Epigenetics and sex differences in the brain: A genome-wide comparison of histone-3 lysine-4 trimethylation (H3K4me3) in male and female mice

Highlights

• We review recent work on the role of epigenetics in brain sexual differentiation.

• A hormone-sensitive brain region was dissected from adult male and female mice.

• ChIP-Seq was used to examine the distribution of H3K4me3 throughout the genome.

• H3K4me3 varied by sex in 248 genes; for 70% of these, peaks were larger in females.

• Genes/loci with more H3K4me3 in females were associated with synaptic transmission.

Abstract

Many neurological and psychiatric disorders exhibit gender disparities, and sex differences in the brain likely explain some of these effects. Recent work in rodents points to a role for epigenetics in the development or maintenance of neural sex differences, although genome-wide studies have so far been lacking. Here we review the existing literature on epigenetics and brain sexual differentiation and present preliminary analyses on the genome-wide distribution of histone-3 lysine-4 trimethylation in a sexually dimorphic brain region in male and female mice. H3K4me3 is a histone mark primarily organized as ‘peaks’ surrounding the transcription start site of active genes. We microdissected the bed nucleus of the stria terminalis and preoptic area (BNST/POA) in adult male and female mice and used ChIP-Seq to compare the distribution of H3K4me3 throughout the genome. We found 248 genes and loci with a significant sex difference in H3K4me3. Of these, the majority (71%) had larger H3K4me3 peaks in females. Comparisons with existing databases indicate that genes and loci with increased H3K4me3 in females are associated with synaptic function and with expression atlases from related brain areas. Based on RT-PCR, only a minority of genes with a sex difference in H3K4me3 has detectable sex differences in expression at baseline conditions. Together with previous findings, our data suggest that there may be sex biases in the use of epigenetic marks. Such biases could underlie sex differences in vulnerabilities to drugs or diseases that disrupt specific epigenetic processes.

Introduction

Many psychiatric disorders and neurological diseases exhibit sex differences in incidence, severity, or response to treatment (Kornstein, 1997, Seeman, 1997, Weinstock, 1999). This is especially true of neurodevelopmental disorders (Zahn-Waxler et al., 2008, Martel et al., 2009), which presumably reflects underlying sex differences in brain development. Chromosomal differences between males and females (XY in males and XX in females) could cause sex differences in the brain (Chen et al., 2009, Arnold, 2012), but most effects of the sex chromosomes on sexual differentiation are thought to be indirect and mediated by gonadal steroid hormones (reviewed in McCarthy et al., 2009a). Sexual differentiation has long been considered “epigenetic,” in reference to the indirect role that sex chromosomes play in hormone-dependent sex differences. More recently, the term “epigenetics” has re-entered the sexual differentiation field, but now to refer much more specifically to changes in chromatin that lead to long-term changes in gene expression without any change in the underlying DNA sequence (McCarthy et al., 2009b, Qureshi and Mehler, 2010, Auger and Auger, 2011, Xu and Andreassi, 2011).

The fundamental structural unit of chromatin is the nucleosome, comprised of about 146 base pairs of DNA wrapped around an octamer of histone proteins. The nucleosomes are further packaged and condensed to different degrees; in general, loose packaging is associated with increased gene expression, whereas more compact states are associated with reduced gene expression. DNA cytosine methylation and hydroxymethylation, and chemical modifications of the nucleosomal histones define chromatin structure and affect gene expression (Felsenfeld and Groudine, 2003, Jiang et al., 2008). For example, DNA cytosine methylation around transcription start sites and promoter elements is typically associated with gene repression. The majority of functionally relevant histone modifications are thought to reside at the flexible N-terminal tails that protrude from the nucleosome. These histone tails undergo a diverse array of covalent modifications such as acetylation, phosphorylation, ADP ribosylation, methylation, and ubiquitination, which correlate with specific transcriptional states (Jenuwein and Allis, 2001, Fischle et al., 2003, Iizuka and Smith, 2003).

