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When compared to women, men have a higher incidence of schizophrenia, with increases in negative and cognitive symptoms, and an overall poorer disease course. Schizophrenia is conceptualized as a disorder of aberrant gene transcription and regulation. Thus, epigenetics, the study of environmentally induced changes in gene regulation, could advance our understanding of the molecular underpinnings of schizophrenia. Peripheral histone methyltransferase (HMT) mRNA levels have been previously shown to be significantly increased in patients with schizophrenia and correlate with symptomology. In this independent study, peripheral lymphocytes were extracted and clinical symptoms were measured on 74 participants, (40 patients with schizophrenia (19 women, 21 men) and 34 healthy individuals (19 women, 15 men)). HMT (G9α, SETDB1 and GLP) mRNA levels and their resulting histone modification H3K9me2 were measured with RT-PCR and ELISA respectively. Plasma estradiol levels were also measured via ELISA and correlated with HMT mRNA. Clinical symptoms were measured utilizing the Positive and Negative Syndrome Scale (PANSS) and the Heinrichs Carpenter Quality of Life Scale (QLS). The results indicate that men with schizophrenia expressed the highest levels of G9α, SETDB1 mRNA and H3K9me2 protein levels. Additionally, higher levels of symptom presentation and an overall poorer quality of life were correlated with higher HMT mRNA and H3K9me2 protein levels in a sex-dependent pattern. These data support the hypothesis of a sex-dependent restrictive epigenome contributing towards the etiology of schizophrenia. The histone methyltransferases measured here could be potential future therapeutic targets for small molecule pharmacology.

Schizophrenia is a chronic and debilitating mental disorder that impacts psychological, social, and cognitive processes across the lifespan (Saha et al., 2005). While the disease greatly disrupts the quality of life for both men and women, there is convincing evidence that sex modulates the clinical presentation and course of the disease. Schizophrenia is less severe in women, with lower incident levels, and a later age of onset (Leung and Chue, 2000 and McGrath et al., 2004). Women also exhibit better premorbid functioning (Allen et al., 2013), less severe negative symptoms, fewer and less frequent acute episodes of psychosis, and a better response to antipsychotic medications compared to men (Halbreich and Kahn, 2003, Leung and Chue, 2000, Riecher-Rossler and Hafner, 2000 and Salem and Kring, 1998). Schizophrenia can be conceptualized as a disorder of aberrant gene regulation, frequently decreases in gene transcription (Torrey et al., 2005). Epigenetics, the study of environmentally induced changes in gene regulation that arise from post-transcriptional modifications to both DNA and DNA interacting proteins (histones), is an opportune avenue to understand the molecular underpinnings of schizophrenia (Wolffe and Hayes, 1999 and Sharma et al., 2012). Epigenetic modifications result in protein assemblies that are commonly described as ‘permissive’ or ‘restrictive’. Restrictive assemblies effectively seal the gene promoter from regulation by transcription factors, often by post-translational methylation of the ninth lysine of histone H3 (H3K9me2; Krauss, 2008 and Lyons and Lomvardas, 2014). In particular, the formation of H3K9me2 is catalyzed by histone methyltransferases (HMTs), including G9α, GLP, and SETDB1 (Krishnan et al., 2011 and Shinkai and Tachibana, 2011). The majority of studies report increases in restrictive chromatin (Chase et al., 2013, Gavin et al., 2008, Gavin et al., 2009b and Sharma et al., 2008) and resulting down-regulation of gene transcription of several candidate genes in patients with schizophrenia (Akbarian et al., 1995, Guidotti et al., 2000, Impagnatiello et al., 1998 and Jindal et al., 2010). The H3K9 methyltransferases (HMTs) G9α, GLP, and SETDB1 and the restrictive histone modification H3K9me2 are increased in patients with schizophrenia, and significantly correlated with clinical symptomology (Chase et al., 2013). Epigenetic mechanisms contribute to various aspects of sex differences in both brain and behavior, particularly during such critical periods as puberty (Morrison et al., 2013). Prepubertal inhibition of DNA methylation or histone deacetylation (both restrictive modifications) result in pubertal