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Brain feminization requires active repression of masculinization via DNA methylation

A Corrigendum to this article was published on 25 May 2017

This article has been updated

Abstract

The developing mammalian brain is destined for a female phenotype unless exposed to gonadal hormones during a perinatal sensitive period. It has been assumed that the undifferentiated brain is masculinized by direct induction of transcription by ligand-activated nuclear steroid receptors. We found that a primary effect of gonadal steroids in the highly sexually dimorphic preoptic area (POA) is to reduce activity of DNA methyltransferase (Dnmt) enzymes, thereby decreasing DNA methylation and releasing masculinizing genes from epigenetic repression. Pharmacological inhibition of Dnmts mimicked gonadal steroids, resulting in masculinized neuronal markers and male sexual behavior in female rats. Conditional knockout of the de novo Dnmt isoform, Dnmt3a, also masculinized sexual behavior in female mice. RNA sequencing revealed gene and isoform variants modulated by methylation that may underlie the divergent reproductive behaviors of males versus females. Our data show that brain feminization is maintained by the active suppression of masculinization via DNA methylation.

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Figure 1: Females have higher Dnmt activity and DNA methylation in the POA, which is reduced by estradiol treatment.
Figure 2: Neonatal Dnmt inhibition increases dendritic spine density on POA neurons and masculinizes behavior.
Figure 3: Conditional Dnmt3a knockout in the POA masculinizes reproductive behavior of female mice.
Figure 4: Dnmt inhibition outside of the critical period increases dendritic spine markers in the POA and masculinizes behavior.
Figure 5: Masculinization of POA gene expression by Dnmt inhibition.

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Change history

  • 08 February 2017

    In the version of this article initially published, the analysis was based on an data set in which one of the female .bam alignment files (FV2) was mistakenly truncated. This file has been removed from NCBI and updated with the raw reads. The realigned raw sequencing reads yielded the same alignment statistics as originally reported in Supplementary Table 5. The reanalysis did not change the main finding that females have significantly more fully methylated CpG sites in the POA than males and estradiol-treated females. In both analyses the reads were filtered for coverage, with a minimum of three reads per site per sample required for inclusion in the analysis. Thus reanalysis with the new, untruncated FV2 file meant that additional CpG and CHG sites became available for analysis across all samples. The analysis of this expanded data set showed a slightly different distribution of sex differences in CpG methylation across genomic regions (Fig. 1d) and chromosomes (Supplementary Fig. 3) than previously reported. After reanalysis incorporating the missing data, Figure 1c,d and Supplementary Figures 2 and 3 have been replaced. In the first Results paragraph, "females had nearly twice the level of fully (100%) methylated CpG sites as males or masculinized females" has been replace by "females had nearly 50% more fully (100%) methylated CpG sites as males or masculinized females"; "sex differences were generally dispersed across chromosomes, although methylation on chromosome 5 and 13 was biased toward females and males, respectively" has been replaced by "sex differences were generally dispersed across chromosomes”; and "The overwhelming majority of CpG sites exhibiting a sex difference in methylation were in intergenic regions (84%), followed by introns (14%), promoter regions (2%) and exons (<1%)" has been replaced by "The overwhelming majority of CpG sites exhibiting a sex difference in methylation were in intergenic regions (69%), followed by introns (26%), exons (5%) and promoter regions (<3%)." In the Figure 1c legend, "F(2,6) = 6.594, P = 0.0306" has been replaced by "F(2,6) = 5.67, P = 0.041.” In the Figure 1d legend, "1,242 sex differences" has been replaced by "2,748 sex differences." In the Supplementary Figure 2 legend, *p < 0.05 has been changed to *p < 0.01. Under Supplementary Figure 2a, "F%meth(9,60) = 709.2" has been changed to "F%meth(9,60) = 1244"; "Fsex(2,60) = 5.047, p = 0.0094" has been changed to "Fsex(2,60) = 2.885, p = 0.0636"; "Males and estradiol-treated females had a greater number of CpG sites 80–90% methylated compared to females (Tukey's HSD, p < 0.0001)" has been changed to "Males (Tukey's HSD, p < 0.0001) and estradiol-treated females (Tukey's HSD, p = 0.0014) had a greater number of CpG sites 80–90% methylated compared to females"; and the final p value has been changed from 0.022 to 0.0055. Similarly, under Supplementary Figure 2b, "F%meth (9,60) = 4787" has been changed to "F%meth (9,60) = 8959"; "Fsex (2,60) = 7.514, p = 0.0012" has been changed to "Fsex (2,60) = 12.73, p < 0.0001"; "male vs. female + e p = 0.0003" has been changed to "male vs. female + e p < 0.0001"; and "Fint(18,60) = 17.16" has been changed to "Fint(18,60) = 23.8." In the Supplementary Figure 3 legend, "Overall, male chromosomes were more hypomethylated relative to females (χ2= 47.83, n=21, p = 0.0004)," has been changed to "There were no overall differences in CpG methylation by chromosome (χ2 = 30.97, n=20, p = 0.0556)." The errors have been corrected in this file as of 8 February 2017.

