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. 2009 Jan;4(1):47-53.
doi: 10.4161/epi.4.1.7288. Epub 2009 Jan 28.

Sex differences in histone modifications in the neonatal mouse brain

Houng-Wei Tsai  1 , Patrick A Grant, Emilie F Rissman
Affiliations

Sex differences in histone modifications in the neonatal mouse brain

Houng-Wei Tsai et al. Epigenetics. 2009 Jan.

Abstract

Sex differences in neural development are established via a number of cellular processes (i.e., migration, death and survival). One critical factor identified is the neonatal rise in testosterone (T) which activates gene transcription via androgen (AR) and, after aromatization to estradiol, estrogen receptors (ERalpha and beta). Recent evidence shows that AR and ERs interact with histone modifying enzymes. Post-translational modifications of histones, including acetylation and methylation, are involved in transcriptional regulation during normal development. Therefore, we hypothesized that acetylation and/or methylation of histone H3 may underlie sexual differentiation, at least in some regions of the brain. We measured levels of acetylated (H3K9/14Ac) and trimethylated (H3K9Me3) H3 in whole neonatal mouse brains and in three regions: preoptic area + hypothalamus, amygdala and cortex + hippocampus (CTX/HIP). Sex differences in H3K9/14Ac and H3K9Me3 (males > females) were noted in the CTX/HIP on embryonic day 18, the day of birth, and six days later. To determine if T mediates these changes in H3 modifications, pregnant dams received vehicle or T for the final four days of gestation; pup brains were collected at birth. Methylation of H3 was sexually dimorphic despite hormone treatment. In contrast, H3 acetylation in the CTX/HIP of females from T-treated dams rose to levels equivalent to males. Thus, H3 modifications are sexually dimorphic in the developing mouse CTX/HIP and acetylation, but not methylation, is masculinized in females by T in utero. This is the first demonstration that histone modification is associated with neural sexual differentiation.

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Figures

Figure 1
Figure 1
Representative immunoblots showing detection of H3K9/14Ac, H3K9Me3 and H4 in the nuclear (N) but not in the cytosolic (C) extract of the mouse whole brain. Nuclear (5 μg) and cytosol lysates (25 μg) were separated on polyacrylamide-SDS gels, transferred to nitrocellulose membranes, and immunoblotted for H3K9/14Ac and H3K9Me3 as well as total H4 as described in the Materials and Methods section. In the left panel, nuclear and cytosolic proteins and recombinant histone H3 and H4 were stained with Coomassie Blue (CB). In the western blot on the right the H4 was from the same recombinant source.
Figure 2
Figure 2
H3K9/14Ac and H3K9Me3 in the cortex/hippocampus of male and female mouse embryos and pups. (A) Top shows a gel and set of representative immunoblot from PN0 males and females. The top row is a western blot showing H3K9/14Ac bands and the bottom row is Coomassie for total H4 from the same individuals. In the graph below we present mean optical densities (±SEM) of H3K9/14Ac in the cortex/hippocampus of male and female embryos and pups were normalized to levels of H4 and expressed as the percentage of females (as 100%). (B) Top shows a representative immunoblot from PN0 males and females. The top row is a western blot showing H3K9Me3 bands and the bottom row is Coomassie for total H4 from the same individuals. Below are mean optical densities (±SEM) of H3K9Me3 in the cortex/hippocampus of male and female embryos and pups were normalized to levels of H4, and expressed as the percentage of females (as 100%). *Females have significantly less protein than males at these time points, p < 0.05. Sample sizes are show in the base of each histogram. E18 = embryonic day 18, PN0 = day of birth, PN6 = postnatal day 6, M = male, F = female.
Figure 3
Figure 3
Testosterone propionate (TP) increased H3K9/14Ac, but not H3K9Me3 in the cortex/hippocampus of female mouse pups. (A) Top shows a set of representative immunoblot from PN0 males and females. The western blots on the top row show H3K9/14Ac bands and the bottom row is total H4 from the same individuals. Mean optical densities (±SEM) of H3K9/14Ac in the cortex/hippocampus of vehicle (V) and TP-treated male (M) and female (F) neonates were normalized to levels of H4, and expressed as the percentage of V-treated F controls (100%). *Females whose dams received vehicle during gestation had significantly lower levels of H3K9/14Ac as compared with the other three groups (p < 0.05). (B) Top shows a representative immunoblot from PN0 males and females. The western blots on the top row show H3K9Me3 bands and the bottom row is total H4 from the same individuals. Mean optical densities (±SEM) of 3mH3 in the cortex/hippocampus of V and TP-treated M and F neonates were normalized to levels of H4, and expressed as the percentage of V-treated females (as 100%). **Females have significantly less H3K9Me3 than males, p < 0.05. Sample sizes are shown in each histogram.
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