Article | Published:

Brain feminization requires active repression of masculinization via DNA methylation

Nature Neuroscience volume 18, pages 690697 (2015) | Download Citation

  • 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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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.

Author information

Affiliations

  1. Program in Neuroscience, University of Maryland School of Medicine, Baltimore, Maryland, USA.

    • Bridget M Nugent
    •  & Margaret M McCarthy
  2. Department of Pharmacology, University of Maryland School of Medicine, Baltimore, Maryland, USA.

    • Bridget M Nugent
    • , Christopher L Wright
    • , Kathryn M Lenz
    •  & Margaret M McCarthy
  3. Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, Maryland, USA.

    • Amol C Shetty
    • , Anup Mahurkar
    •  & Scott E Devine
  4. Neuroscience Department, Mt. Sinai School of Medicine, New York, New York, USA.

    • Georgia E Hodes
    •  & Scott J Russo

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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.

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The authors declare no competing financial interests.

Corresponding author

Correspondence to Bridget M Nugent.

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Supplementary information

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–10

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    Supplementary Methods Checklist

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  1. 1.

    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.

  2. 2.

    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.

  3. 3.

    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.

  4. 4.

    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.

  5. 5.

    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.

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https://doi.org/10.1038/nn.3988