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Acknowledgements

We thank B.K. Krueger and S.M. Thompson for their helpful input on this manuscript. We thank G. Fan (University of California, Los Angeles) for kindly providing the Dnmt3aloxP/loxP mice. This work was supported by grant R01 MH052716 to M.M.M. and F31NS073545-01 to B.M.N., and R21 MH099562 to S.J.R. This work was conducted as part of the doctoral thesis requirements of B.M.N.

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Authors

Contributions

B.M.N. and M.M.M. designed the experiments and wrote the manuscript. B.M.N. performed most of the molecular biology experiments, rat pharmacology and behavioral experiments, analyzed molecular and behavioral data, and performed bioinformatics analysis of whole-genome bisulfite sequencing data. C.L.W. conducted qPCR, repeated Dnmt activity assays and extracted DNA for whole-genome bisulfite sequencing. A.C.S. analyzed RNA-Seq data, and A.M. and S.E.D. provided additional bioinformatics support for RNA-Seq. S.J.R. provided transgenic mice and G.E.H. and M.M.M. performed mouse experiments. K.M.L. performed immunohistochemistry.

Corresponding author

Correspondence to Bridget M Nugent.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Time course of Dnmt activity in the POA.

On PN0, PN1, and PN4 females had significantly higher levels of Dnmt activity in the POA than males (PN0 t(8) = 2.28, p = 0.05; PN1 t(8) = –3.92, p = 0.0044; PN4 t(5) = 2.65, p = 0.0453). Prior to (on Embryonic day 20 (E20; t(5) = 2.23, p = 0.0766)) and after the perinatal androgen surge, there were no significant sex differences in Dnmt activity in the POA (PN7 t(5) = 0.94, p = 0.3919; PN14 t(9) = 0.67, p = 0.5193). Twenty four hours after estradiol injection there was a significant reduction in Dnmt activity in females on PN1 (t(8) = –2.52, p = 0.036), but not PN14 (t(6) = 0.42, p = 0.6928). Data are graphed as percent change from the control males. Statistics were performed on the actual data and no comparisons were made across time points because the values are relative and therefore not comparable across assays. Data shown here were obtained via colormetric assay. Time course was subsequently repeated (Fig.1) with a more-sensitive fluorometric assay. E20: n = 3 female, 4 male. PN0: n = 5 female, 5 male. PN1: n = 5 female, 5 male, 5 female + e; PN4: n = 4 female, 3 male; PN7: n = 3 female, 4 male; PN14: n = 4 female, 6 male, 4 female + e.*p < 0.05, data are represented as mean ± s.e.m.

