Epigenetic control of vasopressin expression is maintained by steroid hormones in the adult male rat brain

Abstract

Although some DNA methylation patterns are altered by steroid hormone exposure in the developing brain, less is known about how changes in steroid hormone levels influence DNA methylation patterns in the adult brain. Steroid hormones act in the adult brain to regulate gene expression. Specifically, the expression of the socially relevant peptide vasopressin (AVP) within the bed nucleus of the stria terminalis (BST) of adult brain is dependent upon testosterone exposure. Castration dramatically reduces and testosterone replacement restores AVP expression within the BST. As decreases in mRNA expression are associated with increases in DNA promoter methylation, we explored the hypothesis that AVP expression in the adult brain is maintained through sustained epigenetic modifications of the AVP gene promoter. We find that castration of adult male rats resulted in decreased AVP mRNA expression and increased methylation of specific CpG sites within the AVP promoter in the BST. Similarly, castration significantly increased estrogen receptor α (ERα) mRNA expression and decreased ERα promoter methylation within the BST. These changes were prevented by testosterone replacement. This suggests that the DNA promoter methylation status of some steroid responsive genes in the adult brain is actively maintained by the presence of circulating steroid hormones. The maintenance of methylated or demethylated states of some genes in the adult brain by the presence of steroid hormones may play a role in the homeostatic regulation of behaviorally relevant systems.

Fig. 1.

Castration effect on AVP mRNA in BST. Relative AVP mRNA expression within the BST in each treatment group. As expected, AVP is significantly reduced by CX and replacement with testosterone-filled capsules reinstates AVP expression in the BST (*P = 0.015; n ≥ 6). Error bars represent SEM.

Fig. 2.

Primer sequences used to assess relative methylation levels. For all primers forward and reverse primers are underlined, targeted CpG sites are highlighted and bolded. Base numbering from GenBank accession no. AF112363.1. HpaII restriction enzyme was used to bind highlighted CCGG site encompassed by primers in A–C. BstUI restriction enzyme was used to bind highlighted CGCG site encompassed by primer in D. (A) AVP promoter primer-targeted CpG site 1. (B) AVP promoter primer-targeted CpG site 2. (C) AVP promoter primer-targeted CpG site 3. (D) AVP promoter primer- targeted CpG site 4. This CGCG site is near a number of transcriptional regulators. Bases in bold lower-case type signify an ERE-half site (*ggtca*), GRE/PRE (*tgtcacaactgtcct*), and CRE (*tcccgtca*). (E) Forward and reverse primers for ERα promoter region are underlined; and the CGCG site at which the BstUI restriction enzyme binds is highlighted and bold. Base numbering from GenBank accession no. NM_012689.1.

Fig. 3.

Effects of CX and testosterone replacement on methylation of the AVP gene promoter in BST. (A) Relative methylation a region of the AVP promoter. Real-time PCR using primer, AVP promoter target site 1, did not reveal any difference in relative methylation levels between any of the treatment groups. (*P = 0.906; n ≥ 9). (B) Primer that encompassed the second target site did reveal a difference between groups, with relative methylation at low levels in control animals (Sham), relative methylation levels increasing with testosterone removal (CX), and relative methylation levels returning back to control levels when hormones were replaced (CX+T) (*P = 0.019; n ≥ 7). (C) Much like the methylation profile using the primer to target site 1, with the AVP promoter primer that encompassed a different target site, target site 3, no differences in relative methylation levels between groups were observed (*P = 0.780; n ≥ 9). (D) With the primer, AVP promoter-target site 4, robust differences in relative methylation between the controls (Sham), castrated (CX), and CX animals replaced with testosterone (CX+T) were observed. Relative methylation levels appeared to be low in control animals, but removal of hormones caused these levels to increasel replacement with hormones caused a decrease in relative methylation levels, which could be suggestive of possible demethylase activity increasing with hormonal replacement (*P < 0.001; n ≥ 8). Error bars represent SEM.

Fig. 4.

Castration (CX) effects on ERα expression and methylation in the BST. (A) Relative expression of ERα mRNA is significantly increased by CX and reduced with testosterone (CX+T) treatment (*P = 0.019; n ≥ 8). (B) Relative methylation of ERα promoter. Methylation of ERα promoter mirrors pattern observed in mRNA levels. CX reduces methylation, and testosterone replacement reinstates methylation to sham CX levels (*P = 0.028; n ≥ 9). Error bars represent SEM.