In the present study, we found significant differences in SLC6A4 promoter methylation between MDD patients and HCs in a large sample. This is in line with recent work showing differences in SLC6A4 methylation level between a small sample of MDD patients and controls (Iga et al, 2016). Furthermore, as assumed in our previous study (Dannlowski et al, 2014) patients with MDD indeed revealed lower SLC6A4 promotor AluJb methylation compared to HCs (Figure 1a). In addition to MDD, AluJb methylation was lower in participants with higher experiences of threatening events during the past year (higher LTE-Q). Since mean LTE-Q was distinctly higher in MDD patients (Table 1), this might explain their distinctly lower mean methylation. A reaction of lowered AluJb methylation when confronted with psychological stressors might lead to the assumption that SLC6A4 AluJb hypomethylation in MDD could represent an underlying endogenous response mechanism to stress. However, since mediation analysis for beta-regression models have not been invented yet to our knowledge, the assumption could not be further tested and stays hypothetical. Global Alu hypomethylation has already been associated with stress exposure, such as for night shift workers (Bollati et al, 2010), individuals with socially disadvantaged status (Subramanyam et al, 2013), and the development of post-traumatic stress disorder after military service (Rusiecki et al, 2012). Interestingly, low Alu methylation was also associated with reduced DNA methyltransferase (DNMT) expression (Deplus et al, 2014) and such reduced DNMT was found in amygdala tissue of postmortem depressed suicides compared to controls (Poulter et al, 2008). Thus, the MDD patients in our present study might reveal lower AluJb methylation compared to HCs also based on reduced DNMT expression. However, this remains speculative since no DNMT levels were examined.

Hypomethylation of Alu elements was generally associated with decreased protein synthesis of nearby genes (cf. Landry et al, 2001), presumably via their function as genomic elements or as transcribed RNA (Wang and Huang, 2014). The AluJb element in the SLC6A4 promoter studied here belongs to the evolutionary early subfamily of AluJb and contains, as expected for this group, numerous transcription-factor-binding sites, particularly that for the repressive PAX6 (Dannlowski et al, 2014). Further, AluJb is located in antisense direction near the transcription start site, allowing possibly exonization, which would lead to alternative SLC6A4 mRNA competing with the original mRNA (Schmitz and Brosius, 2011) and as already shown for an AluJb element in the leptin receptor gene (Huh et al, 2010). Lower SLC6A4 AluJb methylation might allow, eg, more repression of gene expression via PAX6 binding or more alternative (but less original) SLC6A4 mRNA via exonization, both possibly resulting in lower 5-HTT levels (for a detailed discussion see (Dannlowski et al, 2014). Another possibility is the active Alu transcription via RNA polymerase III resulting in free transcribed Alu ncRNAs, which have been demonstrated to inhibit gene expression, and which can be highly adaptive and sensitive to stress (reviewed in (Wang and Huang, 2014). This might also hold true for our SLC6A4 AluJb in case of low or no methylation allowing RNA polymerase III binding. Taken together, recent findings suggest that lower SLC6A4 AluJb methylation could cause inhibition of SLC6A4 gene expression and thus declined levels of 5-HTT. In line with this, MDD patients reveal less 5-HTT-binding compared to HCs (Yeh et al, 2015). However, these considerations remain speculative since no experimental data exists for the impact of AluJb methylation on SLC6A4 expression.

Our results show in agreement with previous studies (Bollati et al, 2010; Subramanyam et al, 2013) lower AluJb methylation associated with high stressful experiences. Interestingly, we also found a gene × environment interaction predicting AluJb methylation: when confronted with high stressful experiences in the past year HTTLPR/rs25531 risk allele carriers revealed lower AluJb methylation compared L A L A homozygotes (Figure 1b). Considering the opposing effect of SLC6A4 AluJb methylation on gene expression (lower methylation related to declined 5-HTT levels as suggested above) compared to CpG island promoter methylation (higher methylation related to declined 5-HTT levels), our results are in line with studies reporting high CpG island methylation associated with high acute as well as chronic stress (Duman and Canli, 2015) in risk allele carriers. In contrast to such current stress, IJzendoorn and colleagues reported responses to past stress: here less unresolved loss or trauma was associated with high SLC6A4 CpG island methylation in risk allele carriers (IJzendoorn et al, 2010), indicating possibly adaptive processes as longterm outcome of stress-related epigenetic changes. The less unresolved loss or trauma might be due to the abovementioned declined 5HTT-levels, which should lead to increased synaptic 5HT availability, comparable to an endogenous SSRI-like effect. Such an adaptive effect might also exist in our MDD patients, since decreased number of depressive episodes were associated with lower AluJb methylation. However, taking an additional performed 4-way interaction with diagnosis × 5-HTTLPR/rs25533 × LTE-Q × sex into account it appeared that sex and diagnosis further impact the gene × environment interaction (see supplementary Table S8 and Supplementary Figure S3). Further, we could replicate a finding of higher SLC6A4 methylation in female compared to male participants (Philibert et al, 2008). Gender specific DNA methylation might reflect hormonal influences during early development stages and possibly contributes to gender specific prevalence of MDD (Uddin et al, 2013). Future studies are needed to deepen the understanding of epigenetic effects on gender differences in the vulnerability for psychiatric disorders.

