Brain feminization requires active repression of masculinization via DNA methylation

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 females. 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. In sexually reproducing species the developing brain is either masculinized or feminized in a manner that assures adult neural physiology and reproductive behavior are in register with the differentiated gonads. In mammals, feminization of the brain is independent of the ovary Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms Correspondence to: [email protected]. The authors of this manuscript have no competing financial interests or other interests that might be perceived to influence this article. DATABASE ACCESSION NUMBERS RNA-Seq reads (accession: GSE66203) have been deposited in NCBI’s Gene Expression Omnibus (GEO). Whole genome bisulfite sequencing reads can be found in NCBI’s Short Read Archive under the BioProject ID:275796. AUTHOR CONTRIBUTIONS BMN and MMM designed the experiments and wrote the manuscript. BMN performed most molecular biology experiments, rat pharmacology and behavioral experiments, analyzed molecular and behavioral data, and performed bioinformatics analysis of WGBS data. CLW conducted the qPCR, some Dnmt activity assays, and extracted DNA for WGBS. ACS analyzed RNA-Seq data, and AM and SD provided additional bioinformatics support for RNA-Seq. SJR provided transgenic mice and GEH and MMM performed mouse experiments. KML performed immunohistochemistry. HHS Public Access Author manuscript Nat Neurosci. Author manuscript; available in PMC 2015 November 01. Published in final edited form as: Nat Neurosci. 2015 May ; 18(5): 690–697. doi:10.1038/nn.3988. A uhor M anscript