Genome-wide alterations in hippocampal 5-hydroxymethylcytosine links plasticity genes to acute stress

Abstract

Environmental stress is among the most important contributors to increased susceptibility to develop psychiatric disorders, including anxiety and post-traumatic stress disorder. While even acute stress alters gene expression, the molecular mechanisms underlying these changes remain largely unknown. 5-hydroxymethylcytosine (5hmC) is a novel environmentally sensitive DNA modification that is highly enriched in post-mitotic neurons and is associated with active transcription of neuronal genes. Recently, we found a hippocampal increase of 5hmC in the glucocorticoid receptor gene (Nr3c1) following acute stress, warranting a deeper investigation of stress-related 5hmC levels. Here we used an established chemical labeling and affinity purification method coupled with high-throughput sequencing technology to generate the first genome-wide profile of hippocampal 5hmC following exposure to acute restraint stress and a one-hour recovery. This approach found a genome-wide disruption in 5hmC associated with acute stress response, primarily in genic regions, and identified known and potentially novel stress-related targets that have a significant enrichment for neuronal ontological functions. Integration of these data with hippocampal gene expression data from these same mice found stress-related hydroxymethylation correlated to altered transcript levels and sequence motif predictions indicated that 5hmC may function by mediating transcription factor binding to these transcripts. Together, these data reveal an environmental impact on this newly discovered epigenetic mark in the brain and represent a critical step toward understanding stress-related epigenetic mechanisms that alter gene expression and can lead to the development of psychiatric disorders.

Introduction

Environmental stress, particularly via its effects on limbic structures such as the hippocampus, is among the most important contributors to increased susceptibility to develop anxiety and depression related disorders (McEwen, 2007, Conrad, 2008). An acute stress response involves activation of the hypothalamic pituitary adrenal (HPA) axis, which results in the sequential release of corticotrophin releasing factor (CRF), adrenocorticotropic hormone, and cortisol. The hippocampus is particularly important because receptors for both CRF and cortisol (e.g., the glucocorticoid receptor) are present in the hippocampus, which suggests that these stress mediators can directly influence neuronal activity and facilitate the onset of neuropsychiatric disorders (Chen et al., 2004a, Yan et al., 1998, Regev and Baram, 2014). The negative feedback inhibition produced upon binding of glucocorticoids to their receptors in the hippocampus is critical for a healthy stress response. Environmentally sensitive epigenetic modifications that are disrupted in response to traumatic experience and stress are emerging as important factors in the long-term biological trajectories leading to stress-related psychiatric disorders (Klengel et al., 2014). The hippocampus was the first brain region to show an epigenetic response to an exogenous stimulus when alterations in histone modifications and gene expression were found following a single acute restraint stress (Gray et al., 2014, Hunter et al., 2009). Elucidating the molecular mechanisms by which an acute stressor contributes to functional and molecular changes in the brain is needed to improve our understanding of complex behaviors and the origins of psychiatric disorders.

DNA methylation is an epigenetic modification with important roles in chromatin remolding, gene silencing, embryonic development, cellular differentiation, and the maintenance of cellular identity (Li et al., 1993, Reik, 2007, Zemach et al., 2010). Traditional studies of DNA methylation have focused on the dynamic variation of a methyl group on cytosine (i.e., 5-methylcytosine; 5mC), which has been linked to neurological disorders as well as psychiatric disorders, including depression, anxiety, post-traumatic stress disorders (PTSD) and schizophrenia (Grayson et al., 2005, Abdolmaleky et al., 2006, Kuratomi et al., 2008, Poulter et al., 2008). We and others have shown that this modification is a key factor in experience-dependent plasticity such as the expression of an anxious temperament disposition, an important risk factor for the development of anxiety and depression (Alisch et al., 2014, Pidsley et al., 2014). Despite these connections of 5mC to human psychiatric disorders, it was recently shown that 5mC can be successively oxidized by the ten-eleven translocation family of proteins (Tet1-3), which converts 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine, and 5-carboxylcytosine following exposure to environmental stimuli (e.g., oxidative stress). Since the detection of DNA methylation using traditional sodium bisulfite methodologies cannot distinguish 5mC and 5hmC, previous interpretations of DNA methylation in health and disease should be reconsidered in future studies. Initial studies found that 5hmC is enriched in neuronal cells and is associated with the regulation of neuronal activity (Yao and Jin, 2014). Genomic studies revealed that 5hmC is located within distal cis-regulatory elements and in the gene bodies of synaptic plasticity-related loci, particularly at intron–exon boundaries, suggesting an important role for 5hmC in coordinating transcriptional activity (Yao and Jin, 2014, Khare et al., 2012). These findings have prompted investigations into the potential role(s) of 5hmC in disease, where it appears to function in neurological disorders (e.g., Rett syndrome and Autism) (Al-Mahdawi et al., 2014, Mellen et al., 2012, Zhubi et al., 2014) and neurodegenerative diseases (e.g., Huntington's and Alzheimer's) (Villar-Menendez et al., 2013, Wang et al., 2013, Chouliaras et al., 2013, Bradley-Whitman and Lovell, 2013). Recently, we reported that acute stress increases 5hmC levels in the glucocorticoid receptor gene, Nr3c1, suggesting a role for 5hmC in stress (Li et al., 2015). Together, with recent links to learning and cognition (Li et al., 2014), studies of 5hmC function(s) have become a significant focus of neuroscience research.

In this study, an established chemical labeling and affinity purification method coupled with high-throughput sequencing technology was used to generate an unbiased genome-wide profile of hippocampal 5hmC following exposure to acute restraint stress. An overlay of these data with genome-wide gene expression data from the same mice found stress-related hydroxymethylation correlated to altered transcript levels. Together, these genomic surveys provide new insight into the potential for functional epigenetic contributions to a stress response and they are intended to serve as a conceptual basis that will facilitate the future study of cellular and brain regional dynamics of 5hmC, especially as it relates to stress and stress recovery. These results demonstrate the power of coupling methylome and transcriptome data to determine the molecular origins of stress-related psychiatric disorders, such as PTSD and anxiety disorders. Here we provide the first genome-wide map of 5hmC in the mouse hippocampus following an acute stress, which reveals known and potentially novel genes contributing to the stress response. These findings establish a role for 5hmC in acute stress and provide insights into the immediate genome-wide neuromolecular response to traumatic events.