Allele-specific FKBP5 DNA demethylation mediates gene-childhood trauma interactions

Abstract

Although the fact that genetic predisposition and environmental exposures interact to shape development and function of the human brain and, ultimately, the risk of psychiatric disorders has drawn wide interest, the corresponding molecular mechanisms have not yet been elucidated. We found that a functional polymorphism altering chromatin interaction between the transcription start site and long-range enhancers in the FK506 binding protein 5 (FKBP5) gene, an important regulator of the stress hormone system, increased the risk of developing stress-related psychiatric disorders in adulthood by allele-specific, childhood trauma-dependent DNA demethylation in functional glucocorticoid response elements of FKBP5. This demethylation was linked to increased stress-dependent gene transcription followed by a long-term dysregulation of the stress hormone system and a global effect on the function of immune cells and brain areas associated with stress regulation. This identification of molecular mechanisms of genotype-directed long-term environmental reactivity will be useful for designing more effective treatment strategies for stress-related disorders.

Figure 1

FKBP5 × childhood abuse interaction on PTSD lifetime diagnosis. Shown is the interaction of child abuse and FKBP5 rs1360780 protective genotype (left) or risk allele carrier status (right) on percentage lifetime PTSD (CAPS). FKBP5 protective genotype: no abuse, N = 133; one type, N = 27; two types, N = 16; FKBP5 risk allele carriers: no abuse; N = 252, one type, N = 69; two types, N = 22 (n.s. P > 0.05, ***P < 0.001).

Figure 2

Genotype- and glucocorticoid receptor–dependent enhancer activities of intron 2 sequences of FKBP5. (a) Representation of the reporter construct. The luciferase gene is driven by a basal human elongation factor-1 alpha (EF-1α, EEF1A1) promoter with a 802-bp fragment of FKBP5 intron 2 containing the putative GRE and rs1360780 cloned in front of the promoter. The rs1360780 risk allele A/T might form a TATA box–like sequence, which would enhance transcriptional activity. (b) Allele specificity of the reporter gene activity in glucocorticoid receptor–free HEK 293 cells. The reporter activity was reduced for the protective allele versus the risk allele (***P < 0.001, Student’s t test, unpaired, two sided). (c) Stimulation with 50 nM dexamethasone significantly enhanced intron 2–driven luciferase activity in glucocorticoid receptor–expressing HeLa cells in a genotype-dependent manner. Reduced activity was observed for the protective allele versus the risk allele (*P = 0.036, Student’s t test, unpaired, two sided). Baseline luciferase activity in HeLa cells without dexamethasone stimulation did not reveal significant differences (data not shown). In the box plots, the box extends indicate lower quartile and upper quartile, and the whiskers denote sample minimum and maximum. The line in the box represents the median and dots represent outliers. (d) Relative binding of recombinant TBP to a double-stranded 60-mer oligonucleotide containing either the A or G allele of rs1360780. The A allele exhibited a stronger binding to TBP than the G allele. (**P = 0.009, Student’s t test, unpaired, two sided). The assay was run in triplicates, background activity was subtracted from the measurement reading. Data are expressed as mean ± s.e.m.

