Molecular underpinnings of the early brain developmental response to differential feeding in the honey bee Apis mellifera

Brain differential morphogenesis in females is one of the major phenotypic manifestations of caste development in honey bees. Brain diphenism appears at the fourth larval phase as a result of the differential feeding regime developing females are submitted during early phases of larval development. Here, we used a forward genetics approach to test the early brain molecular response to differential feeding leading to the brain diphenism observed at later developmental phases. Using RNA sequencing analysis, we identified 53 differentially expressed genes (DEGs) between the brains of queens and workers at the third larval phase. Since miRNAs have been suggested to play a role in caste differentiation after horizontal and vertical transmission, we tested their potential participation in regulating the DEGs. The miRNA-mRNA interaction network, including the DEGs and the royal- and worker-jelly enriched miRNA populations, revealed a subset of miRNAs potentially involved in regulating the expression of DEGs. The interaction of miR-34, miR-210, and miR-317 with Takeout, Neurotrophin-1, Forked, and Masquerade genes was experimentally confirmed using a luciferase reporter system. Taken together, our results reconstruct the regulatory network that governs the development of the early brain diphenism in honey bees.     

Comparative analyses of DNA methylation and sequence evolution using Nasonia genomes

The functional and evolutionary significance of DNA methylation in insect genomes remains to be resolved. Nasonia is well situated for comparative analyses of DNA methylation and genome evolution, since the genomes of a moderately distant outgroup species as well as closely related sibling species are available. Using direct sequencing of bisulfite-converted DNA, we uncovered a substantial level of DNA methylation in 17 of 18 Nasonia vitripennis genes and a strong correlation between methylation level and CpG depletion. Notably, in the sex-determining locus transformer, the exon that is alternatively spliced between the sexes is heavily methylated in both males and females, whereas other exons are only sparsely methylated. Orthologous genes of the honeybee and Nasonia show highly similar relative levels of CpG depletion, despite ~190 My divergence. Densely and sparsely methylated genes in these species also exhibit similar functional enrichments. We found that the degree of CpG depletion is negatively correlated with substitution rates between closely related Nasonia species for synonymous, nonsynonymous, and intron sites. This suggests that mutation rates increase with decreasing levels of germ line methylation. Thus, DNA methylation is prevalent in the Nasonia genome, may participate in regulatory processes such as sex determination and alternative splicing, and is correlated with several aspects of genome and sequence evolution.     

Unmethylated CpG islands associated with genes in higher plant DNA

The genomes of many higher plant species are the most highly methylated among eukaryotes. We report here that in spite of their heavy methylation, genomic DNAs from four plant species contain a fraction that is very rich in non-methylated sites. The fraction was characterized in maize where it represents about 2.5% of the total nuclear genome. In order to establish the genomic origin of the fraction, three maize genes containing clustered CpG were tested for methylation and were found to be non-methylated in the CpG-rich regions. By contrast, tested CpGs were methylated in a gene whose sequence showed no clustering of CpG. These observations suggest that the CpG-rich fraction of plants is at least partially derived from non-methylated regions that are associated with genes. A similar phenomenon has been described in vertebrate genomes. We discuss the evolution of CpG islands in both groups of organisms, and their possible uses in mapping and gene isolation in plants.     

High-throughput assay of DNA methylation based on methylation-specific primer and SAGE

Mapping of genomic DNA methylation is a dispensable part of functional genome. We have developed a novel method based on methylation-specific primer and serial analysis of gene expression, called MSP-SAGE, with potential of high-throughput quantification of genomic DNA methylation. We used a 6-mer methylation-specific primer to extend the methylated CpG sequences other than non-methylated CpG sequences. The 17 bp tags contained methylated CpG sequence, which were obtained from extended methylation sequence by digestion of restriction endonuclease, and then the tags were concatenated and cloned for sequencing. We can identify the locations of methylation according to the sequences of tags and quantify the methylation status from the frequency of the tags. MSP-SAGE has a good linearity in a broad methylation range from 5% to 100% with good accuracy and high precision. The proof-of-principle study shows that MSP-SAGE is a reliable high-throughput assay for quantification of DNA methylation.     

Patterns of DNA methylation in development, division of labor and hybridization in an ant with genetic caste determination

Background

DNA methylation is a common regulator of gene expression, including acting as a regulator of developmental events and behavioral changes in adults. Using the unique system of genetic caste determination in Pogonomyrmex barbatus, we were able to document changes in DNA methylation during development, and also across both ancient and contemporary hybridization events.

