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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Gene Expression Omnibus
NCBI Reference Sequence
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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(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.
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.
(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.
(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.
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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Small cells with big implications: Microglia and sex differences in brain development, plasticity and behavioral health
Progress in Neurobiology (2019)