The Honey Bee Epigenomes: Differential Methylation of Brain DNA in Queens and Workers

Abstract

In honey bees (Apis mellifera) the behaviorally and reproductively distinct queen and worker female castes derive from the same genome as a result of differential intake of royal jelly and are implemented in concert with DNA methylation. To determine if these very different diet-controlled phenotypes correlate with unique brain methylomes, we conducted a study to determine the methyl cytosine (mC) distribution in the brains of queens and workers at single-base-pair resolution using shotgun bisulfite sequencing technology. The whole-genome sequencing was validated by deep 454 sequencing of selected amplicons representing eight methylated genes. We found that nearly all mCs are located in CpG dinucleotides in the exons of 5,854 genes showing greater sequence conservation than non-methylated genes. Over 550 genes show significant methylation differences between queens and workers, revealing the intricate dynamics of methylation patterns. The distinctiveness of the differentially methylated genes is underscored by their intermediate CpG densities relative to drastically CpG-depleted methylated genes and to CpG-richer non-methylated genes. We find a strong correlation between methylation patterns and splicing sites including those that have the potential to generate alternative exons. We validate our genome-wide analyses by a detailed examination of two transcript variants encoded by one of the differentially methylated genes. The link between methylation and splicing is further supported by the differential methylation of genes belonging to the histone gene family. We propose that modulation of alternative splicing is one mechanism by which DNA methylation could be linked to gene regulation in the honey bee. Our study describes a level of molecular diversity previously unknown in honey bees that might be important for generating phenotypic flexibility not only during development but also in the adult post-mitotic brain. The queen honey bee and her worker sisters do not seem to have much in common. Workers are active and intelligent, skillfully navigating the outside world in search of food for the colony. They never reproduce; that task is left entirely to the much larger and longer-lived queen, who is permanently ensconced within the colony and uses a powerful chemical influence to exert control. Remarkably, these two female castes are generated from identical genomes. The key to each female's developmental destiny is her diet as a larva: future queens are raised on royal jelly. This specialized diet is thought to affect a particular chemical modification, methylation, of the bee's DNA, causing the same genome to be deployed differently. To document differences in this epigenomic setting and hypothesize about its effects on behavior, we performed high-resolution bisulphite sequencing of whole genomes from the brains of queen and worker honey bees. In contrast to the heavily methylated human genome, we found that only a small and specific fraction of the honey bee genome is methylated. Most methylation occurred within conserved genes that provide critical cellular functions. Over 550 genes showed significant methylation differences between the queen and the worker, which may contribute to the profound divergence in behavior. How DNA methylation works on these genes remains unclear, but it may change their accessibility to the cellular machinery that controls their expression. We found a tantalizing clue to a mechanism in the clustering of methylation within parts of genes where splicing occurs, suggesting that methylation could control which of several versions of a gene is expressed. Our study provides the first documentation of extensive molecular differences that may allow honey bees to generate different phenotypes from the same genome.