Methylation Patterns of the FKBP5 Gene in Association with Childhood Maltreatment and Depressive Disorders.

Childhood maltreatment is an important risk factor for adult depression and has been associated with changes in the hypothalamic pituitary adrenal (HPA) axis, including cortisol secretion and methylation of the FKBP5 gene. Furthermore, associations between depression and HPA changes have been reported. This study investigated the associations of whole-blood FKBP5 mRNA levels, serum cortisol levels, childhood maltreatment, and depressive symptoms with the whole-blood methylation status (assessed via target bisulfite sequencing) of 105 CpGs at the FKBP5 locus using data from the general population-based Study of Health in Pomerania (SHIP) (N = 203). Both direct and interaction effects with the rs1360780 single-nucleotide polymorphism were investigated. Nominally significant associations of main effects on methylation of a single CpG site were observed at intron 3, intron 7, and the 3'-end of the gene. Additionally, methylation at two clusters at the 3'-end and intron 7 were nominally associated with childhood maltreatment × rs1360780 and depressive symptoms × rs1360780, respectively. The results add to the understanding of molecular mechanisms underlying the emergence of depression and could aid the development of personalised depression therapy and drug development.

Extensive evaluation of DNA methylation of functional elements in the murine Fkbp5 locus using high-accuracy DNA methylation measurement via targeted bisulfite sequencing.

FKBP5 is an important stress-regulatory gene implicated in stress-related psychiatric diseases. Single nucleotide polymorphisms of the FKBP5 gene were shown to interact with early life stress to alter the glucocorticoid-related stress response and moderate disease risk. Demethylation of cytosine-phosphate-guanine-dinucleotides (CpGs) in regulatory glucocorticoid-responsive elements was suggested to be the mediating epigenetic mechanism for long-term stress effects, but studies on Fkbp5 DNA methylation (DNAm) in rodents are so far limited. We evaluated the applicability of high-accuracy DNA methylation measurement via targeted bisulfite sequencing (HAM-TBS), a next-generation sequencing-based technology, to allow a more in-depth characterisation of the DNA methylation of the murine Fkbp5 locus in three different tissues (blood, frontal cortex and hippocampus). In this study, we not only increased the number of evaluated sites in previously described regulatory regions (in introns 1 and 5), but also extended the evaluation to novel, possibly relevant regulatory regions of the gene (in intron 8, the transcriptional start site, the proximal enhancer and CTCF-binding sites within the 5'UTR). We here describe the assessment of HAM-TBS assays for a panel of 157 CpGs with possible functional relevance in the murine Fkbp5 gene. DNAm profiles were tissue-specific, with lesser differences between the two brain regions than between the brain and blood. Moreover, we identified DNAm changes in the Fkbp5 locus after early life stress exposure in the frontal cortex and blood. Our findings indicate that HAM-TBS is a valuable tool for broader exploration of the DNAm of the murine Fkbp5 locus and its involvement in the stress response.

Subjective mental health, incidence of depressive symptoms in later life, and the role of epigenetics: results from two longitudinal cohort studies.

The role of self-perceived general health in predicting morbidity and mortality among older people is established. The predictive value of self-perceived mental health and of its possible biological underpinnings for future depressive symptoms is unexplored. This study aimed to assess the role of mental health-related quality of life (HRQOL) and of its epigenetic markers in predicting depressive symptoms among older people without lifetime history of depression. Data were based on a subgroup (n = 1 492) of participants of the longitudinal ESTHER study. An epigenome-wide association study (EWAS) of mental HRQOL was conducted using DNA from baseline whole blood samples and logistic regression analyses were performed to assess the predictive value of methylation beta values of EWAS identified CpGs for incidence of depressive symptoms in later life. The methylation analyses were replicated in the independent KORA cohort (n = 890) and a meta-analysis of the two studies was conducted. Results of the meta-analysis showed that participants with beta values of cg27115863 within quartile 1 (Q1) had nearly a two-fold increased risk of developing depressive symptoms compared to participants with beta values within Q4 (ORQ1vsQ4 = 1.80; CI 1.25-2.61). In the ESTHER study the predictive value of subjective mental health for future depressive symptoms was also assessed and for 10-unit increase in mental HRQOL scores the odds for incident depressive symptoms were reduced by 54% (OR 0.46; CI 0.40-0.54). These findings suggest that subjective mental health and hypomethylation at cg27115863 are predictive of depressive symptoms, possibly through the activation of inflammatory signaling pathway.