failure (Lomniczi et al., 2013 and Ojeda et al., 2010), demonstrating the marked contribution of epigenetic mechanisms in the regulation of puberty. Promoter methylation and resulting decreased gene expression of the polycomb protein EED, known to interact with histone deacetylases, indicates the commencement of puberty through disinhibition of kisspeptin, an upstream signaling ligand for gonadotropin-releasing hormone (GnRH; Lomniczi et al., 2013). Other sexually dimorphic traits emerge as early as postnatal day 1. When compared to females, males have increased levels of both estrogen receptor promoter methylation and histone deacetylase (HDAC) binding (Kurian et al., 2010 and Murray et al., 2009), effectively decreasing gene transcription. Additionally, MeCP2, a methylated DNA binding repressive scaffold enzyme, demonstrates a sex and age-specific switch in protein levels, with females exhibiting increased amounts in the amygdala and the hypothalamus up until postnatal day 10, after which males exhibit higher levels (Kurian et al., 2007). Lastly, the bed nucleus of the stria terminalis (BNST) contains more cells, and thus is larger in volume in males when compared to female mice. An injection of valproic acid, a histone deacetylase inhibitor, at postnatal days one and two, significantly reduces both cell count and BNST volume in males, eliminating sex differences (Murray et al., 2009). The literature has well documented sex differences on the clinical presentation of schizophrenia, and epigenetic mechanisms of both mental illness and sexual dimorphism separately. However, to our knowledge, few studies to date examine the interplay between these areas of research. Given that histone methyltransferase mRNA levels in peripheral lymphocytes are positively correlated with schizophrenia symptomology (Chase et al., 2013), our primary hypothesis is that men with schizophrenia will have higher levels of these HMT mRNA and resulting H3K9me2 levels when compared to control subjects and women with schizophrenia given the higher incidence of these symptoms in men (Leung and Chue, 2000). The result of this independent study replicates earlier findings, indicating increased restrictive epigenetic measures in schizophrenia (Chase et al., 2013). To supplement these primary analyses, we also investigated the relationship of these epigenetic molecules to individual clinical symptoms in men and women separately.

Three histone methyltransferase genes were examined: G9α, GLP and SETDB1. Participants with a diagnosis of schizophrenia exhibited increased levels of G9α mRNA in the lymphocyte when compared to controls (F(1, 74) = 9.04, p = 0.004). While there were no sex-specific differences in G9α mRNA levels, there was a significant interaction between diagnosis and sex, with male participants with schizophrenia expressing the highest levels of G9α mRNA when compared to women with schizophrenia, and control men and women (F(1, 74) = 3.96, p = 0.05; Fig. 1a). Full-size image (58 K) Fig. 1. Sex and diagnosis differences in both histone methyltransferase mRNA and H3K9me2 ELISA levels. a and b) G9α and SETDB1 mRNA levels were significantly increased in participants with schizophrenia when compared to normal controls, with schizophrenic men expressing the statistically highest levels. c) GLP mRNA expression in lymphocytes was significantly increased in participants with schizophrenia when compared to normal controls. d) H3K9me2 levels were significantly increased in participants with schizophrenia when compared to normal controls. Specifically, men with schizophrenia expressed the highest levels of H3K9me2 in the lymphocyte. *p < 0.05, **p < 0.01. Figure options Lymphocyte mRNA levels of SETDB1 were also significantly increased in participants with schizophrenia when compared to normal controls (F(1, 74) = 4.57, p = 0.03). There were no significant differences found in SETDB1 mRNA between the sexes independently across diagnostic groups. However, further analysis revealed a significant diagnosis by sex interaction, with male patients with schizophrenia again exhibiting the highest amounts of SETDB1 mRNA, a pattern similar to G9α mRNA (F(1, 74) = 4.1, p = 0.04; Fig. 1b). Increases in GLP mRNA were seen in lymphocytes from participants with a diagnosis of schizophrenia compared to normal controls (F(1, 74) = 4.2, p = 0.04; Fig. 1c). There were no independent sex differences or sex by diagnosis interactions seen with GLP mRNA.