Genetic and epigenetic factors underlying sex differences in the regulation of gene expression in the brain

Abstract

There are inherent biological differences between males and females that contribute to sex differences in brain function and to many sex-specific illnesses and disorders. Traditionally, it has been thought that such differences are largely due to hormonal regulation; however, there are also genetic and epigenetic effects caused by the inheritance and unequal dosage of genes located on the X- and Y-chromosomes. Here we discuss the evidence in favor of a genetic and epigenetic basis for sexually dimorphic behavior, as a consequence of underlying differences in the regulation of genes that drive brain function. A better understanding of sex-specific molecular processes in the brain will provide further insight for the development of novel therapeutic approaches for the treatment of neuropsychiatric disorders characterized by gender/sex differences.

Introduction

For the last 50 years, the dominant view regarding the biological phenomenon of sex differences in the brain has focused primarily on the organizational effects of hormonal regulation (Arnold and Gorski 1984; Lillie 1916). For a long time, the study of sexual differentiation focused on only a few robustly dimorphic brain structures, which led to the wider acceptance of a serial model of sex differences. A serial model claims that genetic sex (XX versus XY) determines gonadal sex and gonadal steroids determine sex differences in the brain. This concept of how sex differences arise has survived because, at its most fundamental level, it attempts to unify the evidence supporting both genetic and hormonal influences on brain function, and to describe how they contribute to differing vulnerability to neuropsychiatric disease. Nevertheless, the dominant view that genetic sex stimulates differentiation of the gonads, which later drives the secretion of sex hormones to induce sex differences in function, is unnecessarily restrictive. Several studies have indicated that sex differences are an emergent property of diverse sex-specific signals induced by both environmental and preprogrammed hormonal or genetic cues (Barker et al. 2010; Cox and Rissman 2011; Gatewood et al. 2006; McCarthy and Arnold 2011). Moreover, there is increasing appreciation that any realistic broad-spectrum model of sex-specific differences must integrate the influence of the environment on brain development and function. Hence, a more appropriate model should encompass the role of hormones, as well as the environmental impact mediated by epigenetic regulation driving gene expression for both males and females during development and across the lifespan (McCarthy et al. 2009; Yang and Shah 2014).

In addition to being hormone-centric, studies on cognition and stress have historically tended to focus only on males (Beery and Zucker 2011). There is growing concern that traditionally examination of sex differences has been inherently biased and this could lead to the strengthening of existing stereotypes, thereby further increasing gender inequalities (Eagly and Riger 2014). For example, small sex differences in the size/shape of the corpus callosum have been used to support the argument that sex differences are minimal and unreliable (Fitch et al. 1990). On the contrary, there are extremely large differences in the 3-dmensional “texture” analysis of MRI (magnetic resonance imaging) datasets of white matter, suggesting that sex differences in brain anatomy can be quite significant (Kovalev and Kruggel 2007). Furthermore, clinical studies have shown that sex can influence the degree of susceptibility to diseases or disorders, and that this effect can vary many fold between sexes (Abel et al. 2010; Baba et al. 2005; Breslau et al. 1995). For example, males show a higher propensity for some neurological diseases, including Parkinson's disease (de Lau et al. 2004), Schizophrenia (Jablensky 2000; Satterthwaite et al. 2015), Autism and addiction (Becker and Hu 2008). Females also show higher susceptibility to age-related neurodegenerative diseases e.g. Alzheimer's disease (Andersen et al. 1999) and anxiety-related disorders and depression (Breslau et al. 1995). Comparative studies between males and females may unravel molecular processes involved in neural development and function that would otherwise remain hidden. For example, by ablating the sexually dimorphic progesterone receptor (PR) expressing neuronal population located in the ventromedial hypothalamus sexually distinct behaviors controlled by these neurons has been revealed. In females ablation of PR expressing neurons greatly diminished sexual receptivity and the corresponding ablation in males reduced aggression and mating (Yang et al. 2013). By comparing the effect of the prostaglandin PGE₂ in male and female mice, the function of PGE₂ as a downstream effector of estradiol to permanently masculinize the brain has also been elucidated (Park et al. 2010). Nonetheless, sex differences in brain structure at the molecular and cellular levels, whether caused by sex chromosomes (differential presence in the two sexes, X-chromosome inactivation, dimorphic expression of genes located on the X and Y chromosomes, sex-chromosome variations etc.) or by gonadal hormone levels, can have dual functions (De Vries 2004). The first reflects absolute sexual dimorphism which are species-specific, wherein a particular trait (physiological, behavioral, or morphological) is either exclusively or predominantly present in males or females. For example male-specific courtship displays and male bird courtship singing or female specific nurturing and postpartum aggression. The second is one in which sex-specific differences in one sex may be used to offset the effects of other in order to make both sexes similar. This is demonstrated in the case of the male prairie vole, where in order to compensate for lack of hormonal milieu of pregnancy the neurocircuitry for vasopressin is altered, resulting in enhanced care giving behavior. Male voles have a characteristic dense arginine vasopressin (AVP) fiber network in their septum, whereas female voles have hardly any AVP fibers (Lonstein and De Vries 1999). Interestingly, the example of compensatory effect mentioned above requires quantitative differences in the brain anatomy between sexes in order to make them qualitatively similar. Thus, the sexes may appear to be similar in care giving behavior, but the underlying neurocircuitry regulating the behavior can be markedly different in males and females.

