ObjectiveThe 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.MethodsIn 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.ResultsSignificant 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.ConclusionThese 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.

Abstract Transposable elements (TE) are typically silenced by DNA methylation and repressive histone modifications in differentiated healthy human tissues. However, TE expression increases in a wide range of cancers and is correlated with global hypomethylation of cancer genomes. We assessed expression and DNA methylation of TEs in fibroblast cells that were serially transduced with hTERT, SV40, and HRASR24C to immortalize and then transform them, modeling the different steps of the tumorigenesis process. RNA sequencing and whole-genome bisulfite sequencing were performed at each stage of transformation. TE expression significantly increased as cells progressed through transformation, with the largest increase in expression after the final stage of transformation, consistent with data from human tumors. The upregulated TEs were dominated by endogenous retroviruses [long terminal repeats (LTR)]. Most differentially methylated regions (DMR) in all stages were hypomethylated, with the greatest hypomethylation in the final stage of transformation. A majority of the DMRs overlapped TEs from the RepeatMasker database, indicating that TEs are preferentially demethylated. Many hypomethylated TEs displayed a concordant increase in expression. Demethylation began during immortalization and continued into transformation, while upregulation of TE transcription occurred in transformation. Numerous LTR elements upregulated in the model were also identified in The Cancer Genome Atlas datasets of breast, colon, and prostate cancer. Overall, these findings indicate that TEs, specifically endogenous retroviruses, are demethylated and transcribed during transformation. Significance: Analysis of epigenetic and transcriptional changes in a transformation model reveals that transposable element expression and methylation are dysregulated during oncogenic transformation.

Ovarian cancer (OC) is the fifth leading cause of cancer death in women in the United States. Despite novel therapies and significant advances in our understanding of the disease, the five-year survival rate for OC has not changed in decades. The immunosuppressive microenvironment of OC leads to a low response rate to immunotherapies. We have previously shown that in a murine model of OC epigenetic therapies including DNA methyltransferase inhibitors (DNMTis) and histone deacetylase inhibitors (HDACis) activate type I interferon signaling in tumor cells to drive immune infiltration and sensitization to immunotherapy. This indicates a role for immune control of the tumor, but we did not directly investigate the effects of these epigenetic therapies on the immune cells. We hypothesized that T cells could be activated by epigenetic therapy in this model. We isolated T cells from the spleens of healthy C57/Bl6 mice and treated the cells with a DNMTi, 5-azacytidine (Aza) at 500 nmol/L, and an HDACi, ITF-2357 (ITF) at 100 nmol/L, singly and in combination. Cell pellets were isolated, RNA extracted, and analyzed via qRT-PCR. Cell supernatants were analyzed for cytokine and chemokine production using a Legendplex assay. The treated bulk T cells, CD8+ T cells, and CD4+ T cells showed anti-tumor cytokines, including CCL5, TNF-α and IFN-γ, were upregulated while the pro-tumor cytokine IL-10 was downregulated. These changes were evident at the transcript and secreted cytokine level, pointing to transcriptional upregulation of the target genes. This study demonstrates direct activation of T cells by DNMTis and HDACis and provides mechanistic insight into the role of epigenetic therapies in the murine model of OC.

