Intronic Non-CG DNA hydroxymethylation and alternative mRNA splicing in honey beesReport as inadecuate

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BMC Genomics

, 14:666

Comparative and evolutionary genomics


BackgroundPrevious whole-genome shotgun bisulfite sequencing experiments showed that DNA cytosine methylation in the honey bee Apis mellifera is almost exclusively at CG dinucleotides in exons. However, the most commonly used method, bisulfite sequencing, cannot distinguish 5-methylcytosine from 5-hydroxymethylcytosine, an oxidized form of 5-methylcytosine that is catalyzed by the TET family of dioxygenases. Furthermore, some analysis software programs under-represent non-CG DNA methylation and hydryoxymethylation for a variety of reasons. Therefore, we used an unbiased analysis of bisulfite sequencing data combined with molecular and bioinformatics approaches to distinguish 5-methylcytosine from 5-hydroxymethylcytosine. By doing this, we have performed the first whole genome analyses of DNA modifications at non-CG sites in honey bees and correlated the effects of these DNA modifications on gene expression and alternative mRNA splicing.

ResultsWe confirmed, using unbiased analyses of whole-genome shotgun bisulfite sequencing BS-seq data, with both new data and published data, the previous finding that CG DNA methylation is enriched in exons in honey bees. However, we also found evidence that cytosine methylation and hydroxymethylation at non-CG sites is enriched in introns. Using antibodies against 5-hydroxmethylcytosine, we confirmed that DNA hydroxymethylation at non-CG sites is enriched in introns. Additionally, using a new technique, Pvu-seq which employs the enzyme PvuRts1l to digest DNA at 5-hydroxymethylcytosine sites followed by next-generation DNA sequencing, we further confirmed that hydroxymethylation is enriched in introns at non-CG sites.

ConclusionsCytosine hydroxymethylation at non-CG sites might have more functional significance than previously appreciated, and in honey bees these modifications might be related to the regulation of alternative mRNA splicing by defining the locations of the introns.

KeywordsHoney Bees DNA methylation DNA hydroxymethylation Epigenetics AbbreviationsAHBAfricanized Honey Bee

EHBEuropean Honey Bee

Pvu-seqPvuRts1I digestion of 5hmC sites followed by next generation DNA sequencing

BS-seqBisulfite treatment of DNA followed by next-generation DNA sequencing

ChIP-seqChromatin immunoprecipitation followed by next-generation DNA sequencing

MeDIP-seqImmunoprecipitation of 5mC DNA with anti-5mC antibodies followed by next-generation DNA sequencing

HMeDIP-seqImmunoprecipitation of 5hmC DNA with anti-5hmC antibodies followed by next-generation DNA sequencing

GOGene Ontology

DAVIDDatabase for Annotation, Visualization, and Integrated Discovery

hESCHuman embryonic stem cells



o-eObserved over expected

VCFVariable call format

SAMStatistical analysis of microarrays

SNPSingle nucleotide polymorphism

Amel2The reference genome for Apis mellifera honey bee

MACSModel based analysis for Chip-seq

BCFBinary call format

CGCytosine-guanine dinucleotide

CHHCytosine followed by two nucleotides that are not guanine

CHGCytosine followed by one nucleotide that is not a guanine and then a guanine.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2164-14-666 contains supplementary material, which is available to authorized users.

Pablo Cingolani, Xiaoyi Cao, Radhika S Khetani contributed equally to this work.

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Author: Pablo Cingolani - Xiaoyi Cao - Radhika S Khetani - Chieh-Chun Chen - Melissa Coon - Alya’a Sammak - Aliccia Bollig-Fisch


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