University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Faculty Publications, Department of Statistics Statistics, Department of 2016 Sex-specific hippocampal 5-hydroxymethylcytosine is disrupted in response to acute stress Ligia A. Papale University of Wisconsin, [email protected] Sisi Li University of Wisconsin, [email protected] Andy Madrid University of Wisconsin, [email protected] Qi Zhang University of Nebraska-Lincoln, [email protected] Li Chen Emory University See next page for additional authors Follow this and additional works at: https://digitalcommons.unl.edu/statisticsfacpub Part of the Other Statistics and Probability Commons Papale, Ligia A.; Li, Sisi; Madrid, Andy; Zhang, Qi; Chen, Li; Chopra, Pankaj; Jin, Peng; Keles, Sunduz; and Alisch, Reid S., "Sex- specific hippocampal 5-hydroxymethylcytosine is disrupted in response to acute stress" (2016). Faculty Publications, Department of Statistics. 62. https://digitalcommons.unl.edu/statisticsfacpub/62 This Article is brought to you for free and open access by the Statistics, Department of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Publications, Department of Statistics by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Authors Ligia A. Papale, Sisi Li, Andy Madrid, Qi Zhang, Li Chen, Pankaj Chopra, Peng Jin, Sunduz Keles, and Reid S. Alisch This article is available at DigitalCommons@University of Nebraska - Lincoln: https://digitalcommons.unl.edu/statisticsfacpub/62 Neurobiology of Disease 96 (2016) 54–66 Contents lists available at ScienceDirect Neurobiology of Disease journal homepage: www.elsevier.com/locate/ynbdi Sex-specific hippocampal 5-hydroxymethylcytosine is disrupted in response to acute stress Ligia A. Papale a,1,SisiLia,c,1, Andy Madrid a,c,QiZhangd,LiChene,PankajChoprae,PengJine, Sündüz Keleş b, Reid S. Alisch a,⁎ a Department of Psychiatry, University of Wisconsin, Madison, WI, USA b Department of Statistics, Biostatistics, and Medical Informatics, University of Wisconsin, Madison, WI, USA c Neuroscience Training Program, University of Wisconsin, Madison, WI, USA d Department of Statistics, University of Nebraska, Lincoln, NE, USA e Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA article info abstract Article history: Environmental stress is among the most important contributors to increased susceptibility to develop psychiatric Received 8 July 2016 disorders. While it is well known that acute environmental stress alters gene expression, the molecular mecha- Revised 18 August 2016 nisms underlying these changes remain largely unknown. 5-hydroxymethylcytosine (5hmC) is a novel environ- Accepted 23 August 2016 mentally sensitive epigenetic modification that is highly enriched in neurons and is associated with active Available online 26 August 2016 neuronal transcription. Recently, we reported a genome-wide disruption of hippocampal 5hmC in male mice fol- Keywords: lowing acute stress that was correlated to altered transcript levels of genes in known stress related pathways. fi Sex-specific Since sex-speci c endocrine mechanisms respond to environmental stimulus by altering the neuronal epige- Acute stress nome, we examined the genome-wide profile of hippocampal 5hmC in female mice following exposure to Epigenetics acute stress and identified 363 differentially hydroxymethylated regions (DhMRs) linked to known (e.g., Nr3c1 DNA methylation and Ntrk2) and potentially novel genes associated with stress response and psychiatric disorders. Integration 5-Hydroxymethylcytosine of hippocampal expression data from the same female mice found stress-related hydroxymethylation correlated Gene expression to altered transcript levels. Finally, characterization of stress-induced sex-specific5hmCprofiles in the hippocam- pus revealed 778 sex-specific acute stress-induced DhMRs some of which were correlated to altered transcript levels that produce sex-specific isoforms in response to stress. Together, the alterations in 5hmC presented here provide a possible molecular mechanism for the adaptive sex-specific response to stress that may augment the design of novel therapeutic agents that will have optimal effectiveness in each sex. © 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction healthy brain likely underlie pronounced sex differences in susceptibil- ity, progression, symptom severity, and pathology of neurological disor- Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is ders (Cahill, 2006; Cosgrove et al., 2007; McCarthy et al., 2012). the most common hallmark across all neuropsychiatric diseases (Bale, Consistent with this notion, females are more