Novel therapies are urgently needed for ovarian cancer (OC), the fifth deadliest cancer in women. Preclinical data shows great promise for the treatment of OC with epigenetic therapy. The inhibition of DNMT activates transcription of double-stranded (ds)RNA, including transposable elements. These dsRNAs activate sensors in the cytoplasm and trigger the induction of type I interferon signaling. Adenosine deaminase 1 (ADAR1) is induced by interferon signaling and edits mammalian dsRNA with an A-to-I nucleotide change. These edited dsRNAs cannot be sensed by dsRNA sensors, and thus ADAR1 inhibits the type I interferon response. We show that transposable elements including Alus, LINEs, and ERVs, are edited by ADAR1 in human OC cell lines after DNMTi treatment. We hypothesized that increased ADAR1 editing reduces the DNMTi immune response and treated Adar1 knockdown cells with DNMT1 in vitro and in vivo. Combining ADAR1 knockdown with DNMTi significantly increased pro-inflammatory cytokine production compared to either perturbation alone. We treated an immunocompetent mouse model of OC with DNMTi after implanting them with Adar1 knockdown cells or control cells (ID8 Trp53−/−) and found that DNMTi treatment and Adar1 loss reduces tumor burden and extends survival in this model, and the combination of both is the most effective. Combining Adar1 loss and DNMTi transforms the immune microenvironment, including increased recruitment of natural killer cells and M1 macrophage polarization. This survival benefit is a result of type I interferon signaling and CD8+ T cells. Thus, epigenetically inducing transposable element transcription, followed by inhibition of RNA editing, is a novel therapeutic strategy to reverse immune evasion in OC. Research was supported by the National Cancer Institute R00CA204592 and R37CA251270 (to KBC), the DOD Ovarian Cancer Research Program (W81XWH2010273 to KBC), and by the Marlene and Michael Berman Endowed Fund for Ovarian Cancer Research. JIM was supported by an NCI NRSA Institutional Research Training Program grant (T32 CA 247756). SG was supported by a NRSA Predoctoral Fellowship (NIH/NCI 1F31CA254315-01). The authors would like to acknowledge the Institute for Biomedical Sciences at the George Washington University for graduate student support and training (SG, EEG, TK, VB).

Transgenic expression of bacterial nitroreductase (NTR) enzymes sensitizes eukaryotic cells to prodrugs such as metronidazole (MTZ), enabling selective cell-ablation paradigms that have expanded studies of cell function and regeneration in vertebrates. However, first-generation NTRs required confoundingly toxic prodrug treatments to achieve effective cell ablation, and some cell types have proven resistant. Here we used rational engineering and cross-species screening to develop an NTR variant, NTR 2.0, which exhibits ~100-fold improvement in MTZ-mediated cell-specific ablation efficacy, eliminating the need for near-toxic prodrug treatment regimens. NTR 2.0 therefore enables sustained cell-loss paradigms and ablation of previously resistant cell types. These properties permit enhanced interrogations of cell function, extended challenges to the regenerative capacities of discrete stem cell niches, and novel modeling of chronic degenerative diseases. Accordingly, we have created a series of bipartite transgenic reporter/effector resources to facilitate dissemination of NTR 2.0 to the research community.

BackgroundNovel therapies are urgently needed for ovarian cancer (OC), the fifth deadliest cancer in women. Preclinical work has shown that DNA methyltransferase inhibitors (DNMTis) can reverse the immunosuppressive tumor microenvironment in OC. Inhibiting DNA methyltransferases activate transcription of double-stranded (ds)RNA, including transposable elements. These dsRNAs activate sensors in the cytoplasm and trigger type I interferon (IFN) signaling, recruiting host immune cells to kill the tumor cells. Adenosine deaminase 1 (ADAR1) is induced by IFN signaling and edits mammalian dsRNA with an A-to-I nucleotide change, which is read as an A-to-G change in sequencing data. These edited dsRNAs cannot be sensed by dsRNA sensors, and thus ADAR1 inhibits the type I IFN response in a negative feedback loop. We hypothesized that decreasing ADAR1 editing would enhance the DNMTi-induced immune response.MethodsHuman OC cell lines were treated in vitro with DNMTi and then RNA-sequenced to measure RNA editing. Adar1 was stably knocked down in ID8Trp53-/-mouse OC cells. Control cells (shGFP) or shAdar1 cells were tested with mock or DNMTi treatment. Tumor-infiltrating immune cells were immunophenotyped using flow cytometry and cell culture supernatants were analyzed for secreted chemokines/cytokines. Mice were injected with syngeneic shAdar1 ID8Trp53-/-cells and treated with tetrahydrouridine/DNMTi while given anti-interferon alpha and beta receptor 1, anti-CD8, or anti-NK1.1 antibodies every 3 days.ResultsWe show that ADAR1 edits transposable elements in human OC cell lines after DNMTi treatment in vitro. Combining ADAR1 knockdown with DNMTi significantly increases pro-inflammatory cytokine/chemokine production and sensitivity to IFN-β compared with either perturbation alone. Furthermore, DNMTi treatment and Adar1 loss reduces tumor burden and prolongs survival in an immunocompetent mouse model of OC. Combining Adar1 loss and DNMTi elicited the most robust antitumor response and transformed the immune microenvironment with increased recruitment and activation of CD8+ T cells.ConclusionIn summary, we showed that the survival benefit from DNMTi plus ADAR1 inhibition is dependent on type I IFN signaling. Thus, epigenetically inducing transposable element transcription combined with inhibition of RNA editing is a novel therapeutic strategy to reverse immune evasion in OC, a disease that does not respond to current immunotherapies.

