Early life stress induces age-dependent epigenetic changes in p11 gene expression

Early life stress (ELS) causes long-lasting changes in depression-like behaviors through epigenetic mechanisms. However, little is known about the effects of ELS in adulthood, specically across different age groups. In this study, the epigenetic modications of p11 expression in adult mice subjected to ELS were investigated in different stages of adulthood. Pups experienced maternal separation (MS) for 3 h daily from postnatal day 1 to 21. At young and middle adulthood, behavior phenotypes, hippocampal p11 expression levels, and levels of histone acetylation and methylation and DNA methylation at the hippocampal p11 promoter were measured. Middle-aged, but not young adult, MS mice exhibited depression-like behavior in the forced swimming test. Concurrent with reduced hippocampal p11 levels, mice in both age groups showed decreases in histone acetylation and activating histone methylation as well as increases in repressive histone methylation at the p11 promoter. The extent of the reduction in gene expression and histone acetylation was much higher in middle than in young adulthood. Moreover, DNA methylation analysis of the p11 promoter revealed increased CpG methylation in middle-aged MS mice only. The results highlight the age-dependent deleterious effects of ELS on depression-like behavior and on the epigenetic modications of p11 transcription.


Introduction
Children exposed to early life stress (ELS) such as neglect and abuse have a signi cantly increased risk of developing depression 1,2 . In human and animal studies, ELS has been reported to induce a depressionlike phenotype in adulthood 3,4 . These studies are focused on the epigenetic mechanisms, for example, DNA methylation and histone modi cation, by which ELS may alter the expression of genes involved in the stress response, including brain-derived neurotrophic factor (BDNF), glucocorticoid receptor (GR; NR3C1), and corticotrophin-releasing factor 3,4 . The emphasis of these papers is mainly on the detrimental effects of ELS. However, little is known regarding the effects of ELS on behaviors and epigenetic mechanisms over the lifespan, especially among different adult age groups.
DNA methylation and histone modi cation, two representative epigenetic mechanisms, control gene transcription by affecting chromatin remodeling 5 . DNA methylation at the 5'-C-phosphate-G-3' (CpG) dinucleotide is classically associated with transcriptional repression 6 . Regions with a high density of CpGs are known as CpG islands; they are often found in the gene regulatory regions of promoters.
Methylated CpGs in a promoter can induce gene silencing by blocking transcription factor binding or by attracting proteins that cause chromatin remodeling 6 . Histone modi cations affect chromatin structure via post-translational modi cation, for example, acetylation and methylation, of the lysine (K) residues in the histone tails 7 . Acetylation of histone H3 or H4 relaxes the interaction between DNA and histone, allowing the transcriptional machinery access to the promoter, thereby activating transcription 7 . K4 and K19 on histone H3 are commonly modi ed in gene transcription activation 8 . Histone methylation, in contrast, is associated with both gene activation (H3K4 and H3K36) and repression (H3K9, H3K27, and H4K20), depending on the K residue methylated and its valence state (i.e., mono-, di-, or tri-methylation) 9 .
In animals that had experienced ELS, those in young adulthood exhibit decreased H3K9 dimethylation (me2) at the BNDF IV promoter and, accordingly, increased BDNF IV expression, whereas those in middle adulthood show increased repressive H3K9me2 at the BDNF IV promoter and concurrent decreased BDNF IV expression 10 . Additionally, ELS was found to be associated with an age-dependent decline in cognition. In another study, ELS was demonstrated to be associated with long-term deleterious effects on the regulation of GR expression via histone acetylation and methylation as well as on depression-like behavior 11 . The effects of ELS on the epigenetic regulation of other stress-related genes besides BDNF and GR are less well-known and warrant further investigation. P11 (S100A10) plays an important role in depression and antidepressant action 12 . Patients with depression show reduced p11 levels in the brain, and p11 knockout mice display a depression-like phenotype [13][14][15][16] . Antidepressant therapy, including selective serotonin reuptake inhibitors, enhances p11 expression 15,17,18 . Furthermore, p11 gene transfer therapy effectively reverses depression-like behavior in mice 14 . In addition, p11 is critical in the antidepressant actions of BDNF 18 . However, whether epigenetic regulation of the p11 gene following ELS is altered across the life span is unknown. Maternal separation (MS) is widely used as a model to examine the effects of ELS 19 . In this study, the effects of MS on histone modi cation and DNA methylation at the p11 promoter in the hippocampus was investigated as the animals aged.