It is becoming evident that the effects of sex on neural function is an important variable (Cahill 2006) and conclusions based solely on the study of one sex cannot be assumed to be true in all the cases (Mogil and Chanda 2005). However, one must also recognize that brains of both the sexes also share many similarities. Even then it is true that epigenetic mechanisms are now being implicated in the molecular underpinnings of sex differences in neural gene expression and function (Dunn et al. 2011). Epigenetic mechanisms can integrate intrinsic and extrinsic signals onto the genome thus facilitating the adaptation of an organism to its environment through sustained changes in gene expression (McCarthy et al. 2009). For example, significant insight into the mechanisms of gliogenesis has come from coupling of both extrinsic and intrinsic mechanisms. As a case for an intrinsic mechanism, conditional deletion of DNMT1 from the neural lineage leads to precocious astroglial differentiation (Fan et al. 2005). The requirement of fibroblast growth factor (FGF) and epidermal growth factor (EGF) to establish competence for differentiation is an example of a potent extrinsic factor (Song and Ghosh 2004). However, this epigenetic make up and the integration of stimuli may differ considerably between the sexes and can confer a sex bias towards behavior, disease susceptibilities and treatment responses. Epigenetic modifications offer a plausible mechanism by which sexually dimorphic developmental, hormonal and environmental effects could be softwired into the genome.

Sex differences due to sex chromosomes and imprinting

The genetic differences between the sexes originate from the presence or absence of genes encoded on the Y chromosome, from X-chromosome-linked gene dosage, mosaicism, and genomic imprinting (Arnold and Burgoyne 2004). In mammals, epigenetic mechanisms come into play early in gestation when one of the X chromosomes is transcriptionally silenced through X chromosome inactivation (XCI) in each somatic cell. XCI is transcriptional silencing of one X chromosome in female mammalian cells, which compensate for having twice the genomic dose of X genes from the X chromosome between XX females and XY males (Boumil and Lee 2001). One of the two X chromosomes is transcriptionally silenced by a combination of histone modifications, DNA methylation and noncoding RNA (Avner and Heard 2001; Brown et al. 1991; Lee et al. 1999). Determining which X chromosome undergoes inactivation is a stochastic process, and this makes the female brain a mosaic of cells that express alleles by either X chromosome (Tan et al. 1995). Heterogeneity in female tissues, including the brain, can lead to stochastic, female-specific functional diversity on diverse spatial scales, from neighboring cells to left versus right lateralization of the body (Wu et al. 2014). In some clinical manifestations of X-linked neurological diseases there is also variability in female carriers, as in adrenoleukodystrophy (Jangouk et al. 2012), fragile X-associated tremor/ataxia syndrome (Tassone et al. 2012) and Christianson syndrome (Sikora et al. 2016).