Exposure to stress or glucocorticoids (GCs) is associated with epigenetic and transcriptional changes in genes that either mediate or are targets of GC signalling. FKBP5 (FK506 binding protein 5) is one such gene that also plays a central role in negative feedback regulation of GC signalling 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.

We sought to replicate and expand upon previous work demonstrating antenatal TTC9B and HP1BP3 gene DNA methylation is prospectively predictive of postpartum depression (PPD) with ~80% accuracy. In a preterm birth study from Emory, Illumina MethylEPIC microarray derived 1st but not 3rd trimester biomarker models predicted 3rd trimester Edinburgh Postnatal Depression Scale (EPDS) scores ≥ 13 with an AUC=0.8 (95% CI: 0.63-0.8). Bisulfite pyrosequencing derived biomarker methylation was generated using bisulfite pyrosequencing across all trimesters in a pregnancy cohort at UC Irvine and in 3rd trimester from an independent Johns Hopkins pregnancy cohort. A support vector machine model incorporating 3rd trimester EPDS scores, TTC9B, and HP1BP3 methylation status predicted 4 week to 6 week postpartum EPDS ≥ 13 from 3rd trimester blood in the UC Irvine cohort (AUC=0.78, 95% CI: 0.64-0.78) and from the Johns Hopkins cohort (AUC=0.84, 95% CI: 0.72-0.97), both independent of previous psychiatric diagnosis. Technical replicate predictions in a subset of the Johns Hopkins cohort exhibited strong cross experiment correlation. This study confirms the PPD prediction model has the potential to be developed into a clinical tool enabling the identification of pregnant women at future risk of PPD who may benefit from clinical intervention.

Epigenetic modulation of genes involved in regulating the hypothalamic-pituitary-adrenal (HPA) axis has been hypothesized to be a mechanism explaining the association of early life stress and vulnerability to mental illnesses, including schizophrenia. However, there has been little direct evidence of this mechanism in schizophrenia.

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these animal models. The rat is one particular model animal where an epigenetic tool can complement many pharmacological and behavioral studies to provide insightful mechanistic information. To this end, we adapted the SureSelect Target Capture System (referred to as Methyl-Seq) for the rat, which can assess DNA methylation levels across the rat genome. The rat design targeted promoters, CpG islands, island shores, and GC-rich regions from all RefSeq genes. To implement the platform on a rat experiment, male Sprague Dawley rats were exposed to chronic variable stress for 3 weeks, after which blood samples were collected for genomic DNA extraction. Methyl-Seq libraries were constructed from the rat DNA samples by shearing, adapter ligation, target enrichment, bisulfite conversion, and multiplexing. Libraries were sequenced on a next-generation sequencing platform and the sequenced reads were analyzed to identify DMRs between DNA of stressed and unstressed rats. Top candidate DMRs were independently validated by bisulfite pyrosequencing to confirm the robustness of the platform. Results demonstrate that the rat Methyl-Seq platform is a useful epigenetic tool that can capture methylation changes induced by exposure to stress.