Results
Effects of MS on depression-like behavior and hippocampal p11 expression in young adult and middleaged mice FST was used to evaluate depression-like phenotypes. Control and MS animals in young adulthood did not signi cantly differ in immobility time. Conversely, middle aged-MS animals were signi cantly more immobile than controls, indicating a depression-like phenotype (control = 31.03 ± 7.86 sec, MS = 78.18 ± 10.82 sec; t = 3.411, p = 0.003; Fig. 3A).
Effects of MS on histone acetylation and methylation at the hippocampal p11 promoter of young adult and middle-aged mice Epigenetic histone modi cations at the p11 promoter were examined in control and MS mice in young and middle adulthood. Histone acetylation at the hippocampal p11 promoter was reduced in both young adult (control = 1.00 ± 0.11, MS = 0.73 ± 0.09; t = 3.172, p = 0.005) and middle-aged (control = 1.00 ± 0.09, MS = 0.38 ± 0.13; t = 8.378, p < 0.001) MS mice (Fig. 4A). The reduction was about 45% greater in middleaged MS animals compared to those in young adulthood.
Effects of MS on DNA methylation at the hippocampal p11 promoter of young adult and middle-aged mice CpG methylation at the p11 promoter was examined in the hippocampus of young adult and middle-aged MS and control animals. While DNA methylation at the p11 promoter did not differ between MS and control mice in young adulthood, it was signi cantly increased in the middle-aged MS group (control = 100 ± 11.36%, MS = 141.20 ± 7.89%; t = 3.051, p = 0.006; Fig. 5).

