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. 2016 Jan:77:42-52.
doi: 10.1016/j.yhbeh.2015.05.022. Epub 2015 Jun 27.

Natural variation in maternal care and cross-tissue patterns of oxytocin receptor gene methylation in rats

Annaliese K Beery  1 , Lisa M McEwen  2 , Julia L MacIsaac  2 , Darlene D Francis  3 , Michael S Kobor  2
Affiliations

Natural variation in maternal care and cross-tissue patterns of oxytocin receptor gene methylation in rats

Annaliese K Beery et al. Horm Behav. 2016 Jan.

Abstract

This article is part of a Special Issue "Parental Care". Since the first report of maternal care effects on DNA methylation in rats, epigenetic modifications of the genome in response to life experience have become the subject of intense focus across many disciplines. Oxytocin receptor expression varies in response to early experience, and both oxytocin signaling and methylation status of the oxytocin receptor gene (Oxtr) in blood have been related to disordered social behavior. It is unknown whether Oxtr DNA methylation varies in response to early life experience, and whether currently employed peripheral measures of Oxtr methylation reflect variation in the brain. We examined the effects of early life rearing experience via natural variation in maternal licking and grooming during the first week of life on behavior, physiology, gene expression, and epigenetic regulation of Oxtr across blood and brain tissues (mononucleocytes, hippocampus, striatum, and hypothalamus). Rats reared by "high" licking-grooming (HL) and "low" licking-grooming (LL) rat dams exhibited differences across study outcomes: LL offspring were more active in behavioral arenas, exhibited lower body mass in adulthood, and showed reduced corticosterone responsivity to a stressor. Oxtr DNA methylation was significantly lower at multiple CpGs in the blood of LL versus HL males, but no differences were found in the brain. Across groups, Oxtr transcript levels in the hypothalamus were associated with reduced corticosterone secretion in response to stress, congruent with the role of oxytocin signaling in this region. Methylation of specific CpGs at a high or low level was consistent across tissues, especially within the brain. However, individual variation in DNA methylation relative to these global patterns was not consistent across tissues. These results suggest that blood Oxtr DNA methylation may reflect early experience of maternal care, and that Oxtr methylation across tissues is highly concordant for specific CpGs, but that inferences across tissues are not supported for individual variation in Oxtr methylation.

Keywords: Anxiety behavior; Concordance; Cross-tissue; DNA methylation; Epigenetic; Maternal care; Natural variation; Oxtr; Oxytocin; Oxytocin receptor.

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Figures

Figure 1
Figure 1
Location and components of the target sequence within the Oxtr promoter in rats. (A) The Oxtr gene lies on the reverse (-) strand on chromosome 4. The target region begins approximately 1.2kb upstream and encompasses a CpG island and region of high conservation across vertebrates (alignment data from UCSC genome browser). (B) Epigram of CpGs within the target region, color-coded by mean % methylation in each tissue. Methylation was generally higher in PBMCs than in the brain. Open gray circles represents units providing no data in Sequenom assays because of peak mass; open black circles depict data not analyzed because of >2 CpGs clustered in one unit.
Figure 2
Figure 2
Physiological and behavioral variation as a function of early maternal care. (A) LL offspring were more active in the light-dark box as well as other behavioral tests. (B) HL and LL offspring did not differ in body weight at weaning, but HL offspring of both sexes weighed more in adulthood, including at week 16 (effect of maternal care: p <0.01, effect of sex: p <0.0001). (C) Corticosterone response to a stressor was greater in HL offspring, with similar peak timing but greater overall CORT secretion relative to LL offspring (area under the curve, females not tested). CORT AUC was associated with anxiety-like behaviors (see text). * p < 0.05, ** p < 0.01. Error bars depict ± SEM.
Figure 3
Figure 3
Oxtr methylation across 12 Sequenom units in HL and LL offspring in peripheral blood mononuclear cells (A) or hippocampus (B). In blood, 4 of 12 units examined exhibited significantly higher methylation in HL offspring than LL offspring, representing a combined probability of 0.0003. In brain, 1 of 12 units differed with maternal care, but such a result would be expected by chance (p=.45) and is not meaningful. * = p < 0.05 prior to correction.
Figure 4
Figure 4
Methylation measures across neural tissues. (A) Mean methylation levels were assessed by pyrosequencing across 25 CpGs in three brain tissues, including hippocampus. Methylation differed significantly by both CpG and brain region (each p<0.0001, 2-way ANOVA) and showed both similarities and differences in CpG methylation patterns relative to PBMCs (figure 3B). (B) Methylation levels (measured by pyrosequencing) were highly concordant between hippocampus and other brain regions (striatum and hypothalamus). (C) To a lesser extent, blood measures of methylation were associated with hippocampal methylation (both Sequenom data). Data are shown for each of 25 CpGs (4B) or 12 CpG containing units (4C), averaged across subjects.
Figure 5
Figure 5
Individual methylation values were not correlated across brain tissues, despite tissue concordance at the group level. For each CpG, we computed the Pearson correlation coefficient r between the methylation values for matched samples in pairs of brain regions (bars). Dark and light shaded regions represent 95% and 99% thresholds, respectively, of distributions of possible correlation coefficients determined from 10,000 permutations of the measured values among the individuals. These distributions represent the null hypothesis that an individual methylation value in one brain region does not help to predict the value in another region in the same animal. (A) Correlations based on pyrosequencing data for matched samples passing validation in both hippocampus (HC) and hypothalamus (Hypo). Correlations for individuals at each CpG were either weak (.2< r <.3) or absent (r <.2), and none were significant, even prior to correction for multiple comparisons. (B) Correlations for matched samples passing validation in both hippocampus and striatum (Str). Two correlations (CpG 1 and 11) were individually significant prior to but not following correction, and this result could be expected by chance. Correlations between hippocampus and blood (described in the text) yielded similar results, and no particular CpG yielded consistently high correlation across multiple tissues.
Figure 6
Figure 6
Oxtr expression, quantified by qPCR as fold-change relative to Actb. (A) Relative Oxtr expression varied significantly by brain region with the highest expression levels in the hippocampus and the lowest (but readily detectable levels) in the hypothalamus. Transcript levels were too low to reliably assess in PBMCs (not shown). (B) Relative Oxtr expression was negatively correlated with corticosterone secretion in response to a stressor. **** = p < 0.0001 (Tukey's HSD)
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