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


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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.



  1. 1.

    Estradiol and the developing brain. Physiol. Rev. 88, 91–124 (2008).

  2. 2.

    & Reframing sexual differentiation of the brain. Nat. Neurosci. 14, 677–683 (2011).

  3. 3.

    et al. The nuclear receptor superfamily: the second decade. Cell 83, 835–839 (1995).

  4. 4.

    , , & Roles of estrogen receptors α and β in differentiation of mouse sexual behavior. Neuroscience 138, 921–928 (2006).

  5. 5.

    et al. Brain masculinization requires androgen receptor function. Proc. Natl. Acad. Sci. USA 101, 1673–1678 (2004).

  6. 6.

    & A novel mechanism of dendritic spine plasticity involving estradiol induction of prostaglandin-E2. J. Neurosci. 22, 8586–8596 (2002).

  7. 7.

    , , & The aromatase knock-out mouse provides new evidence that estradiol is required during development in the female for the expression of sociosexual behaviors in adulthood. J. Neurosci. 22, 9104–9112 (2002).

  8. 8.

    et al. Modular genetic control of sexually dimorphic behaviors. Cell 148, 596–607 (2012).

  9. 9.

    Wired for reproduction: organization and development of sexually dimorphic circuits in the mammalian forebrain. Annu. Rev. Neurosci. 25, 507–536 (2002).

  10. 10.

    , & Sexual differentiation of the vertebrate nervous system. Nat. Neurosci. 7, 1034–1039 (2004).

  11. 11.

    et al. Neuronal activity modifies the DNA methylation landscape in the adult brain. Nat. Neurosci. 14, 1345–1351 (2011).

  12. 12.

    , & Zebularine: a new drug for epigenetic therapy. Biochem. Soc. Trans. 32, 910–912 (2004).

  13. 13.

    & Regulation of chromatin structure in memory formation. Curr. Opin. Neurobiol. 19, 336–342 (2009).

  14. 14.

    et al. Discovery of two novel, small-molecule inhibitors of DNA methylation. J. Med. Chem. 49, 678–683 (2006).

  15. 15.

    et al. Neurabin-II/spinophilin. J. Biol. Chem. 273, 3470–3475 (1998).

  16. 16.

    , & Identification of prostaglandin E2 receptors mediating perinatal masculinization of adult sex behavior and neuroanatomical correlates. Dev. Neurobiol. 68, 1406–1419 (2008).

  17. 17.

    Sexual motivation: a neural and behavioural analysis of the mechanisms underlying appetitive and copulatory responses of male rats. Neurosci. Biobehav. Rev. 14, 217–232 (1990).

  18. 18.

    & DNA methyltrasferase is actively retained in the cytoplasm during early development. J. Cell Biol. 147, 25–32 (1999).

  19. 19.

    et al. Dnmt3a regulates emotional behavior and spine plasticity in the nucleus accumbens. Nat. Neurosci. 13, 1137–1143 (2010).

  20. 20.

    & New concepts in DNA methylation. Trends Biochem. Sci. 39, 310–318 (2014).

  21. 21.

    & Mast cells and basophils in acquired immunity. Br. Med. Bull. 56, 936–955 (2000).

  22. 22.

    , , & GnRH, brain mast cells and behavior. Prog. Brain Res. 141, 315–325 (2002).

  23. 23.

    & Induction of PGE2 by estradiol mediates developmental masculinization of sex behavior. Nat. Neurosci. 7, 643–650 (2004).

  24. 24.

    , & Transient sex differences of aromatase (CYP19) mRNA expression in the developing rat brain. Neuroendocrinology 66, 173–180 (1997).

  25. 25.

    et al. Sex differences in the brain: the not so inconvenient truth. J. Neurosci. 32, 2241–2247 (2012).

  26. 26.

    , & Foxp2 mediates sex differences in ultrasonic vocalization by rat pups and directs order of maternal retrieval. J. Neurosci. 33, 3276–3283 (2013).

  27. 27.

    et al. Conserved role of intragenic DNA methylation in regulating alternative promoters. Nature 466, 253–257 (2010).

  28. 28.

    et al. Drebrin-dependent actin clustering in dendritic filopodia governs synaptic targeting of postsynaptic density-95 and dendritic spine morphogenesis. J. Neurosci. 23, 6586–6595 (2003).

