which a pathway analysis showed remarkable enrichment for neurological networks. A subset of genes was validated using Exon Arrays, quantitative RT-PCR and CpG pyrosequencing on bisulfite-treated samples. To our knowledge, this study provides the first genome-wide DNA methylation map of the zebra finch genome as well as a comprehensive set of genes of which transcription is under putative methylation control.

Highlights

Epigenetic modifications, including DNA methylation, have been shown to play an important role in several neurobiological and cognitive processes, such as learning and memory[12,13]
In order to assess a first ‘draft of the zebra finch methylome’, we performed methyl-CpG binding domain protein sequencing (MethylCap-seq) experiments on the G266 as well as the ZFTMA cell line, in presence or absence of the DNA methylation inhibitor 5-aza-2′ -deoxycytidine (AZA) that, when incorporated in the genome, inhibits DNA methyltransferases (DNMTs) resulting in a net demethylation effect[22]
AZA is a chemical analogue of cytidine, that, when incorporated in the genome, inhibits DNMTs resulting in a net demethylation effect[22]

Summary

Introduction

Epigenetic modifications, including DNA methylation, have been shown to play an important role in several neurobiological and cognitive processes, such as learning and memory[12,13]. It is established by DNA methyltransferases (DNMTs) and involves the transfer of a methyl group to cytosine residues at the carbon 5 position, predominantly in a CpG context This methylation mark is known to regulate gene expression and when located in the promoter region, it generally leads to transcriptional silencing of the corresponding gene[15]. In order to assess a first ‘draft of the zebra finch methylome’, we performed methyl-CpG binding domain protein sequencing (MethylCap-seq) experiments on the G266 as well as the ZFTMA cell line, in presence or absence of the DNA methylation inhibitor 5-aza-2′ -deoxycytidine (AZA) that, when incorporated in the genome, inhibits DNMTs resulting in a net demethylation effect[22]. We provide an overview of loci featured by dynamic methylation degrees as well as a list of genes for which expression is putatively regulated by DNA methylation in the promoter region

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