Sex-biased long non-coding RNAs negatively correlated with sex-opposite protein coding gene co-expression networks in Diversity Outbred mouse liver

Sex differences in liver gene expression and disease susceptibility are regulated by pituitary growth hormone secretion patterns, which activate sex-dependent liver transcription factors and establish sex-specific chromatin states. Ablation of pituitary hormone by hypophysectomy (hypox) has identified two major classes of liver sex-biased genes, defined by their sex-dependent positive or negative responses to hypox, respectively; however, the mechanisms that determine the hypox responsiveness of each gene class are unknown. Here, we sought to discover candidate regulatory long noncoding RNAs (lncRNAs) that control hypox responsiveness. First, we used mouse liver RNA-seq data for 30 different biological conditions to discover gene structures and expression patterns for ~15,500 liver-expressed lncRNAs, including antisense and intragenic lncRNAs, as well as lncRNAs that overlap active enhancers, marked by enhancer RNAs. We identified u003e200 robust sex-specific liver lncRNAs, including 157 whose expression is regulated during postnatal liver development or is subject to circadian oscillations. Next, we utilized the high natural allelic variance of Diversity Outbred (DO) mice, a multi-parental outbred population, to discover tightly co-expressed clusters of sex-specific protein-coding genes (gene modules) in male liver, and separately, in female liver. Sex differences in the gene modules identified were extensive. Remarkably, many gene modules were strongly enriched for male-specific or female-specific genes belonging to a single hypox-response classes, indicating that the genetic heterogeneity of DO mice captures responsiveness to hypox. Hypox-responsiveness was shown to be facilitated by multiple, distinct gene regulatory mechanisms, indicating its complex nature. Further, we identified 16 sex-specific lncRNAs whose expression across DO mouse livers showed an unexpected significant negative correlation with protein-coding gene modules enriched for genes of the opposite-sex bias and inverse hypox response class, indicating strong negative regulatory potential for these lncRNAs. Thus, we used a genetically diverse outbred mouse population to discover tightly co-expressed sex-specific gene modules that reveal broad characteristics of gene regulation related to responsiveness to hypox, and generated testable hypotheses for regulatory roles of sex-biased liver lncRNAs that control the sex-bias in liver gene expression.