Interestingly, the X chromosome contains an unusually large number of genes that are involved in nervous system development and function (Zechner et al. 2001), and the polymorphic nature of the X chromosome is an important source of genetic variability. For example, due to the random nature of X chromosome inactivation females can be heterozygous with different alleles being expressed at a locus in different cells or tissues (Smallwood et al. 2003). In addition, X chromosome inactivation is often incomplete and varies according to tissue type and developmental stage, as some X-linked genes escape inactivation and can be expressed at higher levels in females (Lingenfelter et al. 1998). In females, increased genomic dosage of these genes, which are referred to as ‘X escapees’, could lead to fundamental sex differences in brain and behavior (Xu et al. 2002). This can provide a potential source of sex differences in both somatic and neural cells (Brown and Greally 2003; Chen et al. 2009).

The Sex determining region Y (Sry) gene, located on the Y chromosome, has a strong masculinizing effect on the brain. Other than Sry there are at least eight Y-linked genes, which are expressed in the male mouse brain (Xu et al. 2002). Significant associations have been reported between particular Y chromosome haplogroups and male-biased behavioural traits, including aggression (Shah et al. 2009) and alcohol dependence (Kittles et al. 1999). Therefore, males as a group display greater variance on certain brain phenotypes as they express a single hemizygous allele from Y chromosome (Hedges and Nowell 1995; Smallwood et al. 2003).

It has been reported that along with cognitive abilities there is an excess of sex and reproduction related genes on the human X chromosome (Saifi and Chandra 1999). This phenomenon is referred to as ‘the large X-chromosome effect’ (Turelli and Orr 1995). Based on this, it has been proposed that “the large X-chromosome effect” has influenced the development of a specific fertility or cognitive ability in humans. Hence, mosaicism of polymorphic X alleles in the female should contribute towards sex differences in brain function. However, we know that X inactivation is incomplete and varies according to developmental stage and tissue type. Thus, in female brain mosaic of cells expressing alternate alleles at polymorphic loci and the differences in effects of various alleles blunt each other out.

Genomic imprinting is another source of sexual dimorphism in the brain (Bartolomei 2009). Genomic imprinting involves specific suppression of a locus in a chromosome from one parent.Raefski et al. (2005)identified at-least three X-linked genes, X-linked lymphocyte-regulated (Xlr3b, Xlr4b and Xlr4c) in the developing brain, which showed locus-specific imprinting in developing brain (Raefski and O'Neill 2005). These three genes displayed dynamic pattern of tissue and stage specificity and interestingly their imprinting was locus specific and independent of X-chromosome inactivation. Therefore, imprinting is highly complex in its distribution as it exhibits spatial, temporal, cell-specific, sex-specific, and inter-individual variability (Gregg et al. 2010b). The results of imprinting studies by transcriptome sequencing must be interpreted with caution, as statistical modeling of allele-specific expression by sequencing is still an unresolved challenge (DeVeale et al. 2012). Sex-specific parent-of-origin allelic effects have been reported in the adult mouse cortex and hypothalamus. For instance, in the medial preoptic area (mPOA), females express 150 imprinted genes compared to just 48 genes in males (Gregg et al. 2010a). Two candidate genes from this list for which in-depth analysis has been undertaken are mitochondrial ribosomal protein 48 (Mrpl48) and interleukin-18 (Il18). Mrpl48 is one of many Mrpl genes, which regulate translation in mitochondria but are encoded in nuclear DNA (Koc et al. 2001). Mrpl48 shows female-specific paternal expression bias in the mPOA. This indicates parental control over the bioenergetics of neural cells. Il18 encodes a cytokine that modulates neuroinflammation as well as homeostatic processes and behavior (Herrera et al. 2008). Il18 has been linked to multiple sclerosis, a highly sexually dimorphic disease that predominates in women (Herrera et al. 2008). This study reported that ll18 is preferentially expressed from the maternal allele in the female but not the male medial prefrontal cortex or mPOA, which further emphasizes a role for genomic imprinting in sexual dimorphism.

