Combined epigenetic and intraspecific variation of the DRD4 and SERT genes influence novelty seeking behavior in great tit Parus major

Abstract

DNA methylation is one of the main epigenetic mechanisms that can regulate gene expression and is an important means for creating phenotypic variation. In the present study, we performed methylation profiling of 2 candidate genes for personality traits, namely DRD4 and SERT, in the great tit Parus major to ascertain whether personality traits and behavior within different habitats have evolved with the aid of epigenetic variation. We applied bisulphite PCR and strand-specific sequencing to determine the methylation profile of the CpG dinucleotides in the DRD4 and SERT promoters and also in the CpG island overlapping DRD4 exon 3. Furthermore, we performed pyrosequencing to quantify the total methylation levels at each CpG location. Our results indicated that methylation was ∼1-4% higher in urban than in forest birds, for all loci and tissues analyzed, suggesting that this epigenetic modification is influenced by environmental conditions. Screening of genomic DNA sequence revealed that the SERT promoter is CpG poor region. The methylation at a single CpG dinucleotide located 288 bp from the transcription start site was related to exploration score in urban birds. In addition, the genotypes of the SERT polymorphism SNP234 located within the minimal promoter were significantly correlated with novelty seeking behavior in captivity, with the allele increasing this behavior being more frequent in urban birds. As a conclusion, it seems that both genetic and methylation variability of the SERT gene have an important role in shaping personality traits in great tits, whereas genetic and methylation variation at the DRD4 gene is not strongly involved in behavior and personality traits.

Keywords: DRD4; SERT; methylation; novelty seeking; personality traits; urbanization.

Figure 1.

Structure and methylation profile of the DRD4 gene. (a) Schematic representation of the DRD4 gene. Horizontal bars show the number and location of CpG islands and gray boxes represent exons. The location of SNPs is shown by black bars. (b) Methylation status at DRD4 locus in brain and blood-derived DNA samples. The left panel shows the methylation profile of the promoter and the right panel shows the results for the CpG island within exon 3. Each circle represents a single CpG dinucleotide on a DNA strand. (•): methylated cytosine; (○): unmethylated cytosine. (c) Methylation percentage of DRD4 promoter and exon 3 regions comparing urban and forest populations. Differences are significant (see text).

Figure 2.

Structure and methylation profile of the SERT promoter. (a) The Schematic representation of SERT gene. Gray boxes showing the location of CpG sites and the gray box represents the exon. The location of SNPs is shown by black bars. (b) The methylation status at 2 CpG sites in the SERT promoter in brain and blood-derived samples. (c) Variation in methylation percentage of second CpG site in the SERT promoter, according to habitat and SNP290 genotype. The black data points represent forest birds and the gray urban birds. Methylation percentage was both affected by genotype and habitat, but the lack of significant interaction suggests that the difference between habitats was similar for the different genotypes (see text for tests).

Figure 3.

Exploration score of wild great tits, measured as number of movements during 2 min in a standard room with five artificial trees, in relation to methylation level of SERT second CpG site. We provide independent figures for urban (a) and forest (b) great tits (urban: r = 0.37, P = 0.06; forest: r = −0.13, P = 0.40).

Figure 4.

Survivorship plot function for latency to approach a novel object (a penlight battery on the feeder) by wild great tits according to SERT SNP234 genotypes. Black line represents TT birds and dash line represents TA birds. The figure shows the proportion of great tits that do not approach the object (‘surviving’) up to the respective time interval.