Epigenetic regulation of estrogen receptor α contributes to age-related differences in transcription across the hippocampal regions CA1 and CA3

Abstract

The expression of estrogen receptor alpha (ERα) varies across brain regions and changes with age and according to the previous history of estradiol exposure. ERα is regulated by a number of mechanisms including the level of mRNA (Esr1) expression. For this study, we took advantage of regional differences in hippocampal ERα expression to investigate DNA ERα promoter methylation at CpG dinucleotide sites as a potential epigenetic mechanism for regulating gene expression. Young and aged female Fischer 344 rats were ovariectomized, and Esr1 expression and ERα promoter methylation were examined in hippocampal regions CA1 and CA3, either 3 or 14 weeks following surgery. The results indicate that reduced Esr1 expression in region CA1 relative to CA3 was associated with an increase in DNA methylation in region CA1, particularly for the first CpG site. Additionally, differential methylation of distal CpG sites, 11-17, was associated with altered Esr1 expression during aging or following long-term hormone deprivation. The results support the idea that methylation of site 1 may be the primary regulatory region for cross-regional patterns in ERα expression, while distal sites are modifiable across the life span and may act as a feedback mechanism for ERα activity.

Keywords: Aging; DNA methylation; Estrogen receptor alpha; Hippocampus; Transcription.

Figure 1

Experimental research design. Young (3 months) and aged (18 months) F344 female rats were handled for 5 minutes a day for 1 week prior to OVX surgery, and sacrificed following a short-term E2 deprivation time (3 weeks) or a long-term E2 deprivation time (14 weeks).

Figure 2

Illustration of differences in Esr1 expression. The CA1 region from young short-term rats were used as the calibrator sample in calculating the ΔΔCT. For A–C, each bar represents the mean (+SEM) of Esr1 expression for the relevant variable, collapsed across all other variables. Esr1 expression is increased with A) age, B) long-term OVX, and C) in region CA3 relative to region CA1. Asterisks in A–C indicated a significant (p < 0.001) main effect. D) Examination of interactions, where each bar represents the mean (+SEM) of Esr1 expression for young (n=5–6; open bars) and aged (n=6; filled bars), across regions CA1 and CA3 according to OVX duration (short-term: 3 wk, long-term: 14 wk). Asterisk indicates a significant (p < 0.05) increase in region CA1 relative to young short-term OVX CA1. Pound sign indicates a significant (p < 0.05) increase in region CA3 relative to young short-term OVX CA3. For aged animals, long-term OVX was associated with increased expression relative to aged short-term OVX for region CA1 only (α, p < 0.05).

Figure 3

Site specific DNA methylation of CpG sites across CA1 and CA3 regions. A) Each bar represents the mean (+SEM) DNA methylation ratio for sites 1–17 in CA1 (open bars) and CA3 (filled bars). Regional differences in CpG site methylation included increased methylation of site 1 in CA1 and sites 14 and 15 in CA3 (n=20 per region). Asterisk indicates a significant (p<0.05) increase in methylation. B) Hierarchical clustering and heatmap of the clones which contained ≥ 1 methylated site (CA1: 104 clones (51%) & CA3: 84 clones (41%).

Figure 4

ERα promoter DNA methylation is altered in regions CA1 and CA3 during aging. Age-related differences in CpG site methylation for A) area CA1 were observed as an increase in DNA methylation for site 15 for young animals (open bars, n=10) relative to aged animals (filled bars, n=10). B) For area CA3, site 17 contained higher methylation in young rats, and sites 11 and 12 showed higher methylation in aged rats. Due to the influence of OVX duration on transcription, young animals were separated into short-term OVX (Young-s) and long-term OVX (young-l) and compared to all aged animals for sites 11–17. Specific differences were observed in CA1 for C) site 11 and D) site 14. E) For region CA3, a difference was observed for site 13. Bars represent the mean (+SEM) of the methylation ratio. Asterisks indicate a significant (p < 0.05) increase in A and B and a significant (p < 0.05) difference from young short-term in C–E.