Circumstantial evidence suggested that epigenetic modifications might be important for sexual differentiation. For example, a brief exposure to gonadal steroid hormones can have long-lasting or even permanent effects on gene expression, suggesting a cellular memory that is consistent with alterations in the epigenome. In addition, the classical actions of gonadal steroids are mediated by intracellular receptors that recruit co-activators or co-repressors to steroid responsive genes. Many of these co-factors either directly or indirectly cause changes in nearby histone proteins by, for example, increasing or decreasing the acetylation or methylation of histone tails (Spencer et al., 1997, Kim et al., 2001, Stallcup et al., 2003, Privalsky, 2004, Kishimoto et al., 2006, Kininis et al., 2007). An important mechanism by which gonadal steroid hormones activate gene expression may therefore involve a relaxation of chromatin structure following the binding of a receptor–co-activator complex. One steroid receptor co-activator, SRC-1, was linked to sexual differentiation over 10 years ago when it was demonstrated that reducing SRC-1 protein in the developing rat brain interfered with the development of sex differences in behavior and brain morphology (Auger et al., 2000).

Although still in its infancy, the role of epigenetics in sexual differentiation of the brain has recently become an active line of research, with much of the focus on a group of interconnected brain regions including the amygdala, preoptic area of the hypothalamus (POA), and bed nucleus of the stria terminalis (BNST). These are important, steroid-sensitive nodes within neural circuits controlling sexual behavior, the modulation of stress and anxiety, and the processing of olfactory cues, among other functions (Simerly, 2002, Toufexis, 2007). Evidence for both DNA methylation and nucleosomal histone modifications in sexual differentiation of these and other brain areas has been found.

Schwarz et al. (2010) used pyrosequencing to examine DNA methylation in the promoter regions of three genes—estrogen receptor alpha (ERα), ERβ, and the progesterone receptor—that are important for hormone-mediated sex differences in the brain. They found several age- and brain region-specific sex differences at individual CpG sites; some of these were reversed by treating females with estradiol at birth, indicating that they were due to perinatal steroid exposure (Schwarz et al., 2010). The group differences in DNA methylation were relatively small, and it is not known whether they relate to changes in gene expression. However, the findings are important because they were the first to document sex differences in DNA methylation in the brain and to demonstrate that this methylation may be dynamically regulated during development.

DNA methylation is controlled by a family of DNA methyl transferases (DNMTs), two of which (DNMT1 and DNMT3a) are abundantly expressed in the brain. Methylated cytosine recruits proteins, such as methyl CpG binding protein 2 (MeCP2) and nuclear receptor co-repressor (nCOR), which in turn cause chromatin changes that may alter gene expression. Auger and colleagues found that female rats express more DNMT3a, MeCP2 and nCOR than males in the neonatal amygdala (Kolodkin and Auger, 2011, Auger et al., 2011, Auger et al., 2011). Moreover, the sex differences in MeCP2 and nCOR were functionally linked to sex differences in juvenile play behavior (Auger et al., 2011, Auger et al., 2011). Taken together, several proteins related to DNA methylation are expressed in a sexually dimorphic manner in the rodent amygdala during the critical period for sexual differentiation.

The best understood of the histone modifications are acetylation and methylation. Histone acetyltransferases add acetyl groups to histone tails, opening the chromatin structure and increasing access for transcription factors, whereas histone deacetylation is catalyzed by histone deacetylases (HDACs) and is generally associated with reduced transcription (Grunstein, 1997, Cosgrove and Wolberger, 2005). The first study to compare histone modifications in the brains of males and females used immunoblotting to examine histone 3 lysine 9/14 acetylation (H3K9/14Ac) in several brain regions of perinatal mice. While no difference was seen in the highly hormone sensitive POA/hypothalamus, males had greater H3K9/14Ac in the cortex/hippocampus (Tsai et al., 2009). Treating newborn females with testosterone increased (masculinized) H3K9/14Ac in the cortex/hippocampus, suggesting that the sex difference is due to gonadal steroids.

