At the frontier of epigenetics of brain sex differences

Abstract

The notion that epigenetics may play an important role in the establishment and maintenance of sex differences in the brain has garnered great enthusiasm but the reality in terms of actual advances has been slow. Two general approaches include the comparison of a particular epigenetic mark in males vs. females and the inhibition of key epigenetic enzymes or co-factors to determine if this eliminates a particular sex difference in brain or behavior. The majority of emphasis has been on candidate genes such as steroid receptors. Only recently have more generalized survey type approaches been achieved and these promise to open new vistas and accelerate discovery of important roles for DNA methylation, histone modification, genomic imprinting and microRNAs (miRs). Technical challenges abound and, while not unique to this field, will require novel thinking and new approaches by behavioral neuroendocrinologists.

Keywords: amygdala; estrogens; preoptic area; reproductive behavior.

Figure 1

Sexual differentiation of brain and behavior. Sex differences in the brain are established early in development during a critical period. Feminization of the brain proceeds in the absence of exposure to elevated gonadal steroids during the critical period and masculinization occurs when the fetal testis begins production of androgens at the beginning of the critical period, the end of which is defined by the developmental stage at which exogenous administration of androgens to females is ineffective at switching brain development from feminization to masculinization. Gonadal hormones rise again in adulthood and promote sex differences in behavior by acting on a neural substrate that was organized differently in males and females. A central question in behavioral neuroendocrinology has been how early life exposure to androgens exerts an enduring influence on adult brain and behavior.

Figure 2

Two approaches to epigenetics of brain sex differences. One approach is to simply measure known epigenetic marks such as histone acetylation or CpG methylation, and/or the enzymes known to regulate the establishment and maintenance of these marks and ask, are they different in males and females? A second approach is to disrupt the establishment of epigenetic marks early in development by inhibiting the associated enzymes or cofactors such as methyl binding proteins (MBPs) and ask, does this eliminate sex differences in adult brain and behavior?

Figure 3

CpG methylation is modulated by hormones. Cytosines are methylated at the 5′ carbon by a class of enzymes called DNA Methyl Transferases (DNMTs). The number of fully methylated CpGs was quantified in DNA extracted from the preoptic area (POA) of 3 newborn females and 3 females treated with a masculinizing dose of estradiol for the previous 2 days. Estradiol treatment significantly reduced the number of fully methylated sites and analyses of where in the genome these sex differences reside indicated they are predominantly in the intragenic region, where DNA methylation is highest.

Figure 4

DNA de-methylation mediates masculinization of brain and behavior. Treatment of newborn male rat pups with DNMT inhibitors such as Zebularine during the critical period of sexual differentiation reduces DNA methylation and thereby changes gene expression profiles which in turn leads to masculinization of the synaptic patterning of the POA and sexual behavior. Thus masculization requires demethylation while feminization is a repression of masculinization via DNA methylation.

Figure 5

DNA methylation maintains feminization of brain and behavior. Conditional knockout of DNMT3a in the POA of mice demonstrated that reduced DNA methylation outside the critical period could still lead to masculinization of behavior, while treatment with exogenous estradiol could not. This was also found to be true in rats using DNMT inhibitors. During the critical period estradiol reduces DNMT activity but this effect is lost outside of the critical period. Thus the ongoing maintenance of DNA methylation appears essential for continued feminization. These observations reveal a novel source of plasticity in sexually dimorphic behavior.