likely than males to devel- 2015; Martin et al., 2010). The HPA axis is a dynamic system that allows op depression, anxiety, and Alzheimer's disease, while males are more an organism to respond to environmental stress in a sex-specificman- likely to be diagnosed with attention deficit hyperactivity disorder, au- ner by governing the activity of sex-specific endocrine mechanisms in- tism, and Parkinson's disease (Balint et al., 2009; Gillberg et al., 2006; cluding the neuronal epigenome, which can effect gene expression Hebert et al., 2013; Nolen-Hoeksema, 1987; Weissman et al., 1996; (McCarthy et al., 2009; Meaney, 2001). Genomic analyses of human Wooten et al., 2004). The hippocampus is an important brain structure brain tissue showed that ~2.5% of genes are differentially expressed be- to study the effects of glucocorticoids and stress on gene expression as tween males and females (Trabzuni et al., 2013). Thus, fundamental sex studies have shown that known stress-related genes undergo expres- differences in the anatomy and the genetic regulatory network of the sion changes in the hippocampus following acute and chronic stress ex- posure (Cirelli et al., 2006; Gray et al., 2014; McGowan et al., 2009; Roth et al., 2009; Rubin et al., 2014). The negative feedback regulation of the ⁎ Corresponding author at: Department of Psychiatry, University of Wisconsin School of HPA axis produced upon binding of glucocorticoids to their receptors in Medicine, 6001 Research Park Blvd, Madison, WI 53719-1176, USA. the hippocampus is critical for a healthy stress response. Understanding E-mail address: [email protected] (R.S. Alisch). 1 These authors contributed equally to this work. the biological and molecular underpinnings of sex differences in re- Available online on ScienceDirect (www.sciencedirect.com). sponse to stress is likely to be a useful window into the mechanistic http://dx.doi.org/10.1016/j.nbd.2016.08.014 0969-9961/© 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). L.A. Papale et al. / Neurobiology of Disease 96 (2016) 54–66 55 cause of mental illness in both men and women (Altemus et al., 2014; same day to ensure that identical procedures were conducted on both Rutter et al., 2003). sexes. A description of the male mice was described previously (Li et Alterations in environmentally sensitive epigenetic modifications al., 2016). For the female mice, on the day of the experiment seven- are emerging as important factors in the long-term biological trajecto- week old naïve virgin female C57BL/6 mice were randomly divided ries leading to stress-related psychiatric disorders (Hunter et al., 2009; into experimental or control (naïve) groups (N =5and3pergroup,re- Maccari et al., 2016). DNA methylation (5-methylcytosine (5mC)) is spectively). Following a published acute stress paradigm that resulted in an epigenetic modification with important roles in chromatin alterations in epigenetic modifications (including 5hmC in males) and remolding, gene silencing, embryonic development, cellular differentia- gene expression (Gray et al., 2014; Hunter et al., 2009; Li et al., 2016), tion, and the maintenance of cellular identity (Chouliaras et al., 2013; the female experimental mice were restrained for 30 min (2 h after Mellen et al., 2012; Tollervey and Lunyak, 2012). Moreover, changes in lights-on) head first into a 50 ml conical vial that has an 8 millimeter di- 5mC have been linked to neurological disorders as well as psychiatric ameter hole at the tip to allow sufficient oxygen flow. Post restraint an- disorders, including depression, anxiety, post-traumatic stress disorders imals were individually housed in a clean cage for 1 h. After this (PTSD) and schizophrenia (Abdolmaleky et al., 2006; Collishaw et al., recovery period, animals were briefly anesthetized (isoflurane) to min- 2007; Kuratomi et al., 2008; Pidsley et al., 2014; Poulter et al., 2008; imize the stress of handling and sacrificed. At the same time that the ex- Robertson, 2005; Weaver et al., 2004). A recent study found differences perimental mice were taken from the cage to begin the stress paradigm, in 5mC between males and females across all autosomes, some of which the naïve/control mice also were taken from the cage, briefly anesthe- were associated with altered gene expression (Singmann et al., 2015), tized (isoflurane) prior to sacrifice and tissue dissection. Finally, to min- suggesting the potential for a sex-specific role for this DNA methylation imize the effect of parent-to-offspring interaction per litter, a maximum modification. 5mC can be oxidized to 5-hydroxymethylcytosine (5hmC) of