A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure

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 ...

Johns Hopkins School of Medicine

Epigenetic studies that utilize peripheral tissues to identify molecular substrates of neuropsychiatric disorders rely on the assumption that disease-relevant, cellular alterations that occur in the brain are mirrored and detectable in peripheral tissues such as blood. We sought to test this assumption by using a mouse model of Cushing's disease and asking whether epigenetic changes induced by glucocorticoids can be correlated between these tissue types.

Alterations in DNA methylation of Fkbp5 as a determinant of blood-brain correlation of glucocorticoid exposure.

Recent studies have shown that loss of TET1 may play a significant role in the formation of tumors. Because genomic instability is a hallmark of cancer, we examined the potential involvement of 10-11 translocation 1 (TET1) in the DNA damage response (DDR). Here we demonstrate that, in response to clinically relevant doses of ionizing radiation (IR), human glial cells made TET1-deficient with lentiviral vectors displayed greater numbers of colony forming units and lower levels of apoptotic markers compared with glial cells transduced with control vectors; yet, they harbored greater DNA strand breaks. The G/M check point and expression of cyclin B1 were greatly diminished in TET1-deficient cells, and TET1-deficient cells displayed lower levels of γH2A.x following exposure to IR. Levels of DNA-PKcs, which are DNA-PK complex members, were lower in TET1-deficient cells compared with control cell lines. However, levels of ATM were similar in both cell lines. Cyclin B1, DNA-PKcs, and γH2A.x levels were each rescued by reintroduction of the TET1 catalytic domain. Finally, cytosine methylation within intron 1 of PRKDC, the gene encoding DNA-PKcs, was significantly higher upon depletion of TET1. Taken together, this study illustrates the involvement of TET1 in the different arms of the DDR and suggests its loss results in the continued survival of cells with genomic instability.

TET1 deficiency attenuates the DNA damage response and promotes resistance to DNA damaging agents.

Chronic exposure to cortisol is associated with cardiovascular, metabolic, and psychiatric disorders. Although cortisol can be readily measured from peripheral sources such as blood, urine, or saliva, multiple samplings spanning several days to weeks are necessary to reliably assess chronic cortisol exposure levels (referred to as cortisol load). Although cortisol levels in hair have been proposed as a measure of cortisol load, measurement is cumbersome and many people are not candidates due to short hair length and use of hair dyes. To date, there are no blood biomarkers that capture cortisol load. To identify a blood biomarker capable of integrating one-month cortisol exposure levels, 75 healthy participants provided 30+ days of awakening and bedtime saliva cortisol and completed psychosocial measures of anxiety, depression, and stress. Mean daily awakening and bedtime cortisol levels were then compared to CpG methylation levels, gene expression, and genotypes of the stress response gene FKBP5 obtained from blood drawn on the last day of the study. We found a correlation between FKBP5 methylation levels and mean 30+day awakening and bedtime cortisol levels (|r|≥0.32, p ≤ 0.006). We also observed a sex-specific correlation between bedtime cortisol levels and FKBP5 mRNA expression in female participants (r = 0.42, p = 0.005). Dividing the 30-day sampling period into four weekly bins showed that the correlations for both methylation and expression were not being driven by cortisol levels in the week preceding the blood draw. We also identified a female-specific association between FKBP5 mRNA expression and scores on the Beck Anxiety Inventory (r = 0.37, p = 0.013) and Beck Depression Inventory-II (r = 0.32, p = 0.033). Finally, DNA was genotyped at four SNPs, and variation in rs4713902 was shown to have an effect on FKBP5 expression under a codominant model (f = 3.41, p = 0.048) for females only. Our results suggest that blood FKBP5 DNA methylation and mRNA expression levels may be a useful marker for determining general or sex-specific 30-day cortisol load and justifies genome-wide approaches that can potentially identify additional cortisol markers with broader clinical utility.

DNA methylation and sex-specific expression of FKBP5 as correlates of one-month bedtime cortisol levels in healthy individuals.

Through unbiased transcriptomics and multiple molecular tools, transient downregulation of the Orthodenticle homeobox 2 (OTX2) gene was recently causatively associated with the development of depressive-like behaviors in a mouse model of early life stress. The analyses presented in this manuscript test the translational applicability of these findings by examining peripheral markers of methylation of OTX2 and OTX2-regulated genes in relation to measures of depression and resting-state functional connectivity data collected as part of a larger study examining risk and resilience in maltreated children. The sample included 157 children between the ages of 8 and 15 years (χ = 11.4, SD = 1.9). DNA specimens were derived from saliva samples and processed using the Illumina 450 K beadchip. A subset of children (N = 47) with DNA specimens also had resting-state functional MRI data. After controlling for demographic factors, cell heterogeneity, and three principal components, maltreatment history and methylation in OTX2 significantly predicted depression in the children. In terms of the imaging data, increased OTX2 methylation was found to be associated with increased functional connectivity between the right vmPFC and bilateral regions of the medial frontal cortex and the cingulate, including the subcallosal gyrus, frontal pole, and paracingulate gyrus-key structures implicated in depression. Mouse models of early stress hold significant promise in helping to unravel the mechanisms by which child adversity confers risk for psychopathology, with data presented in this manuscript supporting a potential role for OTX2 and OTX2-related (e.g., WNT1, PAX6) genes in the pathophysiology of stress-related depressive disorders in children.

Richard S. Lee

Departments of Psychiatry and Behavioral Sciences

Johns Hopkins School of Medicine

A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure

Richard S. Lee

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Departments of Psychiatry and Behavioral Sciences

Johns Hopkins School of Medicine

A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure

Departments of Psychiatry and Behavioral Sciences