- associated with DNA methylation changes in the honey bee brain. - DNA methylation is stably maintained over time, but also reversible in response to specific environmental conditions, and may thus be a neuromolecular regulator of both of these processes. - A previous study reported DNA methylation differences between aggressive Africanized and gentle European honey bees. - We investigated whether threat-induced aggression altered DNA methylation profiles in the honey bee brain in response to a behavioral stimulus (aggression-provoking intruder bee or inert control). - Results: There were DNA methylation differences between aggressive and control bees for individual cytosine-guanine dinucleotides (CpGs) across the genome. - Eighteen individual CpG sites showed significant difference between aggressive and control bees 120 min post stimulus. - previously published epigenetic differences that distinguish aggressive Africanized and gentle European honey bees, suggesting an evolutionarily conserved use of brain DNA methylation in the regulation of aggression. - Conclusions: There were DNA methylation differences in the brain associated with response to an intruder. - Many DNA methylation differences that occurred in association with the expression of aggression in real time also exist between Africanized bees and European bees, suggesting an evolutionarily conserved role for epigenetic regulation in aggressive behavior.. - There is a well-established relationship between behav- ioral plasticity and dynamic DNA methylation in several model systems. - Recent evidence indicates that DNA methylation under- lies the maintenance of long-term memory [15–18], allowing experiences to stably alter behavior. - The de novo addition and active removal of DNA methylation facilitate transitions between epigenetic states in the brain [19–22], suggesting a compelling molecular mech- anism underlying behavioral plasticity.. - Additionally, differences in brain DNA methylation have been reported between queen and worker bees dur- ing development and in adulthood when they display distinct behaviors [25, 26]. - Early evidence for DNA methylation’s impact on queen / worker development was illustrated by DNMT knockdown during the larval. - Division of labor is a hallmark of eusocial species, and mounting evidence shows that DNA methylation plays a role in this process, possibly by affecting vitellogenin levels and altering lifespan [28]. - In the brain, pharmaco- logical perturbation of DNA methylation affects learning ability, and long-term memory formation appears to require upregulation of DNA methylation modifying enzymes DNMT and Tet [29]. - This evidence, in addition to DNA methylation’s predicted role in regulating gene expression and alternative splicing events, makes DNA methylation an attractive candidate for regulating aggressive behavior in honeybees [30].. - We used a well-established aggression assay to investi- gate the role of DNA methylation in mediating aggres- sive responses to an intruder [4]. - To profile DNA methylation signa- tures associated with aggression in the brain, we per- formed whole genome bisulfite sequencing (WGBS) of DNA from the brains of honey bees that responded aggressively to territorial intrusion, and compared the DNA methylation profiles with those of control bees who experienced an inert stimulus. - DNA methylation was profiled at early and late time points (5 and 120 min, 6 individuals per phenotype) following the intrusion to investigate epigenetic differences between aggressive and control bees.. - Here we show DNA methylation profile signatures of aggressive behavior in honey bees co-localize with gene regulatory elements in cis and are reflected in differences between aggressive Africanized bees and gentle Euro- pean bees. - DNA methylation differences increase over time from an early state immediately after interaction with an intruder to two hours post exposure, which is when memory consolidation typically occurs in other experimental systems [31]. - Out of 3,897,088 CpG sites with an average coverage of at least 5X, we identified 119,557 methylated CpG sites where at least 3 out of 24 samples had DNA methylation levels greater than 10%. - As previously reported in honey bees, most of the DNA methylation resided within exons [25]. - While no individual CpGs were significantly different between aggressive and control bees 5 min after stimu- lus, 18 CpGs distinguished aggressive and control bees 120 min post stimulus (FDR ≤ 0.05). - Additionally, there were two CpGs differentially methylated between 5-min and 120-min control samples, and no differences between 5-min and 120-min aggressive samples. - These results provide evidence for methylation changes in the brain in response to intruder exposure with clear differences 120 min post stimulus.. - Regional analysis of DNA methylation reveals differences resulting from intruder exposure over time. - At 120 min following intruder exposure, there were 50 DMRs between the aggressive and control bees (Additional file 2: Table S2. - This DEG ana- lysis also revealed no expression bias of DNA methyl- transferase enzymes in either phenotype, which is reflected in the fact