These observations suggest that it is important to consider imprinted genes present on both the X chromosome and autosomes in order to understand sex differences in brain and behavior. Linkage analyses have also shown parent-of-origin-specific associations and preferential transmission of neurological disorders from a single parent, demonstrating the relevance of imprinting for brain function and disease (Davies et al. 2008).

Sex differences in epigenetic mechanisms

DNA methylation

Although it has been known for decades that the early social/maternal environment can modify sex differences in behavior, studies implicating specific epigenetic mechanisms in differences in brain function are only beginning to emerge (Kurian et al. 2007; Tsai et al. 2009). DNA methylation in the brain is highly dynamic, particularly in genes associated with neuronal plasticity (Baker-Andresen et al. 2013b). It can be modified by rapid demethylation and de novo methylation occurring in response to a stimulus (Ratnu et al. 2014). DNA methylation is established and maintained by a family of DNA methyl transferases (Dnmts), two of which (Dnmt1 and Dnmt3a) are abundantly expressed in the brain. Female brains have higher levels of DNA methylation, with significantly more methylated CpG sites than males (Nugent et al. 2015), and unpublished data from our group suggest the opposite is true for 5-hydroxymethylcytosine, with higher levels genome-wide in the male prefrontal cortex. DNA methylation regulates gene expression by recruiting methyl CpG binding protein 2 (Mecp2) and nuclear receptor co-repressor (nCor). These methylated-cytosine binding proteins also exert their influence on gene expression by altering the local chromatin environment. Dnmt3a, Mecp2 and nCor are expressed in a sexually dimorphic manner in the neonatal amygdala, with female rats expressing higher levels than males (Auger et al. 2011; Kolodkin and Auger 2011). Rett syndrome, caused by deleterious mutation in the X-linked gene Mecp2 occurs almost exclusively in females (Dragich et al. 2000; Hedges and Nowell 1995). The promoter for brain-derived neurotrophic factor (Bdnf) exon IV is also hypermethylated in female mice, which may influence fear-related learning and memory, given that female mice are resistant to fear extinction (Baker-Andresen et al. 2013a). Sex-specific differences in this form of epigenetic regulation may therefore represent a common mechanism that impacts a variety of behaviors.

It is widely acknowledged that effects of the gonadal steroid hormones on many sex-specific brain features are exerted during the perinatal period. Testosterone is converted into estradiol by aromatase activity within the developing brain, and estradiol influences several developmental sex differences in neuronal anatomy and physiology (Simerly 1989; Simerly 2002). These differences are responsible for sex-specific behavior and sexually dimorphic regulation of gonadotropin secretion (McCarthy 2008). Also, changes in DNA methylation of genes including estrogen receptor alpha (ERα), estrogen receptor beta (ERβ), and PR, which are essential for sex differences in brain differentiation, are hormonally regulated (Schwarz et al. 2010). Therefore, gonadal hormones can profoundly influence DNA methylation during brain development. However, the effect of these hormones in the adult brain has received less attention. It was previously thought that once the perinatal window closes, the brain is permanently wired as either male or female. Within this context, Nugent et al., have demonstrated that effects of steroids on the developing brain is not limited to perinatal stage (Nugent et al. 2015). However, even after the perinatal window closes, pharmacologically inhibiting Dnmts or generating a conditional knockout of Dnmt3a results in a marked decrease in global DNA methylation, release of masculinising genes from epigenetic repression, and masculinisation of female brain and behavior (Nugent et al. 2015).

The sexually dimorphic expression of the ERα in the mPOA is due to differential levels of promoter methylation, starting from development and persisting to adulthood (Yamamoto et al. 2006). For example, in the neonatal rat POA, gender related difference in DNA methylation of the ERα promoter region is observed. Specifically, with greater ERα promoter methylation in males, ERα mRNA and protein levels are lower in males than in females (Kurian et al. 2010). Similarly, maternal grooming impacts sex differences in DNA methylation patterns within the developing amygdala. Males have higher levels of ERα promoter methylation in the amygdala but by increasing simulated maternal grooming in females, ERα expression and gene methylation patterns are masculinized (Edelmann and Auger 2011). However, an earlier study reported increased ERα expression in the POA of female rats raised by high licking mothers (Champagne et al. 2006). The disparity between these studies may be due to experimental design or age or brain areas sampled. Nevertheless, these studies highlight the prominent role of early life experience in driving sex differences in the brain.