Intergenerational Effects of Maternal Holocaust Exposure on FKBP5 Methylation.

OBJECTIVE: There is growing evidence that exposure to trauma prior to conception can affect offspring. The authors have reported that adult offspring of Holocaust survivors showed lower methylation of FK506 binding protein 5 (FKBP5) intron 7, site 6 compared with Jewish comparison volunteers. The present study sought to replicate this finding in a larger sample and to examine parental and offspring correlates of observed effects. METHODS: Cytosine methylation was measured in blood using pyrosequencing. The independent replication sample consisted of 125 Holocaust offspring and 31 control subjects. Additional analyses, performed in a larger sample of 147 offspring and 40 control subjects that included the 31 previously studied participants, examined associations of parental trauma-related variables (i.e., sex of the exposed parent, parental posttraumatic stress disorder, age at Holocaust exposure) and offspring characteristics (i.e., childhood trauma exposure, lifetime psychiatric diagnoses, psychotropic medication use, FKBP5 rs1360780 genotype, FKBP5 gene expression, and neuroendocrine measures) with offspring FKBP5 methylation. RESULTS: FKBP5 site 6 methylation was significantly lower in Holocaust offspring than in control subjects, an effect associated with maternal Holocaust exposure in childhood and with lower offspring self-reported anxiety symptoms. FKBP5 gene expression was elevated in Holocaust offspring. FKBP5 methylation was associated with indices of glucocorticoid sensitivity but not with basal FKBP5 gene expression. CONCLUSIONS: This study replicates and extends the previously observed decrement in FKBP5 intron 7, site 6 methylation in Holocaust offspring. The predominance of this effect in offspring of mothers exposed during childhood implicates maternal developmental programming as a putative mechanism.

Glucocorticoid exposure during hippocampal neurogenesis primes future stress response by inducing changes in DNA methylation.

Prenatal stress exposure is associated with risk for psychiatric disorders later in life. This may be mediated in part via enhanced exposure to glucocorticoids (GCs), which are known to impact neurogenesis. We aimed to identify molecular mediators of these effects, focusing on long-lasting epigenetic changes. In a human hippocampal progenitor cell (HPC) line, we assessed the short- and long-term effects of GC exposure during neurogenesis on messenger RNA (mRNA) expression and DNA methylation (DNAm) profiles. GC exposure induced changes in DNAm at 27,812 CpG dinucleotides and in the expression of 3,857 transcripts (false discovery rate [FDR] ≤ 0.1 and absolute fold change [FC] expression ≥ 1.15). HPC expression and GC-affected DNAm profiles were enriched for changes observed during human fetal brain development. Differentially methylated sites (DMSs) with GC exposure clustered into 4 trajectories over HPC differentiation, with transient as well as long-lasting DNAm changes. Lasting DMSs mapped to distinct functional pathways and were selectively enriched for poised and bivalent enhancer marks. Lasting DMSs had little correlation with lasting expression changes but were associated with a significantly enhanced transcriptional response to a second acute GC challenge. A significant subset of lasting DMSs was also responsive to an acute GC challenge in peripheral blood. These tissue-overlapping DMSs were used to compute a polyepigenetic score that predicted exposure to conditions associated with altered prenatal GCs in newborn's cord blood DNA. Overall, our data suggest that early exposure to GCs can change the set point of future transcriptional responses to stress by inducing lasting DNAm changes. Such altered set points may relate to differential vulnerability to stress exposure later in life.

Identification of dynamic glucocorticoid-induced methylation changes at the FKBP5 locus.