Methodology/Principal Findings

Sodium bisulfite sequencing demonstrated in vivo methylation of symmetric CG dinucleotides in P. barbatus. We also found methylation of non-CpG sequences. This validated two bioinformatics methods for predicting gene methylation, the bias in observed to expected ratio of CpG dinucleotides and the density of CpG/TpG single nucleotide polymorphisms (SNP). Frequencies of genomic DNA methylation were determined for different developmental stages and castes using ms-AFLP assays. The genetic caste determination system (GCD) is probably the product of an ancestral hybridization event between P. barbatus and P. rugosus. Two lineages obligately co-occur within a GCD population, and queens are derived from intra-lineage matings whereas workers are produced from inter-lineage matings. Relative DNA methylation levels of queens and workers from GCD lineages (contemporary hybrids) were not significantly different until adulthood. Virgin queens had significantly higher relative levels of DNA methylation compared to workers. Worker DNA methylation did not vary among developmental stages within each lineage, but was significantly different between the currently hybridizing lineages. Finally, workers of the two genetic caste determination lineages had half as many methylated cytosines as workers from the putative parental species, which have environmental caste determination.

Conclusions/Significance

These results suggest that DNA methylation may be a conserved regulatory mechanism moderating division of labor in both bees and ants. Current and historic hybridization appear to have altered genomic methylation levels suggesting a possible link between changes in overall DNA methylation and the origin and regulation of genetic caste determination in P. barbatus.     

Meta-analysis of human methylation data for evidence of sex-specific autosomal patterns

Background

Several individual studies have suggested that autosomal CpG methylation differs by sex both in terms of individual CpG sites and global autosomal CpG methylation. However, these findings have been inconsistent and plagued by spurious associations due to the cross reactivity of CpG probes on commercial microarrays. We collectively analysed 76 published studies (n = 6,795) for sex-associated differences in both autosomal and sex chromosome CpG sites.

Results

Overall autosomal methylation profiles varied substantially by study, and we encountered substantial batch effects. We accounted for these by conducting random effects meta-analysis for individual autosomal CpG methylation associations. After excluding non-specific probes, we found 184 autosomal CpG sites differentially methylated by sex after correction for multiple testing. In line with previous studies, average beta differences were small. Many of the most significantly associated CpG probes were new. Of note was differential CpG methylation in the promoters of genes thought to be involved in spermatogenesis and male fertility, such as SLC9A2, SPESP1, CRISP2, and NUPL1. Pathway analysis revealed overrepresentation of genes differentially methylated by sex in several broad Gene Ontology biological processes, including RNA splicing and DNA repair.

Conclusions

This study represents a comprehensive analysis of sex-specific methylation patterns. We demonstrate the existence of sex-specific methylation profiles and report a large number of novel DNA methylation differences in autosomal CpG sites between sexes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-981) contains supplementary material, which is available to authorized users.     

Experimental manipulation of ovary activation and gene expression in honey bee (Apis mellifera) queens and workers: testing hypotheses of reproductive regulation

A fundamental issue in sociobiology is to understand how social insect females regulate their individual reproduction to maximize colony and personal fitness. Although the social cues mediating reproductive output within castes of the honey bees (Apis mellifera) are understood at a basic level, the underlying gene regulatory networks are not. In this study, we investigate the expression of 25 genes whose function suggests a role in the gene networks that regulate ovary activation--a functional determinant of reproductive skew. To this end, we used CO2 narcosis to manipulate ovary activation in queens and workers, and then quantified concomitant changes in gene expression using quantitative polymerase chain reaction. Of the 25 genes studied, ten were differentially expressed between treated and control groups in at least one caste. Two of these genes, a ribosomal protein and a tyramine receptor, were differentially expressed between treatments and controls in both castes. We use the expression pattern of all differentially expressed genes to test hypotheses for the caste-specific regulation of ovary activation in honey bees.     

Caste-specific gene expression in the stingless bee <Emphasis Type="Italic">Melipona quadrifasciata</Emphasis> – Are there common patterns in highly eusocial bees?

Summary. Caste polyphenism is a multifaceted phenomenon, most evident in the marked differences in reproductive capacity and longevity between queens and workers. The mechanisms underlying caste differentiation and division of labor are mainly addressed in the honey bee, and recently have been studied at the molecular level. Yet, generalizations drawn from studies on this model organism require validation by comparative studies. We choose Melipona quadrifasciata, a sister-group species to honey bees, to investigate differences in gene expression between newly emerged adult queens and workers. RNA extracts were subjected to a differential display protocol (DDRT-PCR). The putative differentially expressed genes, for which annotation was available, were validated by RT-PCR and hybridization. Differential expression was observed for myosin, projectin, kettin, cytochrome P450, Rab11 and Sas10. Except for kettin, all of these were overexpressed in the worker caste. Projectin and kettin could play roles in caste-specific flight muscle organization. The putative Rab11 and Sas10 homolog genes could be involved in fertility-related cell signaling and in longevity-related gene silencing, respectively. Cytochrome P450 overexpression in Melipona workers corroborates similar findings in the honey bee, thus indicating a common function in the social insect caste syndrome.Received 9 January 2003; revised 10 March 2004; accepted 2 April 2004.     