Supplementary Figure 2 Frequency distribution of CpG and CHG methylation in the neonatal POA

(a) Most CpG sites in the rat POA were highly methylated (2-Way ANOVA, F%meth(9,60) = 1244, p < 0.0001), and females had significantly more CpG sites with methylation at 90–100% compared to males and females treated with e (Fsex(2,60) = 2.885, p = 0.0636; Tukey's HSD, 90–100% female vs. male p < 0.0001, female vs. female + e p < 0.0001. Fint(18,60) = 7.395, p < 0.0001). Males (Tukey's HSD, p < 0.0001) and estradiol-treated females (Tukey's HSD, p = 0.0014) had a greater number of CpG sites 80–90% methylated compared to females. Estradiol-treated females had fewer sites that are 0–10% methylated compared to control females (Tukey's HSD, p = 0.0055). (b) Conversely, most CHG sites had low levels of methylation (F%meth (9,60) = 8959, p < 0.0001), and all groups differed in the number of sites that are 0-10% methylated (Fsex (2,60) = 12.73, p < 0.0001; Tukey's HSD, 0–10% female vs. male p < 0.0001, female vs. female + e p < 0.0001, male vs. female + e p < 0.0001. Fint(18,60) = 23.8, p < 0.0001). Males and females treated with e had more CHG sites methylated 10–20% compared to control females (Tukey's HSD, p < 0.0001). Only sites with at least 3 reads across all 9 samples were included in this analysis. n = 3 female, 3 male, 3 female + e. **p < 0.01, ***p < 0.001, data are represented as mean ± s.e.m.

Supplementary Figure 3 Distribution of sex differences in CpG methylation by chromosome.

Percent methylation in males is plotted relative to percent methylation in females by chromosome. There were no overall differences in CpG methylation by chromosome (χ2 = 30.97, n = 20, p = 0.0556), average percent methylation is based on n = 3 female, n = 3 male.

Supplementary Figure 4 Dnmt enzyme levels in the POA on PN1

Quantification of Dnmt protein levels via western blot on PN1 following 24 hours of oil or e indicates no significant hormonal modulation of enzyme levels (ANOVA, Dnmt1 F(2,13) = 1.188, p = 0.336; Dnmt3a F(2,13) = 0.387, p = 0.687; Dnmt3b F(2,13) = 0.622, p = 0.552). Cropped representative western bands are shown and full length blots are provided in Supplementary Figure 10. Ponceau S staining of proteins was used as a loading control..n= 6 male, 7 female, 3 female + e. Data are represented as mean ± s.e.m,

Supplementary Figure 5 Neonatal Dnmt inhibition masculinizes adult copulatory behavior and Neurabin II protein levels in the POA

(a) RG108 administration on PN0 and PN1 significantly increased the number of mounts (ANOVA, F(3,26) = 5.31, p = 0.0054) and number of thrusts (F(3,27) = 19.97, p < 0.0001) displayed by females in adulthood. Latency to display first mount was not significantly reduced by neonatal RG108 treatment (F(3,26) = 3.104, p = 0.0439, p-value reflects sex difference, not effect of RG108), however the latency to display first thrust behavior was masculinized in females treated with the inhibitor (F(3,20) = 6.60, p = 0.0028). (b) Protein levels of the synaptic marker Neurabin II were significantly higher in RG108-treated females compared to control females (a priori t-test; t(14)=2.301, p = 0.0373.), although there were no main effects of treatment or sex by ANOVA (F(3,26) = 2.008, p = 0.1375). Cropped representative Neurabin II western blot bands with corresponding Ponceau S loading control shown. Full blots are provided in Supplementary Figure 10. (c) Animals treated with RG108 showed no impairments in locomotor activity as measured by grid crosses (F(3,28) = 1.21, p = 0.33) and rears (F(3,28) = 2.15, p = 0.17), however time spent in the center of the open field arena was masculinized by RG108 (F(3,26) = 2.86, p = 0.049). Mounts: n = 9 male, 8 male + RG108, 7 female, 8 female + RG108. Thrusts: n = 9 male, 6 male + RG108, 8 female, 8 female + RG108. Mount latency: n = 9 male, 6 male + RG108, 7 female, 8 female + RG108. Thrust latency: n = 9 male, 6 male + RG108, 3 female, 6 female + RG108 (a), n = 8 male, 7 male + RG108, 8 female, 8 female + RG108 (b), Grid crosses: n = 8 male, 6 male + RG108, 8 female, 7 female + RG108. Rears: n = 8 male, 6 male + RG108, 8 female, 7 female + RG108. Time in center: n = 8 male, 6 male + RG108, 8 female, 7 female + RG108 (c), *p < 0.05, **p < 0.01, ***p < 0.001 compared to control female by ANOVA. #p < 0.05 by t-test. Data are represented as mean ± s.e.m.

