Figure 5. Masculinization of POA gene expression by Dnmt inhibition.

Animals were treated on PN0 and PN1 with Zeb. On PN2, mRNA was extracted and subject to RNA-Seq to identify methylation-dependent changes in gene expression in the female POA. (a) Schematic representation of sex differences in methylation-dependent gene expression in the neonatal POA. Only 70 genes were found to be expressed at different levels in males and females, with 34 higher in males and 36 higher in females. Of these, 70% of genes with higher expression in males were increased in females following Dnmt inhibition indicating methylation-dependent expression. Close to half of the genes expressed at higher levels in females were decreased to male levels following Dnmt inhibition. Overall, more genes significantly changed expression in females compared to males following Zeb treatment, consistent with the higher DNA methylation observed in females. Almost 400 genes became differentially expressed in males and females following Dnmt inhibition in box sexes, and are therefore referred to as convergence genes, meaning expression is maintained at equal levels in males and females by DNA methylation. (b) Heat map of genes with basal sex differences in the POA that were also significantly altered by Zeb treatment in females. Light blue indicates lower relative expression and red indicates higher relative expression across groups. Zeb disrupted basal sex-differences in gene expression in the female POA, creating patterns resembling male gene expression. Arrow indicates Cyp19a1 gene, which codes for the enzyme p450 aromatase, a known contributor to POA masculinization. (c) Heat map of genetic isoforms with basal sex differences that were significantly altered by Zeb treatment in females to create a male-like pattern of isoform expression. (d) Bar plot of all changes in total gene expression induced by Zeb treatment in the neonatal female POA. Grey lines indicate genes without sex differences in expression. Blue lines indicate genes with higher expression in the control male POA vs. the control female POA (putative masculinization genes). Red lines indicate genes with higher expression in the control female POA vs. the control male POA (putative feminization genes). Of the genes with basal sex differences that were altered by Zeb in females, all male-biased genes increased in females following Zeb (blue lines), and all but one of the female-biased genes decreased expression in response to Zeb (red lines), suggesting Dnmt inhibition masculinizes gene expression in the female POA by both increasing male-biased genes and decreasing female-biased genes. Based on data from n = 2 female, 3 Zeb female, 3 male, 3 Zeb male (not represented in plots).