Figure 3

Long-distance interaction of GREs in FKBP5. Chromatin conformation capture confirmed a genotype-dependent interaction of the FKBP5 TSS (TSS) with intron 2 and intron 7 in lymphoblastoid cell lines. (a) Top, FKBP5 genomic locus with the TSS indicated by an arrow and intron 2 and intron 7 indicated by black boxes. The upright thin lines represent EcoRI cutting sites of genomic DNA. Bottom, chromatin conformation capture interaction data. Amplicon primers are indicated by small arrows, and the fragments containing the TSS, intron 2 and intron 7 are emphasized by gray boxes. Data from quantitative PCR are plotted as relative crosslinking frequency on the y axis. A specific interaction was detected as the local peak in interaction frequencies. Thus, intron 7 showed a stronger interaction with the TSS than with fragments upstream and downstream of the intron 7 fragment in both cell lines. Moreover, we observed a strong local peak for the interaction of the TSS with intron 2, but only in the cell line carrying the risk (AA) genotype. This confirmed the physical interaction of the TSS with intron 7 and intron 2 in a genotype-dependent manner. (b,c) Three-dimensional interaction of intronic GREs in intron 2 and 7 of FKBP5 with the TSS. FKBP5 mRNA transcription is induced by cortisol via a three-dimensional interaction and loop formation of predominantly distal enhancer regions (blue) harboring GREs with the core promoter site (PolII = RNA polymerase II). The interaction of intron 2 with the TSS in risk allele carriers leads to an increased FKBP5 induction in response to glucocorticoid receptor activation (represented by a red arrow).

Figure 4

DNA methylation of the FKBP5 locus. (a) Significant DNA methylation was observed in the promoter region, intron 2 and intron 7 of FKBP5, as indicated in green. (b) Single CpG site methylation in the four groups (early trauma × FKBP5 rs1360780 carriers). Data are expressed as mean ± s.e.m.

Figure 5

Differential FKBP5 intron 7 DNA methylation depends on genotype and trauma exposure. Correlation between intron 7 bin 2, mean methylation and log-transformed CTQ scores by FKBP5 rs1360780 genotype in the Grady and Conte cohort are shown. (a) Grady cohort. Risk allele carriers exhibited a strong negative correlation (R = −0.646, P < 0.001) between methylation and CTQ total load compared with carriers of the protective genotype (R = 0.414, P = 0.078) (Fisher z score = −4.23, P < 0.001). (b) Conte cohort. Correlation between methylation and total CTQ in risk allele carriers (R = −0.273, P = 0.124), and in carriers of the protective genotype (R = 0.153, P = 0.485) (Fisher z score = −1.5, P = 0.133). (c) Grady cohort. Negative correlation was found between methylation and the CTQ physical abuse subscore in risk allele carriers (R = −0.586, P < 0.001), but not in carriers of the protective genotype (R = 0.360, P = 0.130) (Fisher z score = −4.49, P < 0.001). (d) Conte cohort. Negative correlation was observed between methylation and the CTQ physical abuse subscore in risk allele carriers (R = −0.397, P = 0.022), but not in carriers of the protective genotype (R = 0.246, P = 0.258) (Fisher z score = −2.33, P = 0.019). (e) Grady cohort. Negative correlation was found between methylation and the CTQ emotional abuse subscore in risk allele carriers (R = −0.685, P < 0.001), but not in carriers of the protective genotype (R = 0.321, P = 0.181) (Fisher z score = −4.1, P < 0.001). (f) Conte cohort. Negative correlation was found between methylation and the CTQ emotional abuse subscore in risk allele carriers (R = −0.397, P = 0.022), but not in carriers of the protective genotype (R = 0.022, P = 0.922) (Fisher z score = −1.53, P = 0.126). (g) Grady cohort. Negative correlation was found between methylation and the CTQ sexual abuse subscore in risk allele carriers (R = −0.656, P < 0.001), but not in carriers of the protective genotype (R = 0.599, P = 0.007) (Fisher z score = −5.17, P < 0.001). (h) Conte cohort. Negative correlation was found between methylation and the CTQ sexual abuse subscore in risk allele carriers (R = 0.118, P = 0.514), and in carriers of the protective genotype (R = 0.305, P = 0.922) (Fisher z score = −0.68, P = 0.496).

