Original article 71 Anxiety is associated with higher levels of global DNA methylation and altered expression of epigenetic and interleukin-6 genes Therese M. Murphya, Aoife O’Donovanb, Niamh Mullinsc, Cliona O’Farrellye, Amanda McCannd and Kevin Malonec Objectives Anxiety is associated with elevated levels of Interestingly, IL-6 gene expression was correlated strongly the inflammatory cytokine interleukin-6 (IL-6) and an with DNMT1/3A/3B and EZH2 expression, highlighting increased risk for diseases with an inflammatory aetiology. a potential relationship between IL-6 and important In cancer, higher levels of IL-6 have been associated with epigenetic regulatory enzymes. increased expression of the epigenetic enzymes DNMT1 Conclusion This study provides novel insight into the and Enhancer of Zeste Homolog 2 (EZH2). However, the relationship between anxiety, epigenetics and IL-6. relationship between IL-6 and DNA methyltransferases Moreover, our findings support the hypothesis that changes (DNMTs) and EZH2 expression has not previously been in DNA methylation profiles may contribute to the biology examined in anxious individuals. of anxiety. Psychiatr Genet 25:71–78 Copyright © 2015 Methods Global DNA methylation levels were measured Wolters Kluwer Health, Inc. All rights reserved. using the Methylflash Methylated DNA Quantification Kit Psychiatric Genetics 2015, 25:71–78 and gene expression levels of the DNMT and EZH2 genes in anxious (n = 25) and nonanxious individuals (n = 22) were Keywords: anxiety, DNA methylation, DNA methyltransferase, inflammation, interleukin-6 compared using quantitative real-time PCR. Specifically, we investigated whether global DNA methylation or aberrant aMedical School, University of Exeter, Royal Devon and Exeter Hospital, Exeter, UK, bDepartment of Psychiatry, San Francisco & San Francisco VA Medical expression of these genes was correlated with IL-6 mRNA Center, University of California, USA, cDepartment of Psychiatry and Mental and protein serum levels in anxious individuals. Health Research, Education and Research Centre, St. Vincent’s University Hospital, dThe UCD Conway Institute of Biomolecular and Biomedical Research, Results Anxious participants had significantly higher levels University College Dublin, UCD School of Medicine and Medical Science, Dublin and eSchool of Biochemistry and Immunology, Trinity College Dublin, Dublin, of global DNA methylation compared with controls Ireland (P = 0.001). There were no differences in the mean mRNA Correspondence to Therese M. Murphy, PhD, Medical School, University of expression levels of the DNMT1/3A/3B, EZH2 and IL-6 Exeter, Royal Devon and Exeter Hospital, Exeter EX2 5DW, UK genes in anxious individuals compared with controls. Tel: + 44 139 272 6429; e-mail: [email protected] However, the expression of DNMT1/3A, EZH2 and IL-6 Received 21 October 2013 Revised 30 May 2014 genes increases with increasing Hospital Anxiety and Accepted 10 September 2014 Depression Scale-Anxiety scores in the anxious cohort only. Introduction turn, high levels of IL-6 increase the risk for cardiovas- Anxiety is associated with an increased risk for many cular and autoimmune diseases as well as cancer diseases with an inflammatory aetiology including cardio- (Kiecolt-Glaser et al., 2003). vascular and autoimmune diseases and some forms of cancer (Kiecolt-Glaser et al., 2002). However, the Epigenetics, defined as the mechanisms that initiate and mechanisms by which anxiety might promote these dis- maintain heritable patterns of gene expression without eases are not well understood. Prolonged upregulation of altering the sequence of the genome (Holliday, 1987), proinflammatory cytokines is thought to contribute to may play an important role in the pathology of psychiatric chronic inflammation in anxious individuals, which may disorders (Poulter et al., 2008; Zhubi et al., 2009) and mediate their increased risk for inflammation-related inflammatory-related diseases, including cancer (Baylin, diseases (Kiecolt-Glaser et al., 2002). Previously, our 2005; Wilson, 2008). The epigenetic mark DNA methy- ′ group has shown that anxious individuals have sig- lation involves the addition of methyl groups to the 5 - nificantly higher serum levels of the systemic proin- position of cytosine rings [5-methylcytosine (5-mC)] at flammatory cytokine, interleukin-6 (IL-6), compared CpG dinucleotides. DNA methylation is orchestrated by – with nonanxious individuals (O’Donovan et al., 2010). In several DNA methyltransferases (DNMTs DNMT1, DNMT3A, DNMT3B, DNMT3L) in combination with polycomb group proteins (Viré et al., 2006). Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this DNMT1 is a maintenance enzyme that binds methyl article on the journal's website (www.psychgenetics.com). groups to hemimethylated DNA during DNA replication 0955-8829 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. DOI: 10.1097/YPG.0000000000000055 Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of the article is prohibited. 72 Psychiatric Genetics 2015, Vol 25 No 2 (Smith et al., 1992). DNMT3A and 3B are de-novo Methods DNMT enzymes that establish methylation patterns Clinical sample collection during embryonic development and genomic imprinting Participants were selected from an anxiety cohort described (Chédin, 2011). DNMT3L interacts with and enhances previously (O’Donovan et al., 2010). Briefly, participants DNMT3A/3B methyltransferase activity in vivo (Chen experiencing high or low levels of psychological distress et al., 2005). Interestingly, the polycomb group protein, were recruited from a large suburban area in Dublin, Ireland. Enhancer of Zeste Homolog 2 (EZH2), is known to The sample cohort included 27 participants with Hospital interact with DNMTs and is required for DNA methy- Anxiety and Depression Scale-Anxiety (HADS-A) scores in lation of EZH2-target promoters (Viré et al., 2006). Thus, the clinical range (Zigmond and Snaith, 1983; Bjelland et al., DNMTs and EZH2 are essential epigenetic regulatory 2002; anxious group, scores > 8) and 29 age-matched parti- enzymes involved in the heritable repression of gene cipants with low scores on the HADS-A (nonanxious group, activity. scores < 8). Exclusion criteria were chronic illness, current or acute illness within the previous 2 weeks, alcoholism, use of Higher levels of IL-6 are associated with increased medication, anaesthesia in the previous 3 months and night expression and activity of DNMT1 and EZH2 in cancer shift work in the previous 2 weeks. The HADS measure (a (Croonquist and Van Ness, 2005; Foran et al., 2010). widely used and well-validated measure of nonsomatic Interestingly, IL-6-dependent miRNAs that regulate symptoms of anxiety and depression) and the self-reported DNMT1 gene expression levels in malignant cholangio- physical health measures used in this study have been cytes have recently been identified (Braconi et al., 2010). described in detail previously (O’Donovan et al., 2010). Therefore, regulation of miRNAs may represent a HADS-D scores and serum IL-6 levels had been previously mechanism by which IL-6 induces the expression of measured for each participant (O’Donovan et al., 2010). DNMT1. Conversely, epigenetic modifications such as Blood samples were collected between 7:30 and 9:30 a.m. DNA methylation are excellent biological candidates for Participants fasted and abstained from smoking and caffeine the molecular arbitration of the immune-dysfunction from midnight, and avoided alcohol and exercise for 24 h phenotype associated with anxiety. DNA methylation before their appointment. The study was approved by the can target genes for transcriptional silencing (Baylin, Vincent’s Healthcare Group Ethics and Medical Research 2005) and is heavily influenced by environmental triggers Committee. such as diet, smoking and traumatic life events (Uddin et al., 2010; Nandakumar et al., 2011; Scoccianti et al., Clinical biological sample collection 2011). In this way aberrant DNA methylation is a hall- Twenty-five participants with HADS-A scores in the mark of a number of human diseases (Robertson, 2005). clinical range (anxious group) and 22 participants with Recently, immune-related genes were identified as tar- low HADS-A scores (nonanxious group) had DNA/RNA gets of aberrant DNA methylation in post-traumatic of sufficient quantity and quality to carry out gene stress disorder, supporting the hypothesis that DNA expression and global methylation analyses. Participants’ methylation contributes to the biology of anxiety-related blood samples (15 ml) were collected in sterile hepar- disorders (Uddin et al., 2010). inized tubes and processed immediately. Whole-blood was overlaid onto a Ficoll-Paque layer and centrifuged for Given the importance that epigenetic regulatory genes 30 min at 400g. Peripheral blood mononuclear cells have in the establishment and maintenance of DNA (PBMCs) were removed from the interface, stained using methylation patterns, it is likely that changes in their trypan blue and counted using a haemocytometer. Cells expression contribute to the molecular aetiology of neu- were subsequently transferred into liquid nitrogen for ropsychiatric disorders including anxiety. Moreover, long-term storage. Genomic DNA and total RNA were altered epigenetic regulatory gene expression has been extracted from the PBMCs using an AllPrep DNA/RNA observed in patients with neuropsychiatric