Discussion
This paper reports that MS in early life exerts persistent effects on depression-like behavior and on epigenetic mechanisms associated with decreased p11 expression in adulthood, and these effects become more pronounced with age. MS had no effect on behavior in young adult mice, but depressionlike behavior appeared in middle age. The distinct behavioral changes observed between young adult and middle-aged MS mice are likely to be due to long-term deleterious effects associated with changes in hippocampal p11 expression via histone modi cation and DNA methylation at its promoter.
In a past study, young adult (2 months) MS mice did not exhibit depression-like behavior in the FST; however, MS mice in middle adulthood (8 months) showed increased immobility time 11 , a nding replicated here. Most MS studies report depression-like behaviors in the FST in young adult animals 20 , although some papers are inconsistent. The ndings seem to depend on the strain of mice used in the study. Similar to the MS model used here, Ruiz et al. (2018) found that MS (3 h daily from PND 1 to PND 14) in rats increased immobility time at two ages; furthermore, middle-aged (10 months) rats had longer immobility times than young adult (4 months) rats 21 . Taken together, early MS seems to induce more severe depressive-like behaviors in middle than in young adulthood.
In this study, MS is associated with a reduction of hippocampal p11 expression at two time points in adulthood. Previous studies have demonstrated that chronic stress in adults induces p11 loss, as well as depression-like behaviors 22,23 . This study is the rst to show an association between ELS and p11 levels. In a study using yeast two-hybrid screening, p11 was initially identi ed as a binding protein to the serotonin 1B (5-HT 1B ) receptor; the 5-HT 1B receptor regulates serotonin neurotransmission 15 . p11 interacts with the 5-HT 1B receptor by increasing its tra cking to the cell surface, where it binds to serotonin released from presynaptic neurons. Consequently, 5-HT 1B receptor signaling e cacy is enhanced. In mice, p11 overexpression increases 5-HT 1B receptor function and is associated with reduced immobility in the tail suspension test. In contrast, in p11 knockout mice, the number of 5-HT 1B receptors is reduced at the cell membrane. Notably, MS (3 h daily from PDN 2 to PND 13) in a rat ELS model reduced hippocampal 5-HT 1B receptor binding when assayed through [ 125 I] cyanopindolol autoradiography 24 . The reduced binding may result from ELS-induced reductions of p11 levels.
Reduced hippocampal p11 expression in young adult and middle-aged MS animals was associated with altered histone modi cations (i.e., signi cant decreases in H3 acetylation and H3K4me3 and a signi cant increase in H3K27me3) at the p11 promoter region. Moreover, p11 expression levels and H3 acetylation after MS were more severely perturbed in middle adulthood. Suri et al. (2014) demonstrated that MS induces opposing age-dependent effects on the expression of histone modifying enzymes such as histone deacetylase (Hdac1 to Hdac11) and histone methyltransferase (G9a and Suv391) in young adulthood (2 months) versus middle adulthood 25 . Nonetheless, the altered expression of these enzymes did not affect overall H3 acetylation, H3K9me2, and H3K9me3 at either of the ages. Although histone modifying enzymes may not globally change histone modi cation across the lifespan, these enzymes may differentially regulate histone modi cations at the promoters of stress-related genes during aging. A loss of balance between epigenetic modi cations during aging is referred to as "epigenetic drift" 26 . The global distribution of many histone acetylation and methylation modi cations (i.e., H3K27me3, H3K56ac, and H4K16ac) has been found to be altered during aging in various organisms 27,28 . Accordingly, MS animals in middle age, but not young adulthood, show histone modi cations associated with decreased BDNF IV expression concurrent with impairments in hippocampal-dependent cognition 10 . A similar agedependent effect on histone acetylation and methylation at the GR exon I 7 promoter has been observed 11 .
Taken together, middle-aged MS animals may exhibit more severe changes in histone modi cation than young adult MS animals due to age-related epigenetic drift.
Increased DNA methylation in p11 promoter regions following MS were only present in middle-aged mice, not those in young adulthood. The increased DNA methylation, along with the altered histone modi cations, could account for the reduced p11 expression in middle-aged MS animals. In accordance, a negative correlation between p11 gene transcription and CpG DNA methylation at the p11 promoter has been reported 29 . Moreover, reduced p11 levels in a genetic rodent model of depression were associated with higher DNA methylation, while escitalopram treatment elevated p11 levels and reduced DNA methylation of the p11 promoter.
In rodents, the absence of maternal care such as licking, grooming, and arched-back nursing, is associated with hypothalamic-pituitary-adrenal (HPA) axis dysregulation as identi ed by increased glucocorticoid levels 30 . The lack of maternal care modi es DNA methylation, resulting in decreased hippocampal GR expression 31 . Accordingly, adults with histories of childhood maltreatment show increased DNA methylation of the GR exon I F promoter and, consequently, increased HPA activity 32,33 . GR acts as a ligand-activated transcription factor. When activated by glucocorticoids, GR translocates to the nucleus and binds to GR binding sites within the promoters of target genes, thereby activating or repressing their expression 34 . p11 is a target gene for GRs. Putative GR binding sites were identi ed using the web-based tools described in the Materials and Methods section; this site includes one CpG site (Fig. 2). Zhang et al. (2008) reported that GR increases p11 promoter activity via the interaction of glucocorticoid-bound GR with GR binding sites 35 . Thus, elevated GR levels may activate p11 transcription. Hippocampal GR expression levels have been found to be reduced in young adult and middle-aged MS animals and, furthermore, the reduction in middle adulthood was remarkably higher than in young adulthood 11 . Thus, the differential reduction in p11 levels in middle-aged MS animals could be due to CpG methylation of the GR binding site, which prevents GR from easily accessing the p11 promoter, resulting in reduced p11 transcriptional activity. Nevertheless, further experiments are needed to address whether MS causes an age-dependent decrease in nuclear GR levels.
In conclusion, this study demonstrates that ELS induces long-term deleterious effects on depression-like behavior and hippocampal p11 expression through altered epigenetic modi cations of the p11 promoter during adulthood. Furthermore, the deleterious effects are more severe with age.