Supplementary Figure 6 Estrous cyclicity was not altered by neonatal Dnmt inhibition

Females were treated with either vehicle or Zeb on PN0 and PN1 and raised to adulthood. (a) Estrous cycle stage was noted for 15 consecutive days. (b) There were no differences in the number of days spent in diestrus (t-test, t(9) = 1.761, p = 0.112), proestrus (t(9)=0.869, p = 0.407), or estrus (t(9) = 1.649, p = 0.1335) across treatment groups and Zeb-treated females appeared to cycle normally. n = 4 vehicle, 7 Zeb.

Supplementary Figure 7 qPCR confirmation of sex and estradiol-mediated differences in aromatase and Neurabin II gene expression patterns in the POA

Rat pups were treated with estradiol or vehicle on PN0 and PN1. mRNA from the POA was collected 6, 48, 96 or 168 hours later. (a) There was a significant effect of sex/hormone treatment on Cyp19a1 (aromatase) mRNA expression in the POA (2-way ANOVA, FSex(2, 49) = 7.264, p = 0.0017), whereby control females had lower levels of aromatase on PN2 compared to males and estradiol-treated females (p = 0.05). On PN7, estradiol-treated females had significantly higher levels of aromatase mRNA compared to control females (p = 0.0325). There was also a significant effect of age on aromatase expression in the POA (FAge(3, 49) = 5.487, p = 0.0025), however there were no significant statistical interactions between age and sex on aromatase expression (FInt(6, 49) = 1.518, p = 0.1920). (b) There were main effects of both sex and age on Ppp1r9b (Neurabin II) mRNA levels in the developing POA (2-way ANOVA, FSex(2, 52) = 7.817, p = 0.0011; FAge(3, 52) = 4.258, p = 0.0092; Fint(6, 52) = 0.8048, p = 0.5708). On PN2, females had significantly lower levels of Neurabin II compared to males and females treated with 100μg E for 24 hours (p = 0.0255) and at PN4 females treated with estradiol had significantly higher levels than both males and control females (p = 0.0109). Cyp19a1 PN0: n = 6 female, 4 male, 5 female + e; PN2: n = 6 female, 5 male, 6 female + e; PN4: n = 5 female, 4 male, 6 female + e; PN7: n = 5 female, 6 male, 3 female + e (a). Ppp1r9b PN0: n = 6 female, 5 male, 5 female + e; PN2: n = 6 female, 5 male, 5 female + e; PN4: n = 5 female, 4 male, 6 female + e; PN7: n = 5 female, 6 male, 5 female + e (b), *p < 0.05 compared to vehicle treated female. Data are represented as mean ± s.e.m.

Supplementary Figure 8 Levels of promoter CpG methylation differ across candidate genes

The methylation status of CpG islands within the promoters of multiple candidate genes was quantified by 454 sequencing following bisulfite conversion on PN2 POA DNA. Most genes exhibited the level of CpG methylation typical of promoter regions, in the 5–10% range, whereas both Ppp1r9b (Neurabin II) and Cyp19a1 (aromatase) were notable for the high levels of CpG methylation, exceeding 50% in the case of aromatase (n = 9; data are the mean of 3 males, 3 females and 3 estradiol-treated females, there were no significant differences between groups in the amount of promoter CpG methylation between groups for a particular gene).

