Sex differences characterize the expression of more than 1,000 genes in mouse, rat, and human liver, affecting a wide range of biological processes, including steroid and lipid metabolism, inflammation, and diseased states (11
). Sex differences in pharmacokinetics and pharmacodynamics have long been recognized and are in part a consequence of the sex-biased expression of cytochrome P450 (CYP) and other drug-metabolizing enzymes (19
). Sex differences in human liver gene expression are widespread (69
) and may contribute to sex differences in cardiovascular disease risk (69
), fatty liver disease (1
), and hepatocellular carcinoma (2
Growth hormone (GH), in particular its sex-dependent pituitary secretory pattern, is the major hormonal determinant of liver sex differences (38
). In rats and mice, GH is secreted by the pituitary gland in a highly pulsatile manner in males, while in females GH secretion is more frequent, such that there is no prolonged GH-free interval between plasma hormone pulses (29
). Ablation of circulating GH by hypophysectomy abolishes liver sex differences globally (61
), and exogenous GH pulses restore male-biased gene expression (27
). Continuous GH infusion in male mice mimics the female GH secretory pattern and induces female-biased genes while repressing male-biased gene expression in the liver (27
). While large numbers of sex-dependent, plasma GH pattern-dependent genes have been identified, little is known about the molecular mechanisms whereby these genes respond robustly to their sex-differentiated hormonal input signals.
The transcription factor STAT5 (25
) plays a prominent role in the transcriptional responses to GH, and it has been implicated in the sex-dependent effects of GH on liver gene expression. Liver STAT5 activity cycles in a dynamic, pulsatile manner in direct response to each sequential plasma GH pulse in male rat liver, whereas in female rat liver, STAT5 activity persists at a low level in response to the more frequent (near-continuous) stimulation by circulating GH (10
). STAT5b, in particular, is required to maintain the expression of ~90% of male-biased genes and for repression of a subset (~60%) of female-biased genes in male mouse liver, as has been shown in mouse knockout models (11
). However, it is unclear whether the sex-biased, STAT5-dependent genes identified are direct targets of STAT5 or whether their dysregulation in STAT5-deficient mice is a secondary response. It is also unclear why some direct STAT5 target genes, such as Igf1
), do not show significant sex-biased expression.
Global gene expression analysis has identified many genes that respond to GH rapidly, several of which are known to be direct targets of STAT5 (60
). These early GH response genes include several transcription factors that show sex-biased expression. One such factor is the transcriptional repressor BCL6 (5
), which shows male-biased expression in liver and is downregulated by the female plasma GH profile (43
). BCL6 can modulate transcriptional responses to cytokines and other factors by binding to STAT response elements, enabling it to regulate a wide range of biological processes, including proliferation, differentiation, and apoptosis (15
). STAT5 and BCL6 have opposing effects on the transcriptional regulation of several GH response genes (6
), suggesting that BCL6 might modulate the sex-biased effects of STAT5 on liver gene expression (43
). Other transcription factors implicated in GH-dependent, sex-differential liver gene expression include the liver-enriched factors HNF4A and HNF6/Onecut1 (4
) and the homeobox CDP family member CUX2 (CUTL2) (35
). However, it is not known whether these factors cooperate with STAT5 to regulate liver sex differences.
Here, we identify STAT5 and BCL6 binding sites genome-wide in male and female mouse liver during both the peak and trough periods of plasma GH-activated STAT5, and we elucidate the interplay between these two factors in the regulation of sex-biased liver gene expression. Quantitative sex differences in STAT5 binding are determined for thousands of STAT5 binding sites and are shown to correlate well with sex-biased expression of STAT5 target genes. The sex-differential binding of STAT5 is also shown to be highly correlated with several sex-dependent epigenetic modifications and sex-differentially enriched motif families, the most prominent of which is HNF6/CDP. In addition, BCL6 binding sites that overlap with STAT5 binding sites are found to be preferentially associated with repression of female-biased genes in male liver. Finally, we identify lipid and cytochrome P450 metabolism among the top functional categories enriched in STAT5 and BCL6 common target genes, highlighting the importance of BCL6 in modulating these GH/STAT5-regulated metabolic processes. This comprehensive analysis of sex-dependent binding of two key GH-regulated transcription factors and their relationship to sex-dependent chromatin modifications provide important new insights into the mechanisms that control sex differences affecting hepatic lipid metabolism and other physiological and pathophysiological processes.