Richard S. Lee

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.

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. We designed probes targeting the glucocorticoid response element (GRE) within the FKBP5 (FK506 binding protein 5) gene, a key regulator of stress response and depressive symptoms, and the promoter region of the MAOA (monoamine oxidase A) gene. In both HEK293 human embryonic kidney 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. 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.

Objective: The impact of chronic exposure to stress or glucocorticoids on psychiatric symptoms has been exemplified by cases of iatrogenic or endogenous hypercortisolism such as Cushing’s syndrome (CS). The amygdala plays an important role in mediating both stress and affective responses, and one of the key factors that link stress response and psychiatric symptoms is the corticotropin-releasing factor (CRF). Epigenetic changes, especially those occurring on CpG dinucleotides in DNA of glucocorticoid target genes in blood, have been previously implicated as potential predictors of glucocorticoid-related events in the central nervous system (CNS). In this study, we examined amygdala volume and mood symptoms in CS patients and aimed at evaluating whether these parameters were associated with blood DNA methylation of CRF.Methods: In this cross-sectional study, 32 CS patients and 32 healthy controls matched for age, sex, and years of education underwent an MRI scan, a Beck Depression Inventory-II, and a State-Trait Anxiety Inventory. Genomic DNA extracted from total leukocytes were used for DNA methylation analysis of several CpG dinucleotides at the CRF promoter region.Results: Significant associations between CRF methylation vs. amygdala volume (CpG-1, P = 0.006) and depression scores (CpG-2, P = 0.01) were found. To assess whether the promoter CpG methylation has functional consequences, we examined RNA and DNA extracted from non-CS, postmortem amygdala tissues. A significant association between CpG methylation and gene expression (CpG-1, P = 0.004) was observed.Conclusion: These results demonstrate that methylation levels of the CRF promoter CpGs are associated with amygdala volume in CS and related mood symptoms. Methylation levels may also be associated with CRF expression. This finding supports the feasibility of using epigenetic patterns in blood as a surrogate for assessing GC-related pathologies in the brain.

Epigenomic annotations for the rat lag far behind those of human and mouse, despite the rat's immense utility in pharmacological and behavioral studies and the need to understand their epigenetic mechanisms. We have designed a targeted-enrichment method followed by next-generation sequencing (Methyl-Seq) to identify DNA methylation (DNAm) signatures across the rat genome. The design reflected an attempt to create a more comprehensive investigation of the rat epigenome, as it included promoters, CpG islands, and island shores of all RefSeq genes. In this study, we implemented the rat Methyl-Seq platform and tested its ability to distinguish differentially methylated regions (DMRs) among three different tissue types, three distinct brain regions, and, in the hippocampus, between males and females. These comparisons yielded DNAm differences of differing magnitudes, many of which were independently validated by bisulfite pyrosequencing, including autosomal regions that were predicted to show the least degree of difference in DNAm between males and females. Quantitative reverse transcription PCR revealed that most genes associated with the DMRs showed tissue-, brain region-, and sex-specific differences in expression. In particular, we found evidence for sex-specific DNAm and expression differences at Tubb6, Lrrn2, Tex26, and Sox5l1, all of which play important roles in neurodevelopment and have been implicated in studies examining sex differences. Our results demonstrate the utility of the rat Methyl-Seq platform and suggest the presence of DNAm differences between the male and female hippocampus. The rat Methyl-Seq has the potential to provide epigenomic insights into pharmacological and behavioral studies performed in the rat.

