Epigenetic regulation of the glucocorticoid receptor promoter 1(7) in adult rats

Abstract

Regulation of glucocorticoid receptor (GR) levels is an important stress adaptation mechanism. Transcription factor Nfgi-a and environmentally induced Gr promoter 1 7 methylation have been implicated in fine-tuning the expression of Gr 1 7 transcripts. Here, we investigated Gr promoter 1 7 methylation and Gr 1 7 expression in adult rats exposed to either acute or chronic stress paradigms. A strong negative correlation was observed between the sum of promoter-wide methylation levels and Gr 1 7 transcript levels, independent of the stressor. Methylation of individual sites did not, however, correlate with transcript levels. This suggested that promoter 1 7 was directly regulated by promoter-wide DNA methylation. Although acute stress increased Ngfi-a expression in the hypothalamic paraventricular nucleus (PVN), Gr 1 7 transcript levels remained unaffected despite low methylation levels. Acute stress had little effect on these low methylation levels, except at four hippocampal CpGs. Chronic stress altered the corticosterone response to an acute stressor. In the adrenal and pituitary glands, but not in the brain, this was accompanied by an increase in methylation levels in orchestrated clusters rather than individual CpGs. PVN methylation levels, unaffected by acute or chronic stress, were significantly more variable within- than between-groups, suggesting that they were instated probably during the perinatal period and represent a pre-established trait. Thus, in addition to the known perinatal programming, the Gr 1 7 promoter is epigenetically regulated by chronic stress in adulthood, and retains promoter-wide tissue-specific plasticity. Differences in methylation susceptibility between the PVN in the perinatal period and the peripheral HPA axis tissues in adulthood may represent an important "trait" vs. "state" regulation of the Gr gene.

Figure 1. Plasma corticosterone levels (A), total Gr levels (B) and sum of methylation levels (C) after exposure to a single acute stressor. Significance levels were in comparison to the no stress group. *p < 0.05. Panels A and B are mean ± SD. Panel C box plots show the 25th percentile, median and 75th percentile.

Figure 2. Quantification of Ngfi-a mRNA (A), 1₇ mRNA (B) and the correlation of 1₇ (C) and total Gr (D) transcripts with the sum of methylation throughout the promoter after acute stress by in situ hybridization in the paraventricular nucleus of the hypothalamus (PVN), the prefrontal cortex (CTX) and in the CA1 area, representing the hippocampus (HIP) and pyrosequencing. Panels A and B are mean +/− SD. Significance levels were in comparison to the no stress group. *p < 0.05.

Figure 3. Methylation levels of individual CpG dinucleotides in the 1₇ promoter in the PVN (A) and the hippocampus (B) after a single acute stressor. Methylation levels are expressed for all animals in columns by color[0% (blue) to 75% (yellow)] and as the group mean ± SD. Significance levels were in comparison to the no stress group. *p < 0.05. CpG sites are numbered according to Weaver et al.

Figure 4. Exposure to 21 d chronic stress alters weight gain (A), relative adrenal gland weight (B) and relative pituitary weight (C). All panels are mean +/− SD. Significance was assessed by ANOVA, and *p < 0.05 for pairwise Tukey post-hoc comparison and correction.

Figure 5. Change in plasma corticosterone response to a single acute stressor after 14 d exposure to chronic stress (A), total Gr mRNA levels (B) and the sum of methylation levels throughout promoter 1₇ (C) after chronic restraint or psychosocial stress. Panels A and B are mean ± SD. Panel C box plots show the 25th percentile, median and 75th percentile.

Figure 6. Methylation levels of individual CpG dinucleotides in the 1₇ promoter in the PVN (A) and the hippocampus (B) after 21 d chronic stress. Methylation levels are expressed for all animals in columns by color [0% (blue) to 75% (yellow)] and as the group mean ± SD. Significance levels were in comparison to the no stress group. *p < 0.05. CpG sites are numbered according to Weaver et al.

Figure 7. Pearson correlation heat maps for individual CpGs in the pituitary gland (A) the adrenal gland (B), and for the sum of methylation between all tissues (C) after exposure to chronic stress. The sum of methylation levels for individual animal in (filled circles, controls; empty circles, restrain stress; triangles, psychosocial stress) (D) Pearson correlation coefficients (panels A−C) are expressed by color [0 (blue) to 1 (yellow)]. *p < 0.01, ◊p < 0.01.