Studies have shown that early life growth conditions and variations in early maternal care can affect the epigenetic state of hypothalamic–pituitary–adrenal (HPA) associated genes in a sexually biased manner and influence the behavior (Rubenstein et al. 2015). Differences in DNA methylation within the HPA associated genes may be particularly important for controlling developmental plasticity. For example, increased DNA methylation of the glucocorticoid receptor (GR) gene promoter is associated with prenatal exposure to maternal depression/anxiety during pregnancy in humans (Oberlander et al. 2008). Male offspring exposed to stress experience early in pregnancy show predisposition to neurodevelopmental disorders and increased GR methylation (Mueller and Bale 2008), and postnatal maternal care in rats alters DNA methylation of the GR promoter and these changes are stably maintained into adulthood and influence how they respond to stressors in life (Weaver et al. 2004). Moreover, there are reports indicating both strain-specific and sex-dependent effects of maternal separation in several behavioral paradigms in early adolescent offspring. Analyses have revealed increased cortical Bdnf expression in both maternally separated male and female Balb/cJ mouse offspring, and decreased hippocampal Bdnf expression in only maternally separated female C57BL/6J mice (Kundakovic et al. 2013). Overall, there appears to be complex interactions between early life stressors, genetic background and sex on the epigenetic state and determination of neurobehavioral outcomes.

However, one must be careful in making simplistic predictions about gene regulation across the entire locus based on changes in DNA methylation at a few CpG sites. Furthermore, the pattern of DNA methylation across multiple CpGs can vary as a function of sex, age, and brain region. In an investigation by Schwarz and colleagues they showed that certain CpG sites in females exhibit no effect of sex or hormone level in the neonate (Schwarz et al. 2010). However, during adulthood these CpG sites exhibit nearly 30% greater methylation. In comparison, there was no hormone- or sex-specific effect on DNA methylation levels within the ERβ promoter, whereas in the hypothalamus of 1-day-old females there was a significantly greater level of methylation than in males at two CpG sites along the ERα promoter. A similar effect was observed by analysis of the progesterone receptor. Surprisingly, at specific CpG sites on the PR promoter, DNA methylation-related sex differences were reversed in adulthood, with males acquiring significantly greater levels of methylation than females (Nugent et al. 2011). DNA methylation is important for the normal development of many regions, including sexually dimorphic brain regions such as mPOA and also regions not typically considered sexually dimorphic, such as the cortex. For example, expression of ERα in the cortex of male and female mice is high in early postnatal development and begins to decline as animals approach puberty before disappearing in the adult (Wilson et al. 2011). This decrease in ERα expression is associated with progressive hypermethylation within at least one of the six promoters of the mouse ERα gene regulatory region at postnatal day (PN) 10 in both male and female mice (Wilson et al. 2008). DNA hypermethylation at specific areas of the ERα promoter region also coincides with higher expression of Dnmt1 (Westberry et al. 2010).

Methylated CpGs can act as an anchor for methyl-binding proteins such as Mecp2 and methyl-CpG binding domain protein (Mbd1, 2, 3, and 4). These proteins can impact gene expression and chromatin structure in a sex-dependent manner by virtue of different levels of expression between males and females. There is significantly higher Dnmt3a expression at post natal (PN1) in the amygdala of female rats, but not within the medial basal hypothalamus or POA (Kolodkin and Auger 2011). Sex differences in the expression of growth arrest and DNA-damage-inducible (Gadd45b) and Mecp2 have also been reported in the amygdala and hypothalamus (Kigar et al. 2016). At PN1, males express significantly lower levels of Mecp2 than females, but this difference largely disappears by PN10 (Kurian et al. 2007). Binding of Mecp2 to methylated DNA is followed by recruitment of co-repressor complexes. The co-repressor molecule, nCor, has organizational effects in the brain, and blunts sex differences in juvenile social play and anxiety-like behavior (Jessen et al. 2010). These data support the notion that epigenetic mechanisms related to DNA modification and gene repression can impact a large number of transcriptional responses and are likely to be, in part, responsible for organizing sex-related differences.