Supplementary Figure 9 qPCR confirmation of RNA-Seq gene expression patterns in the POA

We used qPCR on PN2 POA mRNA to confirm the expression patterns of a subset of genes identified as having sex differences by RNA-Seq. Confirming male-biased gene expression patterns, Nr2f2 (t(10) = 2.175, p = 0.054), Ebf2 (t(10) = 1.976, p = 0.076), and Ebf3 (t(10) = 2.178, p = 0.054), were marginally higher in males compared to females. There were no statistically significant differences in the female-biased gene Darp-32 when measured by qPCR (t(7) = 1.487, p = 0.180). An additional female-biased gene, Adora2a was significantly higher in the female POA compared to males (t(9) = 2.482, p = 0.0349). Nr2f2: n = 6 male, 6 female. Ebf2: n = 6 male, 6 female. Ebf3: n = 6 male, 6 female. Darp-32: n = 4 male, 5 female. Adora2a: n = 5 male, 6 female. *p < 0.05 compared to female. Data are represented as mean ± s.e.m.

Supplementary Figure 10 Full length western blot images.

Western blot films and Ponceau S stained membranes are shown for each protein quantification experiment referenced throughout the paper. Details of western blot protocol, antibody catalog numbers and concentrations used are described in the Methods section. Neurabin II molecular weight ~120 kDa, Dnmt1 ~84 kDa, Dnmt3a ~130kDa, Dnmt3b ~100kDa.

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Sex differences in POA gene expression

(a) Genes with higher expression in control males vs. control females. Male (n=3) and female (n=2) rat pups from two litters were treated with vehicle (1% DMSO) on PN0 and PN1 and RNA was collected from the POA on PN2. RNA-Seq showed that males had 34 genes with significantly higher expression in the POA compared to females. (b) Genes with higher expression in control females vs. control males. We detected 36 genes with significantly higher expression in the female POA compared to males. (XLSX 52 kb)

Methylation-dependent masculinization candidate genes.

Male and female rat pups from two litters were treated with vehicle (1% DMSO; n=3 females, n=3 males) or Zeb (300ng, n=3 females) on PN0 and PN1 and RNA was collected from the POA on PN2. Of the 34 genes that were significantly higher in control males relative to control females (Supplementary Table 1a), 24 were significantly increased by Zeb treatment in females. (XLSX 52 kb)

Gene isoforms exclusive to one sex.

(a) Gene isoforms exclusive to the male POA. Male (n=3) and female (n=2) rat pups from two litters were treated with vehicle (1% DMSO) on PN0 and PN1 and RNA was collected from the POA on PN2. RNA-Seq showed that there were 32 gene isoforms expressed in the male POA, but not the female POA. (b) Gene isoforms exclusive to the female POA. We detected 37 gene isoforms expressed in the female POA, but not the male POA. (XLSX 61 kb)

Methylation-dependent masculinization and feminization gene isoforms.

(a) Male biased gene isoforms increased in females following DNMT inhibition. Male and female rat pups from two litters were treated with vehicle (1% DMSO; n=3 females, n=3 males) or Zeb (300ng, n=3 females) on PN0 and PN1 and RNA was collected from the POA on PN2. Gene isoforms that were significantly higher in males compared to females, which were significantly increased in females with Zeb are listed. (b) Female biased gene isoforms decreased in females following DNMT inhibition. Gene isoforms that were significantly higher in females compared to males, which were significantly decreased in females with Zeb are listed. (XLSX 59 kb)

WGBS library stats.

Male (n=3) and female (n=3) rat pups were treated with vehicle (sesame oil), and an additional group of females (n=3) were treated with estradiol (100 μg) on PN0 and PN1. POA DNA was collected on PN4, bisulfite converted and subject to whole genome bisulfite sequencing. Bismark processing statistics are reported for each library. (XLSX 34 kb)

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Nugent, B., Wright, C., Shetty, A. et al. Brain feminization requires active repression of masculinization via DNA methylation. Nat Neurosci 18, 690–697 (2015). https://doi.org/10.1038/nn.3988

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