Figure 6

Effects of glucocorticoid receptor agonist treatment on FKBP5 DNA methylation in hippocampal progenitor cells. (a) Dexamethasone treatment induced demethylation in the human hippocampal progenitor cell line HPC03A/07 in the proliferation and differentiation phase in intron 7 (***P < 0.001), but not intron 2. Data are expressed as mean ± s.e.m. (b) Dexamethasone treatment of human hippocampal progenitor cells in proliferation and differentiation phase resulted in significant demethylation of CpGs in FKBP5 intron 7, bins 2 and 3 (***P < 0.001, **P = 0.01, n.s. P > 0.05, Student’s t test, unpaired, two-sided). Dexamethasone treatment in the proliferation phase only resulted in similar demethylation of CpGs in FKBP5 intron 7 in comparison with the differentiation phase (data not shown). A subsequent washout of dexamethasone and an additional 20-d incubation revealed similar results, supporting a long-lasting demethylation in bins 2 and 3. Data are expressed as mean ± s.e.m. (c) Treatment in proliferation and differentiation phase resulted in an immediate demethylation of intron 7 bin 2 (***P < 0.001, Student’s t test, unpaired, two-sided). Treatment in the proliferation and differentiation phase and subsequent washout phase in steriod-devoid medium for 20 d revealed a stable demethylation by glucocorticoid receptor activation. In contrast, dexamethasone treatment after proliferation and differentiation did not induce a long-lasting demethylation in intron 7 bin 2 (n.s. P = 0.063, Student’s t test, unpaired, two-sided), suggesting a sensitive period of time for glucocorticoids-mediated epigenetic changes. Data are expressed as mean ± s.e.m.

Figure 7

Functional effects of FKBP5 intron 7 methylation. (a) Representation of the reporter construct. A 514-bp fragment of FKBP5 intron 7 containing three putative GREs was cloned in front of the EF-1α promoter and the luciferase gene, acting as transcriptional regulator. Only the FKBP5 insert can be methylated in vitro by MSssI, as the vector is CpG free. Circled M denotes putative methylation sites. Two of them are located in consensus GRE sequences. (b) Stimulation by 50 nM dexamethasone was attenuated by DNA methylation (*P = 0.035, n.s. P = 0.146, Student’s t test, unpaired, two-sided). Baseline luciferase activity without dexamethasone stimulation did not reveal significant differences (P > 0.05; data not shown). In the box plots, the box extends indicate lower quartile and upper quartile, and the whiskers denote sample minimum and maximum. The line in the box represents the median and dots represent outliers. (c) Methylation in intron 7 was correlated with dexamethasone-mediated inhibition of lipopolysaccharide-induced interleukin 6 production (DEX IC₅₀) in peripheral blood monocytes ex vivo (R = −0.531, P = 0.002). This correlation was stronger in rs1360780 risk allele carriers (N = 19, R = −0.691, P = 0.001) than carriers of the protective genotype (N = 13, R = −0.119, P = 0.698). This suggests that lower DNA methylation in intron 7 leads to stronger FKBP5 induction and to glucocorticoid receptor resistance. (d) Exposure to early trauma enhanced FKBP5 genotype–dependent differences in glucocorticoid receptor–dependent gene expression in peripheral blood. Top, R scores of the correlation of peripheral blood gene expression and cortisol for the 76 transcripts with significant genotype-dependent correlation differences in individuals with high levels of early trauma (cases, N = 55) and stratified by FKBP5 genotype (risk allele: N = 40, CTQ total score = 67.88 ± 14.71; protective genotype: N = 15, CTQ total = 72.44 ± 17.68). For all transcripts, the correlation with cortisol, and therefore the presumable responsiveness to the glucocorticoid receptor, was higher in FKBP5 protective genotype carriers (black squares) with a mean R of 0.74 than in carriers of the risk allele (open squares), with a mean R of 0.23. Bottom, correlation between gene expression levels and cortisol of the same transcripts, but in individuals that were not exposed to early trauma (controls, N = 74; risk allele: N = 60, CTQ total = 28.42 ± 2.96; protective genotype: N = 14, CTQ total = 28.79 ± 3.21). In this group, no genotype-dependent differences of the correlation were observed, with mean R scores of 0.31 and 0.35 for risk and protective genotypes, respectively. x axis represents individual transcripts 1 to 76.