A single nucleotide polymorphism (SNP: rs1360780) in FKBP5 (FK506 Binding Protein 5) has been shown to interact with exposure to childhood adversity to promote loss of methylation and increase in gene expression in adults. We asked whether rs1360780 can influence FKBP5 intronic methylation in the context of exposure to maternal affective disorders in utero. Sixty cord blood DNA samples from the Boston Birth Cohort were genotyped at rs1360780 and studied for methylation changes as they relate to genotype and exposure to affective disorders during pregnancy. Linear regression was employed to contrast the risk (TT) genotype to the heterozygous (CT) and homozygous (CC) genotypes with adjustment for potential confounders. The recessive genotype (TT) was associated with increased methylation at multiple CpGs in the FKBP5 intron 5 region (p < 0.01). These findings were enhanced among cases exposed to maternal affective disorders (p = 0.02). A human cell line treated with cortisol showed that changes in intron 5 CpG methylation and FKBP5 expression were inversely associated. These findings suggest that rs1360780 can influence FKBP5 intronic methylation by acting in cis as a methylation quantitative locus and highlight the impact of genotypic risk on methylation in utero. Additionally, prenatal stress exposure compounded with the risk genotype may lead to a compensatory increase in methylation.

Chronic exposure to glucocorticoids (GCs) can lead to psychiatric complications through epigenetic mechanisms such as DNA methylation (DNAm). We sought to determine whether epigenetic changes in a peripheral tissue can serve as a surrogate for those in a relatively inaccessible tissue such as the brain. DNA extracted from the hippocampus and blood of mice treated with GCs or vehicle solution was assayed using a genome-wide DNAm platform (Methyl-Seq) to identify differentially methylated regions (DMRs) induced by GC treatment. We observed that ∼70% of the DMRs in both tissues lost methylation following GC treatment. Of the 3,095 DMRs that mapped to the same genes in both tissues, 1,853 DMRs underwent DNAm changes in the same direction. Interestingly, only 209 DMRs (<7%) overlapped in genomic coordinates between the 2 tissues, suggesting tissue-specific differences in GC-targeted loci. Pathway analysis showed that the DMR-associated genes were members of pathways involved in metabolism, immune function, and neurodevelopment. Also, changes in cell type composition of blood and brain were examined by fluorescence-activated cell sorting. Separation of the cortex into neuronal and non-neuronal fractions and the leukocytes into T-cells, B-cells, and neutrophils showed that GC-induced methylation changes primarily occurred in neurons and T-cells, with the blood tissue also undergoing a shift in the proportion of constituent cell types while the proportion of neurons and glia in the brain remained stable. From the current pilot study, we found that despite tissue-specific epigenetic changes and cellular heterogeneity, blood can serve as a surrogate for GC-induced changes in the brain.

FKBP5 encodes a co-chaperone of HSP90 protein that regulates intracellular glucocorticoid receptor sensitivity. When it is bound to the glucocorticoid receptor complex, cortisol binds with lower affinity to glucocorticoid receptor. Cushing's syndrome is associated with memory deficits, smaller hippocampal volumes, and wide range of cognitive impairments. We aimed at evaluating blood DNA methylation of FKBP5 and its relationship with memory and hippocampal volumes in Cushing's syndrome patients. Polymorphism rs1360780 in FKBP5 has also been assessed to determine whether genetic variations can also govern CpG methylation. Thirty-two Cushing's syndrome patients and 32 matched controls underwent memory tests, 3-Tesla MRI of the brain, and DNA extraction from total leukocytes. DNA samples were bisulfite treated, PCR amplified, and pyrosequenced to assess a total of 41CpG-dinucleotides in the introns 1, 2, 5, and 7 of FKBP5. Significantly lower intronic FKBP5 DNA methylation in CS patients compared to controls was observed in ten CpG-dinucleotides. DNA methylation at these CpGs correlated with left and right HV (Intron-2-Region-2-CpG-3: LHV, r = 0.73, p = 0.02; RHV, r = 0.58, p = 0.03). Cured and active CS patients showed both lower methylation of intron 2 (92.37, 91.8, and 93.34 %, respectively, p = 0.03 for both) and of intron 7 (77.08, 73.74, and 79.71 %, respectively, p = 0.02 and p < 0.01) than controls. Twenty-two subjects had the CC genotype, 34 had the TC genotype, and eight had the TT genotype. Lower average DNA methylation in intron 7 was observed in the TT subjects compared to CC (72.5vs. 79.5 %, p = 0.02) and to TC (72.5 vs. 79.0 %, p = 0.03). Our data demonstrate, for the first time, a reduction of intronic DNA methylation of FKBP5 in CS patients.

