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

Authors

Pablo Cingolani, Wayne State UniversityFollow
Xiaoyi Cao, Institute for Genomic Biology, University of IllinoisFollow
Radhika S. Khetani, Institute for Genomic Biology, University of IllinoisFollow
Chieh-Chun Chen, Institute for Genomic Biology, University of IllinoisFollow
Melissa Coon, Wayne State UniversityFollow
Alya'a Sammak, Wayne State UniversityFollow
Aliccia Bollig-Fischer, Wayne State UniversityFollow
Susan Land, Wayne State UniversityFollow
Yun Huang, La Jolla Institute for Allergy & ImmunologyFollow
Matthew E. Hudson, Institute for Genomic Biology, University of IllinoisFollow
Mark D. Garfinkel, University of Alabama in HuntsvilleFollow
Sheng Zhong, Institute for Genomic Biology, University of IllinoisFollow
Gene E. Robinson, Institute for Genomic Biology, University of IllinoisFollow
Douglas M. Ruden, Wayne State UniversityFollow

Document Type

Article

Abstract

Abstract

Background

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.

Results

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.

Conclusions

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.

Disciplines

Environmental Health | Medical Genetics | Obstetrics and Gynecology | Oncology

Recommended Citation

Cingolani et al.: Intronic Non-CG DNA hydroxymethylation and alternative mRNA splicing in honey bees. BMC Genomics 2013 14:666.