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Open Access Highly Accessed Research article

Intronic Non-CG DNA hydroxymethylation and alternative mRNA splicing in honey bees

Pablo Cingolani12, Xiaoyi Cao3, Radhika S Khetani3, Chieh-Chun Chen3, Melissa Coon1, Alya’a Sammak1, Aliccia Bollig-Fischer5, Susan Land1, Yun Huang6, Matthew E Hudson34, Mark D Garfinkel7, Sheng Zhong3, Gene E Robinson389 and Douglas M Ruden110*

Author Affiliations

1 Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI 48201, USA

2 School of Computer Science & Genome Quebec Innovation Centre, McGill University, Montreal, QC, Canada

3 Institute for Genomic Biology, University of Illinois, Urbana, IL 61801, USA

4 Department of Crop Sciences, University of Illinois, Urbana, IL 61801, USA

5 Department of Oncology, Wayne State University, Detroit, MI 48201, USA

6 La Jolla Institute for Allergy & Immunology, La Jolla, CA 92037, USA

7 Department of Biological Sciences, University of Alabama in Huntsville, Huntsville, AL, USA

8 Department of Entomology, University of Illinois, Urbana, IL 61801, USA

9 Neuroscience Program, University of Illinois, Urbana, IL 61801, USA

10 Institute of Environmental Health Sciences, Wayne State University, Detroit, MI 48201, USA

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BMC Genomics 2013, 14:666  doi:10.1186/1471-2164-14-666

Published: 30 September 2013



Previous 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.


We 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.


Cytosine 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.

Honey Bees; DNA methylation; DNA hydroxymethylation; Epigenetics