BACKGROUND: Epigenetic mechanisms may play a major role in the biological embedding of early-life stress (ELS). One proposed mechanism is that glucocorticoid (GC) release following ELS exposure induces long-lasting alterations in DNA methylation (DNAm) of important regulatory genes of the stress response. Here, we investigate the dynamics of GC-dependent methylation changes in key regulatory regions of the FKBP5 locus in which ELS-associated DNAm changes have been reported. RESULTS: We repeatedly measured DNAm in human peripheral blood samples from 2 independent cohorts exposed to the GC agonist dexamethasone (DEX) using a targeted bisulfite sequencing approach, complemented by data from Illumina 450K arrays. We detected differentially methylated CpGs in enhancers co-localizing with GC receptor binding sites after acute DEX treatment (1 h, 3 h, 6 h), which returned to baseline levels within 23 h. These changes withstood correction for immune cell count differences. While we observed main effects of sex, age, body mass index, smoking, and depression symptoms on FKBP5 methylation levels, only the functional FKBP5 SNP (rs1360780) moderated the dynamic changes following DEX. This genotype effect was observed in both cohorts and included sites previously shown to be associated with ELS. CONCLUSION: Our study highlights that DNAm levels within regulatory regions of the FKBP5 locus show dynamic changes following a GC challenge and suggest that factors influencing the dynamics of this regulation may contribute to the previously reported alterations in DNAm associated with current and past ELS exposure.

Epigenetic upregulation of FKBP5 by aging and stress contributes to NF-κB-driven inflammation and cardiovascular risk.

Aging and psychosocial stress are associated with increased inflammation and disease risk, but the underlying molecular mechanisms are unclear. Because both aging and stress are also associated with lasting epigenetic changes, a plausible hypothesis is that stress along the lifespan could confer disease risk through epigenetic effects on molecules involved in inflammatory processes. Here, by combining large-scale analyses in human cohorts with experiments in cells, we report that FKBP5, a protein implicated in stress physiology, contributes to these relations. Across independent human cohorts (total n > 3,000), aging synergized with stress-related phenotypes, measured with childhood trauma and major depression questionnaires, to epigenetically up-regulate FKBP5 expression. These age/stress-related epigenetic effects were recapitulated in a cellular model of replicative senescence, whereby we exposed replicating human fibroblasts to stress (glucocorticoid) hormones. Unbiased genome-wide analyses in human blood linked higher FKBP5 mRNA with a proinflammatory profile and altered NF-κB-related gene networks. Accordingly, experiments in immune cells showed that higher FKBP5 promotes inflammation by strengthening the interactions of NF-κB regulatory kinases, whereas opposing FKBP5 either by genetic deletion (CRISPR/Cas9-mediated) or selective pharmacological inhibition prevented the effects on NF-κB. Further, the age/stress-related epigenetic signature enhanced FKBP5 response to NF-κB through a positive feedback loop and was present in individuals with a history of acute myocardial infarction, a disease state linked to peripheral inflammation. These findings suggest that aging/stress-driven FKBP5-NF-κB signaling mediates inflammation, potentially contributing to cardiovascular risk, and may thus point to novel biomarker and treatment possibilities.

HAM-TBS: high-accuracy methylation measurements via targeted bisulfite sequencing.

BACKGROUND: The ability to accurately and efficiently measure DNA methylation is critical to advance the understanding of this epigenetic mechanism and its contribution to common diseases. Here, we present a highly accurate method to measure methylation using bisulfite sequencing (termed HAM-TBS). This novel method is able to assess DNA methylation in multiple samples with high accuracy in a cost-effective manner. We developed this assay for the FKBP5 locus, an important gene in the regulation of the stress system and previously linked to stress-related disorders, but the method is applicable to any locus of interest. RESULTS: HAM-TBS enables multiplexed analyses of up to 96 samples and regions spanning 10 kb using the Illumina MiSeq. It incorporates a triplicate bisulfite conversion step, pooled target enrichment via PCR, PCR-free library preparation and a minimum coverage of 1000×. TBS was able to resolve DNA methylation levels with a mean accuracy of 0.72%. Using this method, we designed and validated a targeted panel to specifically assess regulatory regions within the FKBP5 locus that are not covered in commercially available DNA methylation arrays. CONCLUSIONS: HAM-TBS represents a highly accurate, medium-throughput sequencing approach for robust detection of DNA methylation changes in specific target regions.

Corrigendum: Dynamic Changes in DNA Methylation Occur during the First Year of Life in Preterm Infants.

[This corrects the article on p. 158 in vol. 7, PMID: 28018293.].

Early life stress, FK506 binding protein 5 gene (FKBP5) methylation, and inhibition-related prefrontal function: A prospective longitudinal study.

