Targeted DNA Methylation Using Modified DNA Probes: A Potential Therapeutic Tool for Depression and Stress-Related Disorders

Abstract

Epigenetic modifications play a crucial role in gene regulation and have been implicated in various physiological processes and disease conditions. DNA methylation (DNAm) has been implicated in the etiology and progression of many stress-related psychiatric behaviors, such as depression. The ability to manipulate DNAm may provide a means to reverse and treat such disorders. Although CRISPR-based technologies have enabled locus-specific DNAm editing, their clinical applicability may be limited due to immunogenicity concerns and off-target effects. In this study, we introduce a novel approach for targeted DNAm manipulation using single-stranded methylated DNA probes. The probes were designed against the GRE of FKBP5 and the promoter region of MAOA. In both human embryonic kidney HEK293 and mouse pituitary AtT-20 cells, transfection with their respective methylated probes significantly increased DNAm at targeted CpG sites in a persistent and dose-dependent manner. Importantly, the induced methylation effectively attenuated glucocorticoid-induced upregulation of FKBP5 gene expression. Alteration of methylation was specific to single-stranded probes, as double-stranded methylated probes and unmethylated probes showed no significant effects. Some limitations include the need to further characterize factors that influence probe efficiency, such as probe length and CpG density; develop an efficient in vivo probe delivery system; and perform a more extensive consideration of possible off-target effects. Despite these limitations, our findings suggest that methylated DNA probes have the potential to function as a simple tool for targeted epigenetic manipulation and serve as a safer alternative to CRISPR-based epigenome editing tools for the treatment of stress-related disorders such as depression.

Keywords: DNA methylation (DNAm); FK506 binding protein 5 (FKBP5); cortisol; epigenetics; gene expression; glucocorticoid response element (GRE); monoamine oxidase A (MAOA).

Figure 1

Genomic organization of FKBP5. The human FKBP5 locus is located on the negative strand of chromosome 6. For this study, three intronic regions indicated by thin horizontal gray lines have been tested. Two sets of large black arrows represent the outside and inside PCR primers used for bisulfite pyrosequencing. These black arrows flank the primers used for generating the probe (red arrows), which cannot be amplified by the pyrosequencing primers. The intron 5 GREs are composed of the main GRE formed by GRE1 and GRE2, for which the DNA probe was designed, and an adjacent GRE that was tested as a negative control region.

Figure 2

Dose-dependent DNAm and gene expression changes following transfection of methylated DNA probes against FKBP5. (A) 293HEK cells were transfected with a single-stranded unmethylated probe (500 ng − SssI), a single-stranded methylated probe at two different concentrations (250 ng and 500 ng + SssI), or a double-stranded methylated probe (500 ng + SssI +DS). Untransfected cells served as controls (Control). DNAm levels of five CpG sites at the conserved glucocorticoid response element (GRE) of human FKBP5 intron 5 were analyzed by bisulfite pyrosequencing. Data for the first two CpGs are shown. (B) Typical pyrograms obtained from bisulfite pyrosequencing are shown for each group at CpG-1. The percent DNAm determination occurs when R (or A/G) is dispensed, and it corresponds to the reverse complement of T/C (T for unmethylated and C for methylation CpG, respectively). Each pyrogram represents % methylation from one sample. (C) FKBP5 expression was measured by qRT-PCR in the same groups of 293HEK cells as in (A) treated with 1 μM dexamethasone (DEX) for four hours prior to collection. Bar graphs represent the mean ± SEM, and unpaired two-tailed Student’s t-tests were performed with n = 4 per group. *** p < 0.001, ** p < 0.01, and * p < 0.05.

Figure 3

Persistence and accumulation of DNA methylation. (A) 293HEK cells were transfected with the unmethylated (−SssI) and methylated (+SssI) FKBP5 DNA probe at Week 1 (W1), after which the cells were cultured for an additional four weeks (W4) before analysis by bisulfite pyrosequencing. (B) 293HEK cells were transfected with unmethylated (−SssI) and methylated (+SssI) FKBP5 DNA probes consecutively every three days and expanded, while 50% of the cells were collected for analysis. Bar graphs represent the mean ± SEM, and unpaired two-tailed Student’s t-tests were performed with n = 4 per group. *** p < 0.001, ** p < 0.01, and * p < 0.05.

Figure 4

DNAm analysis of human FKBP5 GREs at additional intronic regions. Experimentally verified glucocorticoid response elements (GREs) at intron 7 (A), intron 5 (B), and 1 (C) were evaluated in probe-transfected 293 HEK samples for non-specific epigenetic effects. There were no significant differences in cells transfected with unmethylated vs. methylated DNA probes, except at CpG-1 of Intron 1 (C). Bar graphs represent the mean ± SEM, and unpaired two-tailed Student’s t-tests were performed with n = 4 per group. ** p < 0.01.

Figure 5

DNAm and gene expression analysis following transfection of methylated DNA probes against mouse Fkbp5. (A) AtT-20 cells were transfected with single-stranded unmethylated probe (500 ng − SssI), single-stranded methylated probe (500 ng + SssI), or double-stranded methylated probe (500 ng + SssI +DS). Untransfected cells served as controls (Control). DNAm levels of four CpG sites at the conserved glucocorticoid response element (GRE) of mouse Fkbp5 intron 5 were analyzed by bisulfite pyrosequencing. Data for all four GRE CpGs are shown. (B) Fkbp5 expression was measured by qRT-PCR in the same groups of AtT-20 cells as in (A) treated with 1 μM dexamethasone (DEX) for four hours prior to collection. Bar graphs represent the mean ± SEM, and unpaired two-tailed Student’s t-tests were performed with n = 4 per group. *** p < 0.001, ** p < 0.01, and * p < 0.05.

Figure 6

DNAm and gene expression analysis following transfection of methylated DNA probes against MAOA. (A) 293HEK cells were transfected with a single-stranded unmethylated probe (-SssI) or a single-stranded methylated probe (+SssI). DNAm levels of 14 CpG sites at a regulatory region of human MAOA were analyzed by bisulfite pyrosequencing. (B) MAOA expression was measured by qRT-PCR in the same groups of 293HEK cells. Bar graphs represent the mean ± SEM, and unpaired two-tailed Student’s t-tests were performed with n = 4 per group. * p < 0.05 and ** p < 0.01. A one-way ANOVA was used to test for overall differences across all 14 CpGs, followed by Tukey’s post hoc test for pairwise comparisons.