A possible connecting link between AluJb methylation, stress and MDD clinical appearance are DNMTs. DNMT expression in the nucleus accumbens promotes depression-like behavior in rodents (LaPlant et al, 2010) and increased global DNA methylation as well as genetic variation in DNMTs has already been associated with suicide attempts in psychiatric patients (Murphy et al, 2013b). Furthermore, DNMT inhibition has been shown to downregulate Alu methylation (Deplus et al, 2014), enhance resilience to chronic stress, induce antidepressant effects, and changes in neuronal stress-induced DNA methylation in rodents (Sales and Joca, 2016). However, it remains possible, that not DNMTs but antidepressant treatment changed the AluJb methylation, although extensive subsequent analysis of type and duration of medication revealed no significant association neither with AluJb methylation nor amygdala reactivity.

On a neurofunctional level, we observed an interaction of AluJb methylation and the diagnosis of MDD in the right amygdala responsiveness to emotional faces. Again, assuming the opposing effect of AluJb methylation on gene expression compared to CpG island methylation in the SLC6A4 promoter, our findings are in line with recent studies reporting SLC6A4 CpG island methylation as a predictor for amygdala reactivity in HC and MDD patients (Frodl et al, 2015; Nikolova et al, 2014). Further, lower AluJb methylation in MDD patients was strongly associated with reduced amygdala reactivity. Our results are pointing in the same direction as studies in rodents reporting changes of retrotransposon expression in the basolateral amygdala after stress exposure (Ponomarev et al, 2010). However, the spatial resolution of BOLD fMRI together with individual variations in amygdala morphometry does not allow direct conclusions about single nuclei. Endogenous SSRI-like effects, as the abovementioned possible consequence of lower AluJb methylation, might cause such decreased amygdala reactivity in response to emotional faces, an effect already shown after pharmacological treatment with SSRIs in MDD patients (Ruhé et al, 2014). In HCs, an association between amygdala reactivity and AluJb methylation might have been absent due to lower LTE-Q levels and/or more successful coping strategies in combination with sufficient demethylation when confronted with stress. However, since no neuronal 5-HTT mRNA levels can be collected in living participants, this interpretation remains speculative.

Analysis of joint effects of 5-HTTLPR/rs25531 and AluJb methylation on amygdala reactivity revealed no significant associations. However, we could replicate a controversially discussed finding of risk allele carriers exhibiting greater threat-related amygdala reactivity relative to non-carriers (Murphy et al, 2013a). Based on a large sample, meeting the need for high statistical power (Murphy et al, 2013a), our data adds notable evidence to the hypothesis that 5-HTTLPR/rs25531 represents an important player in the neural activity during emotion processing.

Our results predicting amygdala reactivity were limited to the right amygdala since weaker association in the left amygdala did not withstand FWE correction. This corroborates the fMRI paradigm used in our study, which mainly increases activation in the right amygdala (Lanteaume et al, 2007). Further it might reflect a functional asymmetry of the amygdala demanding further investigations (Baas et al, 2004), since the majority of studies analyzing the amygdala reported lateralized results.

Some limitations have to be acknowledged. First, DNA methylation was measured via whole EDTA-blood. Inter-subject heterogeneity in blood cell type proportions might potentially confound methylation levels and the following PCA analysis in our study (Jaffe and Irizarry, 2014). Furthermore, measuring methylation in the periphery of the body, a direct correlation to methylation levels in the brain cannot be determined. However, recent work indicates substantial correlations between SLC6A4 methylation of CpG sites in peripheral blood leukocytes and SLC6A4 methylation in post mortem amygdala tissue in healthy controls and patients with MDD (Riese et al, 2014). In addition, SLC6A4 promoter methylation based on peripheral blood cells has already been mapped onto individual differences in serotonin biosynthesis in vivo (Wang et al, 2012). Besides, influences of adjacent CpG sites and other functional polymorphisms could be biologically relevant and potentially confound our results. BDNF, for instance, has frequently been shown to interact with SLC6A4 on behavioral, transcriptional, and epigenetic levels (Ignácio et al, 2014). More research is needed to examine further genetic and epigenetic factors involved in the association of MDD and SLC6A4 AluJb methylation rates in detail. Although physical parameters like smoking status or physical exercises revealed no effects on SLC6A4 methylation previously (Alexander et al, 2014; Olsson et al, 2010) interfering influences cannot be excluded. Methylation rates did not differ with regard to medication status in our MDD sample, yet, influences of several drugs could not be conclusively clarified regarding the high heterogeneity of psychotropic drugs. Besides, amygdala reactivity has frequently been shown to be influenced by medication intake (Ruhé et al, 2014), which could not be determined in our sample, but should be taken into account in future replication studies.

Despite these limitations, our study provides new insights in the role of SLC6A4 AluJb methylation in MDD and amygdala reactivity and its associations with 5-HTTLPR/rs25531 and stress. Lowered AluJb methylation in MDD patients might represent an underlying endogenous response mechanism to higher stress levels. In conjunction with a shorter history of MDD and decreased amygdala reactivity, AluJb hypomethylation might even point to an adaptive epigenetic process, maybe via theoretically possible endogenous antidepressant-like effects by decreasing 5-HTT expression rates. The present study therefore is the first, which highlights the possible role of repetitive (Alu) elements in MDD and which might indicate new promising targets for therapeutic interventions.