Histone modifications

Histone modifications modulate gene regulation by either affecting electrostatic links between DNA and histone proteins or by changing histone-histone interactions (Baker-Andresen et al. 2013b). This results in reorganization of chromatin states that can either enhance or reduce access of the transcriptional machinery to DNA, respectively (Jenuwein and Allis 2001). Neonatal male mice exhibit increased histone 3 lysine 9 trimethylation (H3K9me3) and acetylation on lysine residues 9 and 14 of histone 3 (H3K9/14Ac) in the cortex and hippocampus, compared with females (Tsai et al. 2009). In both young and middle-aged females 17β-estradiol specifically increases histone H3 acetylation at the Bdnf promoters pII and pIV in the dorsal hippocampus and alters memory formation (Fortress et al. 2014; Frick et al. 2015; Zhao et al. 2012). Changes in histone 3 lysine 4 trimethylation (H3K4me3) in the rodent forebrain are also associated with learning and memory (Gupta et al. 2010). However, there has been only one genome-wide study detailing sex-specific differences in H3K4me3 levels, which is surprising considering the demonstration of sexually dimorphic gene expression associated with H3K4-methyltransferase and demethylase enzymes in various brain regions. In a study by Schneider et al., in frontal cortex and hippocampus they have demonstrated a sexually dimorphic effect of stress (lead exposure and prenatal stress) on post translational histone modifications (H3K9/14Ac and H3K9Me3) (Schneider et al. 2016). They also suggest that, on the hippocampus this effect might be greater in males than in females.

The trimethylation of histone 3 at lysine 4 (H3K4me3) is associated with active gene transcription (Berger 2007). On a genome-wide scale this modification broadly correlates with active transcription and is enriched at transcriptional start sites of genes. Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) has been used in different cell types of primate brain (human and non-human) to examine the genome-wide distribution of H3K4me3 (Cheung et al. 2010). However, surprisingly there are very few studies exploring sex-specific regulation of mono-, di and tri-methyl H3K4 (H3K4me1/H3K4me2/H3K4me3) marks on a genome-wide scale (Shen et al. 2015). This is particularly intriguing, given that several studies have reported subtle sex-dependent differences in the expression of H3K4- demethylase and methyltransferase enzymes for various brain regions. For example, expression of lysine (K)-specific methyltransferase (Kmt2a) in adult human cerebral cortex, is higher in females as compared to males (Huang et al. 2007), while family of H3K4-specific demethylases, are expressed at higher levels in females (Xu et al. 2002; Yang et al. 2010). The majority of loci throughout the genome exhibit very similar H3K4me3 peaks in male and female mice (Shen et al. 2015). However, at ∼200 loci there are significant sex difference in the peak size. Surprisingly, the majority of genes with large peaks were in females and most of the peaks in females were related to brain function. This was true whether or not X-chromosome linked genes were included in the analysis. However, there was no significant sex difference in gene expression, which should have been predicted by the presence of H3K4me3. This finding is similar to that of Ghahramani et al (2014) who demonstrated that over 80% of the genes which exhibited a sex difference in DNA methylation were those with greater DNA methylation in males, although this did not correlate with sex differences in gene expression (Ghahramani et al. 2014).