Individuals who have experienced high levels of childhood stress are at increased risk for a wide range of behavioral problems that persist into adulthood, yet the neurobiological and molecular mechanisms underlying these associations remain poorly understood. Many of the difficulties observed in stress-exposed children involve problems with learning and inhibitory control. This experiment was designed to test individuals' ability to learn to inhibit responding during a laboratory task. To do so, we measured stress exposure among a community sample of school-aged children, and then followed these children for a decade. Those from the highest and lowest quintiles of childhood stress exposure were invited to return to our laboratory as young adults. At that time, we reassessed their life stress exposure, acquired functional magnetic resonance imaging data during an inhibitory control task, and assayed these individuals' levels of methylation in the FK506 binding protein 5 (FKBP5) gene. We found that individuals who experienced high levels of stress in childhood showed less differentiation in the dorsolateral prefrontal cortex between error and correct trials during inhibition. This effect was associated only with childhood stress exposure and not by current levels of stress in adulthood. In addition, FKBP5 methylation mediated the association between early life stress and inhibition-related prefrontal activity. These findings are discussed in terms of using multiple levels of analyses to understand the ways in which adversity in early development may affect adult behavioral adaptation.

Dynamic Changes in DNA Methylation Occur during the First Year of Life in Preterm Infants.

BACKGROUND: Preterm birth associates with a substantially increased risk of later cardiovascular disease and neurodevelopmental disorders. Understanding underlying mechanisms will facilitate the development of screening and intervention strategies to reduce disease risk. Changes in DNA methylation have been proposed as one mechanism linking the early environment with later disease risk. We tested the hypothesis that preterm birth associates with altered DNA methylation in genes encoding insulin-like growth factor 2 (IGF2) and FK506-binding protein 5 (FKBP5), which appear particularly vulnerable to early life adversity. METHODS: Fifty preterm infants were seen and assessed at birth, term equivalent age, 3 months and 1-year corrected ages; 40 term infants were seen at birth, 3 months and 1 year. Saliva was collected for DNA extraction at birth, term, and 1 year. Pyrosequencing of bisulfite-converted DNA was performed to measure DNA methylation at specific CpG sites within the IGF2 and FKBP5 loci. RESULTS: Weight and head circumference was reduced in preterm infants at all time points. Preterm infants had a higher percentage body fat at term-corrected age, but this difference was not persistent. DNA methylation at the differentially methylated region (DMR) of IGF2 (IGF2DMR2) and FKBP5 was lower in preterm infants at birth- and term-corrected age compared to term infants at birth. IGF2DMR2 and FKBP5 methylation was related to birthweight SD score in preterm infants. Among preterm infants, social deprivation was an independent contributor toward reducing DNA methylation at IGF2DMR2 at birth- and term-corrected age and maternal smoking was associated with reduced DNA methylation at FKBP5 at birth. There were no persistent differences in DNA methylation at 1 year of age. CONCLUSION: Changes in DNA methylation were identified at key regions of IGF2/H19 and FKBP5 in preterm infants in early life. Potential contributing factors include maternal smoking and social deprivation. However, these changes did not persist at 1 year of age and further longitudinal studies are required to determine any associations between altered DNA methylation in the perinatal period of individuals born preterm and their long-term health.

Gene-Stress-Epigenetic Regulation of FKBP5: Clinical and Translational Implications.

Stress responses and related outcomes vary markedly across individuals. Elucidating the molecular underpinnings of this variability is of great relevance for developing individualized prevention strategies and treatments for stress-related disorders. An important modulator of stress responses is the FK506-binding protein 51 (FKBP5/FKBP51). FKBP5 acts as a co-chaperone that modulates not only glucocorticoid receptor activity in response to stressors but also a multitude of other cellular processes in both the brain and periphery. Notably, the FKBP5 gene is regulated via complex interactions among environmental stressors, FKBP5 genetic variants, and epigenetic modifications of glucocorticoid-responsive genomic sites. These interactions can result in FKBP5 disinhibition that has been shown to contribute to a number of aberrant phenotypes in both rodents and humans. Consequently, FKBP5 blockade may hold promise as treatment intervention for stress-related disorders, and recently developed selective FKBP5 blockers show encouraging results in vitro and in rodent models. Although risk for stress-related disorders is conferred by multiple environmental and genetic factors, the findings related to FKBP5 illustrate how a deeper understanding of the molecular and systemic mechanisms underlying specific gene-environment interactions may provide insights into the pathogenesis of stress-related disorders.