Traditional treatments for obesity have poor long-term outcomes. Currently, the most successful method of sustained weight loss is bariatric surgery. Despite the indisputable effectiveness of bariatric surgery, there remains significant inter-individual variability in the treatment response. The mechanisms by which these bariatric procedures lead to successful weight loss are only partially understood and are thought to include biological mechanisms that are subject to genetic variation. The aim of our study was to identify genetic predictors that may explain variation in weight loss efficacy following bariatric surgery.

The oxytocin receptor (OXTR) is a key regulator of stress and anxiety and may be regulated by both psychosocial risk factors and gonadal hormones, making it an attractive candidate for study in postpartum depression (PPD). The objective of this study was to investigate both serum hormone and PPD specific DNA methylation variation in the OXTR. Illumina HM450 microarray data generated in a prospective PPD cohort identified significant associations (P = 0.014) with PPD in an intronic region in the OXTR located 4 bp proximal to an estrogen receptor (ER) binding region. Pyrosequencing confirmed moderate evidence for an interaction of CpGs in the region with childhood abuse status to mediate PPD. These CpGs located on chr3 at positions 8810078 and 8810069 exhibited significant associations with postpartum depression scores from an independent cohort of 240 women with no prior psychiatric history. Hormone analysis suggested a PPD specific negative correlation of DNA methylation in the region with serum estradiol levels. Estradiol levels and OXTR DNA methylation exhibited a significant interaction to associate with the ratio of allopregnanolone to progesterone. Cumulatively, the data corroborate our previous hypotheses of a PPD specific increased sensitivity of epigenetic reprogramming at estrogen target genes and suggests that OXTR epigenetic variation may be an important mediator of mood relevant neuroactive steroid production.

Emerging evidence suggests that early-life experiences and exposure to environmental factors are strong determinants of behavior. One of the most studied and well-characterized environmental factors is stress. Exposure to stress and its primary agent cortisol is a strong risk factor for many behavioral disorders. Studies show that stress and cortisol can influence gene function through epigenetic mechanisms, which may lead to persistent changes in behavior. In this chapter, we explain how stress and our perception of the stressor activate our hypothalamic–pituitary–adrenal (HPA) axis and how exposure to chronic stress influences behaviors such as mood and addiction to drugs. Specifically, we will cover how chronic stress impacts the HPA axis itself and genes that are critical for brain function, especially those that regulate the activity of neurotransmitters. Together, evidence suggests that stress exposure affects multiple neurotransmitter systems, and behavioral disorders likely arise from a combination of deficiencies in multiple systems.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

DNA methylation variation at HP1BP3 and TTC9B is modified by estrogen exposure in the rodent hippocampus and was previously shown to be prospectively predictive of postpartum depression (PPD) when modeled in antenatal blood. The objective of this study was to replicate the predictive efficacy of the previously established model in women with and without a previous psychiatric diagnosis and to understand the effects of changing hormone levels on PPD biomarker loci. Using a statistical model trained on DNA methylation data from N=51 high-risk women, we prospectively predicted PPD status in an independent N=51 women using first trimester antenatal gene expression levels of HP1BP3 and TTC9B, with an area under the receiver operator characteristic curve (AUC) of 0.81 (95% CI: 0.69–0.92, p<5 × 10−4). Modeling DNA methylation of these genes in N=240 women without a previous psychiatric diagnosis resulted in a cross-sectional prediction of PPD status with an AUC of 0.81 (95% CI: 0.68–0.93, p=0.01). TTC9B and HP1BP3 DNA methylation at early antenatal time points showed moderate evidence for association to the change in estradiol and allopregnanolone over the course of pregnancy, suggesting that epigenetic variation at these loci may be important for mediating hormonal sensitivity. In addition both loci showed PPD-specific trajectories with age, possibly mediated by age-associated hormonal changes. The data add to the growing body of evidence suggesting that PPD is mediated by differential gene expression and epigenetic sensitivity to pregnancy hormones and that modeling proxies of this sensitivity enable accurate prediction of PPD.