Unlike H3K4me3, trimethylation of histone 3 at lysine 27 (H3K27me3) is associated with repressed gene expression (Cao et al. 2002). The presence of both H3K4me3 and H3K27me3 at the same gene promoter creates a poised condition for activation and is termed as “bivalent state” (Bernstein et al. 2006). In particular, the H3K27me3 mark is associated with an inactive X chromosome (Chow and Heard 2009). Genes that escape X inactivation are depleted in the repressive histone H3K27me3 mark (Yang et al. 2010). Specific enzymes such as the histone demethylases play an important role in removing H3K27me3 at escape genes, and different histone demethylases can target specific lysine residues and methylation states (mono-, di-, or trimethyl). Some enzymes specifically demethylate distinct methyl-lysine residues (H3K9me3, H3K36me3 and H3K4me3) on histone H3 (Klose and Zhang 2007) whereas other enzymes like JmjC-domain proteins (JMJD3) and ubiquitously transcribed tetratricopeptide repeat gene on the X chromosome (Utx) specifically demethylate di- and trimethyl-lysine 27 (H3K27me2/3) on histone H3 (Agger et al. 2007; De Santa et al. 2007; Lan et al. 2007; Lee et al. 2007). X-chromosome-encoded histone demethylases, including Utx and jumonji AT-rich interactive domain 1c (Jarid1c) are interesting as both are present on the X chromosome and escape X-inactivation. Furthermore, some X-escapees have paralogs on the Y chromosome (Xu et al. 2008a). The function of Uty, which is a paralog of Utx, and shares 84% amino acid sequence similarity with Utx, is unknown. The expression of Utx is higher in female mice than in males in all brain regions except the amygdala. Uty is expressed preferentially in the paraventricular nucleus of the mouse hypothalamus (Xu et al. 2008b). These differential expression levels between X and Y paralogs in specific brain areas may lead to sex differences in brain function. In a preliminary series of studies, we have observed that overexpressing Utx in the medial prefrontal cortex of male mice influences their ability to acquire extinction of cued fear memory (unpublished data).

Similarly, Jarid1c is a H3K4me3-specifc demethylase that plays an important role in brain development (Lingenfelter et al. 1998). Compared to the Jarid1c allele present on active X chromosome, sex difference in the expression of Jarid1c on inactive X chromosome varies from 20% to 100% depending on the type of tissue. Expression of the Y paralog Jarid1c does not compensate for the female bias. Mutations in Jarid1c cause X-linked mental retardation, seizure, high agitation, and autism-like behavior. Knockdown of Jarid1c in the dorsal hippocampus also results in impaired episodic memory formation in mice (Xu and Andreassi 2011).

Taken together, there is increasing evidence for sex differences in the epigenetic modulation of histone modifiers and histone modifications in specific brain regions, as well as evidence for an epigenetic underpinning of processes involved in neural development. Histone modifications further emphasize the potential for enduring widespread effects of epigenetic modulation on sex differences in the brain.

Noncoding RNAs

Noncoding RNAs (ncRNAs) are pervasively transcribed in the vast majority of mammalian genomes and comprise almost 98% of the transcripts (Mattick 2001). There are numerous classes of ncRNAs, including but not limited to microRNAs (miRNAs), short-interfering RNAs (siRNAs), small nucleolar RNAs (snoRNAs), P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs), enhancer RNAs (eRNAs) and long ncRNAs (lncRNAs; i.e., transcripts longer than 200 base pairs). These ncRNAs are expressed in a developmental stage-specific manner and impact nearly every biological process (Amaral et al. 2008). An increasing number of studies are reporting important roles for ncRNAs in sexual dimorphism in the brain. X-linked lncRNAs are known to be involved in maintaining the sexually dimorphic transcriptional state of the X-chromosomes through the initiation, establishment and maintenance of X-chromosome silencing (Chow and Heard 2009). LncRNA X inactive specific transcript (XIST) is an RNA transcribed from the X inactivation center (Xic) on the X chromosome destined to be inactivated (Xi). XIST RNA directs chromatin and transcriptional change by binding to the Polycomb repressive complex 2 (PRC2), and targeting PRC2 to the Xi in cis. The antisense partner of XIST, TSIX, is a lncRNA encoded within the Xic and represses its activity. This determines which one of the X-chromosomes will remain active. Many of these diseases are known to involve X-linked genes, and some of these are expressed more in one sex then other, which may explain why autism and mental retardation are more common in males (Skuse 2005; Skuse et al. 1997). Genes that invariably escape XCI perhaps contribute towards greater phenotypic variation among females (Carrel and Willard 2005). For example, deletion of the Xic-encoded lncRNA, testis specific X-linked gene (Tsx), in male mice leads to a reduction in fear and enhancement of short-term hippocampal memory (Anguera et al. 2011). Although the underlying mutations or causes are not known in every instance, many neurological and psychiatric diseases differ in the incidence of, or severity between, the sexes (Bangasser and Valentino 2014; Zagni et al. 2016). These observations suggest that it is important to consider how many and in what tissues imprinted genes (on both the X chromosome and autosomes) may contribute to sex differences in brain and behavior.

