This hypothesis was supported, in part, by the finding that MeCP2 and HDAC2 are colocalized in the NAc ( Figure 5A). The interactions of MeCP2 and HDAC2 were assessed using IP-Western blot analysis of vSTR proteins. We found that CUMS increased the formation of MeCP2-HDAC2 complexes in stressed BALB mice. This effect was reversed by continuous IMI treatment ( Figure 5B). Next, to investigate the effect of CUMS on the binding of MeCP2-HDAC2 complexes at the Gdnf promoter, we performed re-ChIP assays using an antibody for HDAC2 on the vSTR samples that were initially immunoprecipitated with an antibody for MeCP2. The re-ChIP assays indicated that the Gdnf promoter-containing DNA fragments of stressed BALB mice, see more but not B6 mice,
were significantly enriched compared with those of nonstressed mice, and this effect was reversed by continuous IMI treatment ( Figure 5C). These results suggest that the CUMS-induced binding of MeCP2-HDAC2 complexes to the Gdnf promoter silences this website its transcription. To investigate the role of DNA methylation in the CUMS-induced suppression of Gdnf expression and on depression-like behaviors, zebularine (ZEB), a DNA methyltransferase inhibitor, was continuously delivered into the NAc of BALB mice by an osmotic pump.
The experimental design is shown in Figure S1E. Five days after surgery, mice were subjected to 4 weeks of CUMS, followed by behavioral and expression analyses. We found that the social interaction times and sucrose preferences of stressed mice receiving ZEB (100 μM) were significantly higher compared with those times and preferences of vehicle-treated mice ( Figures 6A and B). In the novelty-suppressed feeding test, the latency to feed was significantly decreased in stressed mice receiving ZEB compared with vehicle-treated controls ( Figure 6C). In the forced swim test, the immobility times were significantly shorter in stressed and nonstressed mice receiving ZEB compared with
the Phosphoprotein phosphatase times of vehicle-treated mice ( Figure 6D). Furthermore, the mRNA levels of Gdnf in ZEB-treated mice were greater than the levels in vehicle-treated mice ( Figure 6E) in stressed conditions. These findings confirm that there is less DNA methylation of CpG site 2 at the Gdnf promoter in stressed mice treated with ZEB compared with vehicle-treated mice ( Figure 6F). We also tested whether intra-NAc delivery of RG108, a potent, nonnucleoside inhibitor of DNA methylation, could reverse the increased depression-like behaviors in BALB mice. Similar to the effects of ZEB, continuous delivery of RG108 (100 μM) directly into the NAc increased the social interaction time ( Figure 6G) and sucrose preference ( Figure 6H) of mice in the stressed condition. Furthermore, we found that CUMS increased the mRNA expressions for DNA methyltransferase 1 (DNMT1) and DNMT3a, but not DNMT3b, in the vSTR of stressed mice. This effect was reversed by continuous intra-NAc delivery of ZEB and RG108 ( Figure 6I).