Exposure to stress or glucocorticoids (GCs) is associated with epigenetic and transcriptional changes in genes that either mediate or are targets of GC signaling. FKBP5 (FK506 binding protein 5) is one such gene that also plays a central role in negative feedback regulation of GC signaling and several stress-related psychiatric disorders. In this study, we sought to examine how the mouse Fkbp5 gene is regulated in a neuronal context and identify requisite factors that can mediate the epigenetic sequelae of excess GC exposure. Mice treated with GCs were used to establish the widespread changes in DNA methylation (DNAm) and expression of Fkbp5 across four brain regions. Then two cell lines were used to test the persistence, decay, and functional significance of GC-induced methylation changes near two GC response elements (GREs) in the fifth intron of Fkbp5. We also tested the involvement of DNMT1, cell proliferation, and MeCP2 in mediating the effect of GCs on DNAm and gene activation. DNAm changes at some CpGs persist while others decay, and reduced methylation states are associated with a more robust transcriptional response. Importantly, the ability to undergo GC-induced DNAm loss is tied to DNMT1 function during cell division. Further, GC-induced DNAm loss is associated with reduced binding of MeCP2 at intron 5 and a physical interaction between the fifth intron and promoter of Fkbp5. Our results highlight several key factors at the Fkbp5 locus that may have important implications for GC- or stress-exposure during early stages of neurodevelopment.

Children born to women who experience stress during pregnancy have an increased risk of cancer in later life, but no previous animal studies have tested such a link. We questioned whether prenatal stress (PS) in A/J mice affected the development of lung tumors after postnatal response to tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Timed-bred A/J mice were randomly assigned on gestation day 12.5 to PS by restraint for 5 consecutive days or control (no restraint). Adult offspring of control and stressed pregnancies were all treated with three NNK injections (50 mg/kg every other day) and euthanized 16 weeks later to examine their lungs. Compared with controls, PS dams exhibited significantly increased levels of plasma corticosterone, increased adrenal weights and decreased fetus weights without fetal loss. Prenatally stressed litters had a significantly higher neonatal death rate within first week of life, and surviving male and female offspring developed lung epithelial proliferations with increase multiplicity, increased area and aggressive morphology. PS also induced more advanced atypical adenomatous hyperplasia lesions. We found no difference in lung NNK-derived methyl DNA adducts, but PS did significantly enhance CD3+ T cell and Foxp3+ T cell tumor infiltration. PS significantly increases multiplicity, area of NNK-induced lung tumors and advanced morphology. PS did not affect production of NNK-derived methyl DNA adducts but did increase lymphocytic infiltration of lung tumors. To our knowledge, this is the first animal model of PS with evaluation of cancer development in offspring.

Body weight is substantially determined by eating behaviors, which are themselves driven by biological factors interacting with the environment. Previous studies in young children suggest that genetic influences on dopamine function may confer differential susceptibility to the environment in such a way that increases behavioral obesity risk in a lower socioeconomic status (SES) environment but decreases it in a higher SES environment. We aimed to test if this pattern of effect could also be observed in adolescence, another critical period for development in brain and behavior, using a novel measure of predicted expression of the dopamine receptor 4 (DRD4) gene in prefrontal cortex. In a sample of 76 adolescents (37 boys and 39 girls from Baltimore, Maryland/US, aged 14-18y), we estimated individual levels of DRD4 gene expression (PredDRD4) in prefrontal cortex from individual genomic data using PrediXcan, and tested interactions with a composite SES score derived from their annual household income, maternal education, food insecurity, perceived resource availability, and receipt of public assistance. Primary outcomes were snack intake during a multi-item ad libitum meal test, and food-related impulsivity assessed using a food-adapted go/no-go task. A linear regression model adjusted for sex, BMI z-score, and genetic ethnicity demonstrated a PredDRD4 by composite SES score interaction for snack intake (p = 0.009), such that adolescents who had lower PredDRD4 levels exhibited greater snack intake in the lower SES group, but lesser snack intake in the higher SES group. Exploratory analysis revealed a similar pattern for scores on the Perceived Stress Scale (p = 0.001) such that the low PredDRD4 group reported higher stress in the lower SES group, but less stress in the higher SES group, suggesting that PredDRD4 may act in part by affecting perceptions of the environment. These results are consistent with a differential susceptibility model in which genes influencing environmental responsiveness interact with environments varying in obesogenicity to confer behavioral obesity risk in a less favorable environment, but behavioral obesity protection in a favorable one.