Along with lncRNAs, small ncRNAs are also known to show dimorphic expression and function. Bale and colleagues surveyed 250 miRNAs in the neonatal mouse brain and, despite using whole brain, found that almost two-thirds of them are expressed at different levels in males and females (Morgan and Bale 2012). This is surprising as the brain is a highly heterogeneous and functionally compartmentalized structure, and suggests that there is a global regulation of gene expression that differs purely based on sex. However, it is of interest to explore whether gene expression is regulated differentially in males and females in distinct regions of the brain. Koturbash et al study revealed that miRNAs are expressed in a sexually dimorphic manner in various regions of the adult murine brain, including the hippocampus, cerebellum and frontal cortex (Koturbash et al. 2011). For example, hippocampal dendritic spine morphology and plasticity are sexually dimorphic, suggesting that miRNA-329, which is upregulated in the male hippocampus, may play a role in mediating these differences. Similarly, the human SRY transcript is a predicted target of let-7a and let-7e, members of the let-7 family of miRNAs that are highly expressed in the brain (Betel et al. 2008).

Conclusion

Clearly there is a need for further investigation of sex differences in the brain, as increasing evidence indicates there are significant differences between males and females during both homeostatic and disease states. Scientific conclusions based on the study of one sex have limited value in understanding some phenomena in the other sex. Epigenetic mechanisms provide a platform that represents convergence between the combined effect of hormonal, genetic, and environmental influences on sex differences in the brain. However, many challenges and opportunities remain for the study of the combinatorial effects of genetics, epigenetic mechanisms and hormones on sexual dimorphism. Indeed, the brain is a heterogeneous tissue where the vast variety of neuronal subtypes and diverse environmental factors may confound discovery of differences that can be causally attributed to sex. One must also be careful in interpreting in vitro studies of sex differences, as it cannot exactly recapitulate hormone biology or other in vivo factors that expose meaningful differences between sexes. There is greater need to recognize that genes/RNAs/proteins/hormones are all part of complex molecular/biochemical network pathways. Therefore, a comparative analysis and integration of multi-dimensional data sets is required for a holistic assessment of the biological interaction network involved in sexual dimorphism. The availability of sophisticated technology tools like: such as transposase-accessible chromatin sequencing (ATAC-Seq) (Kukurba et al. 2016), next-generation (NG) Capture-C for high resolution and high throughput chromosome conformation analysis (Davies et al. 2016) or live imaging of transgenic mice to capture brain-wide view of neural activity (Wekselblatt et al. 2016) will facilitate deconstructing complex networks underlying biological processes like sex differences. Finally, it is inevitable that bioinformatics and statistical methods will play crucial role in future studies with an epigenetic and genomics component. These new tools will allow us to better understand the enormous combinatorial complexity of epigenetic mechanisms and hormonal factors. With that, we will gain a deeper understanding of how male and female brains process information, leading to dimorphism with respect to vulnerability and resilience to disorders of the brain.

Significance.

It is becoming increasingly evident that the influence of sex differences on neural function are often as important as the effects of any other important factor, and conclusions based primarily on one sex cannot be generalized for the other. In particular, epigenetic mechanisms are now being implicated in the molecular underpinnings of sex differences in neural gene expression and function. Epigenetic mechanisms are especially important because it can integrate intrinsic and extrinsic signals onto the genome. We have tried to highlight that this epigenetic make up and the integration of stimuli can differ considerably between the sexes and can confer sex bias towards brain anatomy, behavioral responses and treatment responses. Therefore, we would like to emphasize on the need to include both male and female in future neuroscience studies in order to gain deeper understanding of the cognitive function, sexual dimorphism and vulnerability to disorders of the brain