that we observe both hyper- and hypo- methylation within the 120 min DMRs. - 120-min DMRs include most (11/16) CpGs from scaffold 2.11 that were identified independently, and confirm that this is a hotspot of epigenetic change, with 21 out of 50 DMRs residing on this scaffold (Fig. - Increasing numbers of DMRs from 5 to 120 min also strengthens our finding of change over time within individual CpGs and strongly suggests that the DNA methylation changes are a direct response to intruder exposure.. - Genes associated with 120-min DMRs include those involved with RNA processing, chromatin remodeling and hormone signaling, processes that have been previ- ously implicated in brain transcriptomic analyses of the bee aggression [4, 7]. - Overall, genes associated with 120-min DMRs may help facilitate large-scale gene expression response to intruder and alter signaling path- ways that heighten the response to a new intruder in the future, because the presence of one intruder often pre- sages the presence of more [37].. - Since other forms of behavioral plasticity that involve neuronal remodeling, such as memory formation, re- quire coordinated gene expression changes, we investi- gated the potential impact of DNA methylation on gene regulation across the genome. - On the cellular level, DNA methylation is known to play a critical role in the late-phase of long-term potentiation (L-LTP. - To explore whether DNA methylation might work on a similar time scale in the context of aggression, we related the present results to previously published transcriptomic profiles in Shpigler et al. - aggressive and control bees 5 min post exposure, and 4323 CpGs 120 min post exposure. - 1 Regional DNA Methylation Differences Distinguish Aggressive And Control Bees. - Aggressive bees (red) and control bees (blue) show distinct DNA methylation profiles. - The region depicted spans the ~ 90 kb hotspot of DNA methylation change and contains many signaling genes. - b-c) Top panel presents DNA methylation level for a short segment of scaffold 11.16, with dots for individual CpGs and smoothed lines for average methylation levels for each phenotype. - This analysis revealed that only 120-min DEGs have a significant overlap with suggestive CpGs (both P-value. - Surprisingly, both 120-min and 5-min CpGs have significant overlaps with 120-min DEGs, often co-localizing in the same gene (Fig. - A total of 289 120-min DEGs overlapped 5-min CpGs. - and 319 120-min DEGs overlapped 120-min CpGs.. - We also observed a significant co-localization of 120-min and 5-min suggestive CpGs in 140 of the 120-min DEGs (overlap p by hypergeometric test). - Although 120-min DEGs overlapped both 5-min and 120-min CpGs, these CpG groups were largely distinct, with only 11 common CpGs within 120-min DEGs across time points. - Together, these findings reveal a significant number of methylation differences marking 120-min DEGs, both immediately after and 2 h post exposure to an. - Gene expression differences between aggressive and control bees that arise 120 min after interaction with an intruder are often marked with differences in DNA methylation.. - For both (a) and (b), the top panel depicts boxplots of DNA methylation levels at both time points for aggressive and control bees. - Note that different sets of CpGs have significant differences at different time points, thus marking gene before and after gene expression change at 120 min with distinct CpGs. - (c) Summary of overlap between 120-min DEGs and suggestive CpG ’ s. - Both 5-min and 120-min suggestive CpGs have significant overlaps with120-minute DEGs, with a high degree of co-localization within a subset of genes. - Here, we found of 120-min DEGs marked by suggestive CpGs at 5 min were replaced by other suggestive CpGs at 120 min, potentially switching from a poised state to locking in the gene expression change post exposure.. - DMRs and suggestive CpGs overlap DNA methylation between Africanized and European honey bees, suggesting an evolutionary conserved role for epigenetics in regulating aggression. - We found a highly significant overlap between 120-min DMRs (and suggestive CpGs) and AHB / EHB dif- ferences (both P-value <. - This overlap suggests a conserved role for DNA methylation in modulating aggres- sive behavior across genetic backgrounds.. - DNA methylation co-localizes with TFBS that regulate neuroplasticity and neurodevelopment. - To further understand the molecular role of DNA methylation in gene regulation, we assessed the co- occupation of transcription factor binding sites (TFBS) and suggestive CpGs within promoters across the gen- ome. - DNA methylation can directly block TFBS in pro- moters, as seen in mammalian systems [40]. - To identify transcription factors that might interact with DNA methylation to mediate the transcriptional response to an intruder, we calculated hypergeometric tests for the overlap of putative TFBSs and 120-min suggestive CpGs within promoters (2 kb) as defined by the OGS 3.2 gene set (Additional file 5: Table S4). - Outside of the promoter, we observed strong enrichment of DNA methylation and TFBS within genes, potentially serving a protective role against spurious TF binding in gene