BDNF exon IV promoter methylation and antidepressant action: a complex interplay

Abstract

Background: BDNF exon IV promoter methylation is a potential biomarker for treatment response to antidepressants in MDD. We have previously shown CpG-87 methylation as a successful biomarker for the prediction of non-response to monoaminergic antidepressants like the SSRI Fluoxetine or the SNRI Venlafaxine. This study aimed to dissect the biological evidence and mechanisms for the functionality of CpG-87 methylation in a cell culture model.

Results: We observed a significant interaction between methylation and antidepressant-mediated transcriptional activity in BDNF exon IV promoter. In addition, antidepressant treatment increased the promoter methylation in a concentration-dependent manner. Further single CpG methylation of -87 did not change the promoter activity, but methylation of CREB domain CpG-39 increased the transcriptional activity in an antidepressant-dependent manner. Interestingly, DNMT3a overexpression also increases the BDNF exon IV transcription and more so in Venlafaxine-treated cells.

Conclusions: The study strengthens the previously reported association between antidepressant treatment and BDNF exon IV promoter methylation as well as hints toward the mechanism of action. We argue that potential CpG methylation biomarkers display a complex synergy with the molecular changes at the neighboring CpG positions, thus highlighting the importance of epiallele analyses.

Keywords: Antidepressants; BDNF exon IV promoter; CREB; MeCP2; Methylation; Site-directed mutagenesis.

Fig. 1

BDNF exon IV promoter schematics. Sequence of the BDNF exon IV promoter analyzed for methylation. Primers (blue) and the CpGs (red) are highlighted. CpG-39 and -35 encompasses the cAMP response element (CRE). CpGs are numbered with respect to BDNF exon IV promoter

Fig. 2

a Effect of antidepressants on BDNF exon IV promoter activity. The antidepressants at the given concentration decrease the promoter activity: Venlafaxine (50 µM, P < 0.001), Fluoxetine (10 µM, P < 0.05), Mirtazapine (50 µM, P < 0.05) and Lithium carbonate (3 mM, P < 0.01). Overall, methylation reduces the promoter activity (ANOVA, main effect of methylation: P = 0.0008). There is no significant reduction in promoter activity on AD treatment in methylated fragment

Fig. 3

Effect of single CpG methylation on BDNF exon IV promoter activity. Methylation at CpG position -35, -87 and -39 does not show any significant change in promoter activity in control condition. In the cells treated with ADs, CpG-39 methylation significantly increases the promoter activity (P < 0.0001). Methylation at CpG-35 and -87 had no significant effect

Fig. 4

Antidepressants increase BDNF exon IV promoter methylation. Venlafaxine at 50 µM significantly increases BDNF exon IV promoter methylation in comparison with 10 µM (^*P < 0.0001) and 10 µM Fluoxetine in comparison with 5 µM (^#P = 0.025)

Fig. 5

Effect of DNMT overexpression on BDNF exon IV promoter activity. a DNMT 3a overexpression significantly increases BDNF exon IV promoter activity (P < 0.0001). DNMT3b and DNMT1 do not have any significant effect. b Venlafaxine further increases the DNMT3a-mediated increase in promoter activity (P < 0.01). Fluoxetine has no significant effect

Fig. 6

Proposed molecular mechanism for methylation-specific BDNF exon IV transcriptional activation. CREB, a known transcriptional factor binds to cAMP response element (CRE) on BDNF exon IV promoter and upregulate transcription by recruiting co-activators like CREB-binding protein (CBP). MeCP2 binds across genome, preferably to methylated CpGs and functions as chromatin modifier. Site-specific methylation of BDNF exon IV promoter increases MeCP2 occupancy at the methylated CpGs. MeCP2 and CREB form a transcriptional activating complex. No increase in CREB–CRE interaction was observed, if methylated CpG is far from CRE as with CpG-87 (a) or at the 3′ end of CRE as observed with CpG-35 (b). Methylation at CpG-39 allows the recruitment of MeCP2-CREB protein complex and increases the transcriptional activity at BDNF exon IV promoter (c). Illustration was created with BioRender.com