To more directly test whether changes in histone acetylation are required for hormone-dependent sexual differentiation, Murray et al. (2009) treated mice with an HDAC inhibitor during the critical neonatal period. Newborn male, female, and androgenized female mice were given saline or valproic acid and effects on the principal nucleus of the BNST (BNSTp) were determined at weaning (Murray et al., 2009). The BNSTp exhibits a number of morphological and neurochemical sex differences in rodents and humans (Guillamon et al., 1988, Hines et al., 1985, Forger et al., 2004, Allen and Gorski, 1990), and in mice these differences can be eliminated by giving females a single injection of testosterone propionate on the day of birth (Hisasue et al., 2010). Valproic acid treatment transiently increased histone acetylation in the brain and prevented masculinization of BNSTp cell number in both males and testosterone-treated females (Murray et al., 2009). There was no effect on the BNSTp of control females or on two non-sexually dimorphic brain regions, suggesting that a transient blockade of histone deacetylation prevented the masculinizing actions of testosterone without a generalized effect in non-dimorphic brain regions.

Matsuda et al. (2011) used a similar approach to ask whether the masculinization of sexual behavior in rats requires alterations in histone acetylation. When the HDAC inhibitor, trichostatin A, was infused into the cerebral ventricles of newborn males, these animals showed impairments in sexual behavior in adulthood. Similar behavioral effects were seen when antisense oligonucleotides to specific HDACs were infused neonatally, supporting the conclusion that the effects seen after trichostatin A administration were due to effects on HDAC activity (Matsuda et al., 2011). Taken together, the studies by Murray et al. (2009) and Matsuda et al. (2011) suggest that masculinization of neuroanatomy and behavior in rodents normally requires hormone-dependent reductions in histone acetylation. Because reductions in histone acetylation are generally associated with reduced gene expression, some genes may need to be silenced in males for normal masculinization, although specific gene targets underlying the effects of the HDAC inhibitors are not known.

The addition of methyl groups to lysine or arginine residues of histone tails is another well-studied epigenetic modification. Histone methylation can repress or activate transcription depending on the position of the modification and the number of methyl groups added at a given site (mono-, di- or tri-methylation). To date, the only study we are aware of comparing histone methylation in the brains of males and females used immunoblotting to examine the overall level of trimethylated histone 3 at lysine 9 (H3K9me3) in several brain regions of neonatal mice and found higher levels in the cortex/hippocampus of males than of females (Tsai et al., 2009).

The trimethylation of histone 3 at lysine 4 (H3K4me3) has not yet been compared in males and females and is of particular interest because this modification is enriched at transcriptional start sites of genes and, on a genome-wide scale, broadly correlates with active transcription (Santos-Rosa et al., 2002, Bernstein et al., 2005, Berger, 2007). Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) has successfully been used to examine the genome-wide distribution of H3K4me3 in different cell types of the human and non-human primate brain (Cheung et al., 2010). Furthermore, in the rodent forebrain, H3K4me3 is subject to dynamic changes in the context of hippocampal learning and memory (Gupta et al., 2010) and exposure to dopaminergic drugs (Aguilar-Valles et al., 2014, Huang et al., 2007). To the best of our knowledge, sex-specific regulation of H3K4me3 and two related marks, mono- and di-methyl H3K4 (H3K4me1/H3K4me2), has not yet been explored on a genome-wide scale. This is surprising given that subtle sex-specific differences in the expression of H3K4-methyltransferase and demethylase enzymes have been reported for various brain regions. For example, in adult human cerebral cortex, expression of MLL1/KMT2A methyltransferase is higher in females as compared to males (Huang et al., 2007), while other genes, including KDM5C/SMCX/JARID1C encoding an H3K4-specific demethylase, are X-linked genes that escape X-inactivation in some brain regions and therefore may also be expressed at higher levels in females (Xu et al., 2008a, Yang et al., 2010). Here, we report initial analyses of a ChIP-Seq experiment designed to identify genome-wide sex differences in H3K4me3. We focus on the BNST and POA, two forebrain structures that show sex-specific differences in mammals, including humans (McCarthy et al., 2009a, Forger, 2009).