Maternal separation (MS)
Litters were divided randomly into control and MS groups. Pups in the MS group were separated from their mothers for 3 h daily starting on postnatal day (PND) 1 to PND 21 as described previously 37 . Pups in the control group were left undisturbed except during routine animal facility handling. Male control and MS mice were assayed at either 2 months old (young adulthood) or at 8 months old (middle adulthood; Fig. 1).

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To assess depression-like behavior, the FST was performed in control and MS mice at either young (n = 10-12/group) or middle (n = 11-13/group) adulthood as described previously 37 . Mice were individually placed in transparent plastic cylinders (25 cm height × 10 cm diameter) containing 12 cm of water (23-25 °C) for 7 min and recorded on video. After an initial 2-min habituation period, the time spent immobile during the remaining 5 min was analyzed.
Measurement of mRNA levels using quantitative real-time polymerase chain reaction (qRT-PCR) Following the FST, whole brains were extracted (n = 10-12/group, young adulthood; n = 11-13/group, middle adulthood). The hippocampus was dissected from the brain; RNA isolation, cDNA synthesis, and qRT-PCR were performed on the hippocampal tissue as described previously 37 . Gene-speci c primers (Table 1)   Chromatin immunoprecipitation (ChIP) assays Chromatin was extracted from the isolated hippocampus using a standard protocol (SimpleChIP® Plus Enzymatic Chromatic IP Kit; Cell Signaling, Beverly, MA, USA) as described previously (n = 10-12/group, young adulthood; n = 11-13/group, middle adulthood) 37 . Primers were designed around a putative p11 promoter region (Table 1 and Fig. 2). Because the p11 promoter is not well characterized in mice, the region of interest was created based on previous data on epigenetic alterations in the rat p11 promoter 29,38 . The putative proximal promoter of the p11 gene (~ 700 base-pair upstream region) includes transcription factor binding sites generated by PROMO 39,40 and MotifMap 41,42 , two freely available web-based tools that identify presumptive transcription factor binding sites (Fig. 2).

Methylation-speci c polymerase chain reaction (MSP) analysis
Genomic DNA was extracted from the hippocampus using a QIAGEN DNA prep kit (51036; Valencia, CA, USA) and treated with bisul te using an EpiTect® Bisul te kit (59104; QIAGEN). To determine the DNA methylation status of CpG in the p11 promoter region, a qRT-PCR was performed on the same amount of bisul te-treated DNA using an EpiScope® MSP kit (#R100A; TaKaRa, Otsu, Japan) containing SYBR green (TaKaRa). Speci c primers for methylated or unmethylated p11 promoters were designed using MethPrimer 43 . Primers include either a 150 base-pair (methylation-speci c) or 155 base-pair (unmethylation-speci c) region with 5 CpGs sites in the p11 promoter region (Fig. 2). Primer sequences are listed in Table 1. The p11 promoter region was con rmed to be ampli ed with methylation-and unmethylation-speci c primers ( Figure S2). The qPCR reaction conditions were as follows: initial denaturation at 95 °C for 30 sec, followed by 40 cycles of denaturation at 98 °C for 5 sec, annealing at 53 °C for 30 sec, and extension at 72 °C for 1 min. Ct values were normalized to GAPDH, and differences in methylation and unmethylation between control and MS groups (n = 10-12/group, young adulthood; n = 11-13/group, middle adulthood) were calculated using the 2 −△△Ct comparative Ct method. Relative levels of methylated DNA (%) were calculated according to the following formula: methylated rate (%) = methylated DNA / (methylated DNA + unmethylated DNA) × 100.

Statistical analysis
GraphPad Prim 8.0 (La Jolla, USA) was used for statistical analysis. All data are presented as the mean ± standard error of the mean (SEM). Because control and MS groups were compared in young or middle adulthood, the data were analyzed using unpaired Student's t-tests.

Data Availability
The data ate availability from the corresponding author upon request.