  29. 29.

    et al. Epigenetics in alternative pre-mRNA splicing. Cell 144, 16–26 (2011).

  30. 30.

    et al. Distribution, recognition and regulation of non-CpG methylation in the adult mammalian brain. Nat. Neurosci. 17, 215–222 (2014).

  31. 31.

    et al. Global epigenomic reconfiguration during mammalian brain development. Science 341, 1237905 (2013).

  32. 32.

    et al. Cortical DNA methylation maintains remote memory. Nat. Neurosci. 13, 664–666 (2010).

  33. 33.

    et al. Zebularine partially reverses GST methylation in prostate cancer cells and restores sensitivity to the DNA minor groove binder brostallicin. Epigenetics 8, 656–665 (2013).

  34. 34.

    et al. Mechanistic insights on the inhibition of C5 DNA methyltransferases by zebularine. PLoS ONE 5, e12388 (2010).

  35. 35.

    et al. Tissue distribution of 5-hydroxymethylcytosine and search for active demethylation intermediates. PLoS ONE 5, e15367 (2010).

  36. 36.

    , & DNA methylation: TET proteins-guardians of CpG islands? EMBO Rep. 13, 28–35 (2012).

  37. 37.

    & Active DNA demethylation by Gadd45 and DNA repair. Trends Cell Biol. 22, 220–227 (2012).

  38. 38.

    & Tet family proteins and 5-hydroxymethylcytosine in development and disease. Development 139, 1895–1902 (2012).

  39. 39.

    et al. Growth arrest and DNA-damage-inducible, beta (GADD45b)-mediated DNA demethylation in major psychosis. Neuropsychopharmacology 37, 531–542 (2012).

  40. 40.

    Mounting behavior in the female rat during the estrous cycle, after ovariectomy, and after estrogen or testosterone administration. Horm. Behav. 3, 307–320 (1972).

  41. 41.

    , & A functional circuit underlying male sexual behaviour in the female mouse brain. Nature 448, 1009–1014 (2007).

  42. 42.

    et al. Somatic sex reprogramming of adult ovaries to testes by FOXL2 ablation. Cell 139, 1130–1142 (2009).

  43. 43.

    et al. Inactivation of dnmt3b in mouse embryonic fibroblasts results in dna hypomethylation, chromosomal instability, and spontaneous immortalization. J. Biol. Chem. 280, 17986–17991 (2005).

  44. 44.

    , & Prostaglandin-E2: a point of divergence in estradiol-mediated sexual differentiation. Horm. Behav. 48, 512–521 (2005).

  45. 45.

    , & Enduring consequences of neonatal treatment with antisense oligodeoxynucleotides to estrogen receptor messenger ribonucleic acid on sexual differentiation of rat brain. Endocrinology 133, 433–439 (1993).

  46. 46.

    R Core Team. R: a language and environment for statistical computing. (R Foundation for Statistical Computing, 2014).

  47. 47.

    & Analysis of thick brain sections by obverse-reverse computer microscopy: application of a new, high clarity Golgi-Nissl stain. J. Neurosci. Methods 4, 117–125 (1981).

  48. 48.

    , & A functional circuit underlying male sexual behavior in the female mouse brain. Nature 448, 1009–1014 (2007).

  49. 49.

    , , , & Androgen- and estrogen-independent regulation of copulatory behavior following castration in male B6D2F1 mice. Horm. Behav. 56, 254–263 (2009).

  50. 50.

    & Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics 27, 1571–1572 (2011).

  51. 51.

    et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

  52. 52.

    et al. MethylKit: a comprehensive R package for the analysis of genome-wide DNA methylation profiles. Genome Biol. 13, R87 (2012).

  53. 53.

    , & TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111 (2009).

  54. 54.

    & Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010).

  55. 55.

    et al. gplots: various R programming tools for plotting data. R package version 2.14.1. (2014).

Download references


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


  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


  1. Search for Bridget M Nugent in:

  2. Search for Christopher L Wright in:

  3. Search for Amol C Shetty in:

  4. Search for Georgia E Hodes in:

  5. Search for Kathryn M Lenz in:

  6. Search for Anup Mahurkar in:

  7. Search for Scott J Russo in:

  8. Search for Scott E Devine in:

  9. Search for Margaret M McCarthy in:


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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Bridget M Nugent.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–10

  2. 2.

    Supplementary Methods Checklist

Excel files

  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.

About this article

Publication history