Effects of Early Life Stress on Epigenetic Changes of the Glucocorticoid Receptor 17 Promoter during Adulthood

Abstract

Growing evidence suggests that early life stress (ELS) has long-lasting effects on glucocorticoid receptor (GR) expression and behavior via epigenetic changes of the GR exon 1₇ promoter. However, it remains unclear whether ELS regulates histone modifications of the GR exon 1₇ promoter across the life span. We investigated the effects of maternal separation (MS) on histone acetylation and methylation of GR exon 1₇ promoter in the hippocampus, according to the age of adults. Depression-like behavior and epigenetic regulation of GR expression were examined at young and middle adulthood in mice subjected to MS from postnatal day 1 to 21. In the forced swimming test, young adult MS mice showed no effect on immobility time, but middle-aged MS mice significantly increased immobility time. Young adult and middle-aged MS mice showed decreased GR expression. Their two ages showed decreased histone acetylation with increased histone deacetylases (HDAC5) levels, decreased permissive methylation, and increased repressive methylation at the GR exon 1₇ promoter. The extent of changes in gene expression and histone modification in middle adulthood was greater than in young adulthood. These results indicate that MS in early life causes long-term negative effects on behavior via histone modification of the GR gene across the life span.

Keywords: depression; early life stress; epigenetic; glucocorticoid receptor; hippocampus; histone modification.

Figure 1

Schematic representation of the experimental design.

Figure 2

Effects of maternal separation (MS) on depression-like behavior in young and middle adulthood. Animals with MS and their age-matched controls were tested in young (PND 60) and middle (PND 240) adulthood using the FST. Data are expressed as the mean ± SEM (n = 7–13/group, ** p < 0.01, unpaired Student t-test).

Figure 3

Age-dependent changes in total glucocorticoid receptor (GR) expression following MS exposure. Levels of total GR mRNA were analyzed in young adulthood (PND 60) and middle (PND 240) adulthood mice exposed to MS using qRT-PCR. All quantities were normalized to GAPDH. Data are expressed as values relative to the CON group using the 2^−△△ct method, and represent the mean ± SEM (n = 10–13/group, ** p < 0.01, unpaired Student t-test).

Figure 4

Age-dependent changes in the level of histone H3 acetylation at the GR promoter 1₇ and HDAC5 expression following MS exposure. (A) ChIP assays were used to measure the level of acetylated histone H3 (Acetyl-H3) at the GR promoter 1₇ in the hippocampus using an antibody to Acetyl-H3. Data were normalized to input DNA (n = 10–13/group). (B) The HDAC5 mRNA level in the hippocampus was measured using qRT-PCR. All quantities were normalized to GAPDH (n = 9–13/group). These levels were analyzed in young (PND 60) and middle (PND 240) adulthood, respectively. Data were expressed as values relative to the CON group using the 2^−△△ct method and represent the mean ± SEM (* p < 0.05, ** p < 0.01, unpaired Student t-test).

Figure 4

Age-dependent changes in the level of histone H3 acetylation at the GR promoter 1₇ and HDAC5 expression following MS exposure. (A) ChIP assays were used to measure the level of acetylated histone H3 (Acetyl-H3) at the GR promoter 1₇ in the hippocampus using an antibody to Acetyl-H3. Data were normalized to input DNA (n = 10–13/group). (B) The HDAC5 mRNA level in the hippocampus was measured using qRT-PCR. All quantities were normalized to GAPDH (n = 9–13/group). These levels were analyzed in young (PND 60) and middle (PND 240) adulthood, respectively. Data were expressed as values relative to the CON group using the 2^−△△ct method and represent the mean ± SEM (* p < 0.05, ** p < 0.01, unpaired Student t-test).

Figure 5

Age-dependent changes in the levels of histone H3 methylation at the GR promoter 1₇ following MS exposure. ChIP assays were used to measure the level of acetylated histone H3 trimethylated K4 (H3K4me³, A) and K27 (H3K27me³, B) at GR promoter 1₇ in the hippocampus using antibodies to H3K4me³ and H3K27me³. These levels were analyzed in young (PND 60) and middle (PND 240) adulthood, respectively. Data were normalized to input DNA and are expressed as values relative to the CON group using the 2^−△△ct method, and represent mean ± SEM (n = 9–13/group, * p < 0.05, ** p < 0.01, unpaired Student t-test).

Figure 5

Age-dependent changes in the levels of histone H3 methylation at the GR promoter 1₇ following MS exposure. ChIP assays were used to measure the level of acetylated histone H3 trimethylated K4 (H3K4me³, A) and K27 (H3K27me³, B) at GR promoter 1₇ in the hippocampus using antibodies to H3K4me³ and H3K27me³. These levels were analyzed in young (PND 60) and middle (PND 240) adulthood, respectively. Data were normalized to input DNA and are expressed as values relative to the CON group using the 2^−△△ct method, and represent mean ± SEM (n = 9–13/group, * p < 0.05, ** p < 0.01, unpaired Student t-test).