Intronic Parent-of-Origin Dependent Differential Methylation at the Actn1 Gene Is Conserved in Rodents but Is Not Associated with Imprinted Expression

Parent-of-origin differential DNA methylation has been associated with regulation of the preferential expression of paternal or maternal alleles of imprinted genes. Based on this association, recent studies have searched for parent-of-origin dependent differentially methylated regions in order to identify new imprinted genes in their vicinity. In a previous genome-wide analysis of mouse brain DNA methylation, we found a novel differentially methylated region in a CpG island located in the last intron of the alpha 1 Actinin (Actn1) gene. In this region, preferential methylation of the maternal allele was observed; however, there were no reports of imprinted expression of Actn1. Therefore, we have tested if differential methylation of this region is common to other tissues and species and affects the expression of Actn1. We have found that Actn1 differential methylation occurs in diverse mouse tissues. Moreover, it is also present in other murine rodents (rat), but not in the orthologous human region. In contrast, we have found no indication of an imprinted effect on gene expression of Actn1 in mice: expression is always biallelic regardless of sex, tissue type, developmental stage or isoform. Therefore, we have identified a novel parent-of-origin dependent differentially methylated region that has no apparent association with imprinted expression of the closest genes. Our findings sound a cautionary note to genome-wide searches on the use of differentially methylated regions for the identification of imprinted genes and suggest that parent-of-origin dependent differential methylation might be conserved for functions other that the control of imprinted expression.     

Extensive methylation of a part of the CpG island located 3.0-4.5 kbp upstream to the chicken alpha-globin gene cluster may contribute to silencing the globin genes in non-erythroid cells

Here, we show that in the chicken genome, the domain of alpha-globin genes is preceded by a CpG island of which the downstream part ( approximately 0.65 kbp) is heavily methylated in lymphoid cells; it is either non-methylated or undermethylated in erythroid cells. Recombinant plasmids were constructed with the corresponding DNA fragment (called "uCpG") placed upstream to a reporter CAT gene expressed from the promoter of the alpha(D) chicken globin gene. Selective methylation of CpG dinucleotides within the uCpG fragment suppressed fivefold the expression of the CAT gene, when neither this gene itself nor the alpha(D) promoter were methylated. Methylation of CpG dinucleotides within the alpha(D) gene promoter did not modify the suppression effect exerted by methylated uCpG. We interpret these results within the frame of the hypothesis postulating, that methylation of the upstream CpG island of the chicken alpha-globin gene domain may play an essential role in silencing the alpha-globin genes in non-erythroid cells.     

Altered DNA methylation in leukocytes with trisomy 21

The primary abnormality in Down syndrome (DS), trisomy 21, is well known; but how this chromosomal gain produces the complex DS phenotype, including immune system defects, is not well understood. We profiled DNA methylation in total peripheral blood leukocytes (PBL) and T-lymphocytes from adults with DS and normal controls and found gene-specific abnormalities of CpG methylation in DS, with many of the differentially methylated genes having known or predicted roles in lymphocyte development and function. Validation of the microarray data by bisulfite sequencing and methylation-sensitive Pyrosequencing (MS-Pyroseq) confirmed strong differences in methylation (p<0.0001) for each of 8 genes tested: TMEM131, TCF7, CD3Z/CD247, SH3BP2, EIF4E, PLD6, SUMO3, and CPT1B, in DS versus control PBL. In addition, we validated differential methylation of NOD2/CARD15 by bisulfite sequencing in DS versus control T-cells. The differentially methylated genes were found on various autosomes, with no enrichment on chromosome 21. Differences in methylation were generally stable in a given individual, remained significant after adjusting for age, and were not due to altered cell counts. Some but not all of the differentially methylated genes showed different mean mRNA expression in DS versus control PBL; and the altered expression of 5 of these genes, TMEM131, TCF7, CD3Z, NOD2, and NPDC1, was recapitulated by exposing normal lymphocytes to the demethylating drug 5-aza-2'deoxycytidine (5aza-dC) plus mitogens. We conclude that altered gene-specific DNA methylation is a recurrent and functionally relevant downstream response to trisomy 21 in human cells.     

A nuclear protein that binds preferentially to methylated DNA in vitro may play a role in the inaccessibility of methylated CpGs in mammalian nuclei

The effects of DNA methylation on gene expression and chromatin structure suggest the existence of a mechanism in the nucleus capable of distinguishing methylated and non-methylated sequences. We report the finding of a nuclear protein in several vertebrate tissues and cell lines that binds preferentially to methylated DNA in vitro. Its lack of sequence-specific requirements makes it potentially capable of binding to any methylated sequence in mammalian nuclei. An in vivo counterpart of these results is that methylated CpGs are inaccessible to nucleases within nuclei. In contrast, non-methylated CpG sites, located mainly at CpG islands, and restriction sites not containing this dinucleotide, are relatively accessible. The possibility that DNA methylation acts through binding to specific proteins that could alter chromatin structure is discussed.