bodies, as observed in termites [48]. - Across the genome, we also observed that TFBS of Cf2, CG8281, jigr, lola, fkh, vvl, and CG10267/Zif were enriched over 120-min suggestive CpG sites by AME analysis [49]. - For these TFBS enriched over suggestive CpGs, DNA methylation may be acting directly and altering the binding affinity for the TF. - We found far more methylation differences between aggressive and control bees at 120 min than at 5 min after intruder exposure, which implies that DNA methylation could be assisting in altering neurogenomic states to maintain an aroused and vigilant state.. - The observed brain epigenetic differences had a significant overlap with both gene expression differences in response to an intruder and with DNA methylation dif- ferences across genetic strains of honey bees that differ drastically in response to intruders.. - This hypothesized link between epigenetic regulation and neural plasticity is further strengthened by the highly significant overlap we found between DNA methylation differences and binding sites for neurobiologically relevant transcrip- tion factors. - This evidence suggests a mechanism whereby DNA methylation can affect brain gene regulation over a very short time period by blocking or enhancing the action of transcription factors.. - We also provide evidence for DNA methylation mark- ing genes prior to expression changes, thus potentially poising genes in a ready state that can more immediately respond to an encounter with an intruder. - In humans, multiple genes including IL-6 interleukin 6 and the serotonin transporter SLC6A4 (solute carrier family 6 member 4) have altered DNA methylation levels in their promoter regions (reviewed in Waltes et al. - We showed that differences exist between the brain DNA methylation profiles of aggressive and con- trol honey bees, indicating a possible epigenetic basis for. - This study provides the first evidence that changes in brain DNA methylation occur in response to territorial intru- sion, supporting the conclusion that there is an epigen- etic basis to behavioral plasticity in aggression.. - Collections were done either 5 min or 120 min following initial intruder exposure. - Two bees were collected from each control group at random either 5 min or 120 min following initial exposure to the tube.. - Shotgun DNA libraries were made using the KAPA Library Preparation Kit, then bisulfite treated using the Zymo EZ DNA Methylation-Lightning Kit. - The bismark program was also used to mark duplicates and extract DNA methylation levels for each CpG.. - To find differences in DNA methylation between aggressive and control bees over time, we first arcsine transformed percentage measurements derived from ratios of reads containing methylated and unmethylated cytosines in the CpG dinucleotide context. - To assess the genome-wide impact of brain DNA methylation on brain gene expression, we used gene expression data from the Shpigler et al. - This study identified brain gene expression differences between aggressive and control bees at 30, 60, and 120 min after the introduction of an intruder bee to a petri dish containing test bees. - Significance of over- lap between 5-min and 120-min suggestive CpGs within 120-min DEGs was calculated by hypergeometric test.. - A similar analysis was performed on DNA methylation data from Cingolani et al. - Co-localization of DNA methylation and TFBS in promoters Locations of putative transcription factor binding sites (TFBS) were found by mapping TFBS motifs identified in Drosophila melanogaster to the Amel 4.5 version of the honey bee genome using the program CisGenome [40]. - Hypergeometric tests were performed to calculate p-values for overlap between DNA methylation and TFBS within promoters, and FDRs were calculated from p-values using the qvalue package in R [54, 55]. - For each suggestive CpG identified at 120-min post intruder exposure, 20 base pairs of sequence up and down stream of cytosine was recorded (total 41 bp for each suggestive CpG). - DMRs between control and aggressive bees at 120 min post interaction. - Early-life stress reduces DNA methylation of the Pomc gene in male mice. - Dynamic DNA methylation programs persistent adverse effects of early-life stress. - Resilience to social stress coincides with functional DNA methylation of the Crf gene in adult mice. - DNA methylation and memory formation. - DNA methylation in memory formation: emerging insights. - Cortical DNA methylation maintains remote memory. - DNA methylation changes in plasticity genes accompany the formation and maintenance of memory. - Stress-induced gene expression and behavior are controlled by DNA methylation and methyl donor availability in the dentate gyrus.. - DNA methylation regulates neuronal glutamatergic synaptic scaling.. - DNA methylation regulates associative reward learning. - DNA methylation dynamics, metabolic fluxes, gene splicing, and alternative phenotypes in honey bees. - Nutritional control of reproductive status in honeybees via DNA methylation. - Drugging the methylome: DNA methylation and memory. - DNA methylation in insects: on the brink of the epigenomic era
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