The growth hormone-regulated transcription factors STAT5 and BCL6 coordinately regulate sex differences in mouse liver, primarily through effects in male liver, where male-biased genes are upregulated and many female-biased genes are actively repressed. Here we investigated whether CUX2, a highly female-specific liver transcription factor, contributes to an analogous regulatory network in female liver. Adenoviral overexpression of CUX2 in male liver induced 36% of female-biased genes and repressed 35% of male-biased genes. In female liver, CUX2 small interfering RNA (siRNA) preferentially induced genes repressed by adenovirus expressing CUX2 (adeno-CUX2) in male liver, and it preferentially repressed genes induced by adeno-CUX2 in male liver. CUX2 binding in female liver chromatin was enriched at sites of male-biased DNase hypersensitivity and at genomic regions showing male-enriched STAT5 binding. CUX2 binding was also enriched near genes repressed by adeno-CUX2 in male liver or induced by CUX2 siRNA in female liver but not at genes induced by adeno-CUX2, indicating that CUX2 binding is preferentially associated with gene repression. Nevertheless, direct CUX2 binding was seen at several highly female-specific genes that were positively regulated by CUX2, including A1bg, Cyp2b9, Cyp3a44, Tox, and Trim24. CUX2 expression and chromatin binding were high in immature male liver, where repression of adult male-biased genes and expression of adult femalebiased genes are common, suggesting that the downregulation of CUX2 in male liver at puberty contributes to the developmental changes establishing adult patterns of sex-specific gene expression. S ex differences in liver gene expression are widespread and affect a broad range of physiological processes, including steroid and drug metabolism, pheromone binding, and lipid metabolism. Hepatic sex-biased genes are regulated by growth hormone (GH) (35, 49), which is secreted by the pituitary gland in a sex-specific manner in rats, mice, and humans (16,28,44,53). Pituitary GH secretion is highly pulsatile in adult male rats and mice, where strong plasma peaks of GH are followed by periods when GH levels are below detection, whereas GH secretion is more frequent in females, resulting in a more continuous exposure to circulating GH. These sexually dimorphic plasma GH patterns stimulate sex differential patterns of tyrosine phosphorylation/activation and nuclear translocation of the transcription factors STAT5a and STAT5b (collectively, STAT5) (50). Thus, STAT5 activation is persistent in female liver but is intermittent in male liver, where it coincides with the onset of each plasma GH pulse (4, 43, 54). STAT5 positively regulates ϳ90% of male-biased genes and negatively regulates ϳ60% of female-biased genes in male mouse liver (5). STAT5 binding sites are found near 35 to 40% of sex-specific genes, suggesting they are directly regulated by STAT5 (54). However, a majority of sex-specific genes do not respond rapidly to GH-activated STAT5, suggesting indirect regulatory mechani...

BackgroundEarly liver development and the transcriptional transitions during hepatogenesis are well characterized. However, gene expression changes during the late postnatal/pre-pubertal to young adulthood period are less well understood, especially with regards to sex-specific gene expression.MethodsMicroarray analysis of male and female mouse liver was carried out at 3, 4, and 8 wk of age to elucidate developmental changes in gene expression from the late postnatal/pre-pubertal period to young adulthood.ResultsA large number of sex-biased and sex-independent genes showed significant changes during this developmental period. Notably, sex-independent genes involved in cell cycle, chromosome condensation, and DNA replication were down regulated from 3 wk to 8 wk, while genes associated with metal ion binding, ion transport and kinase activity were up regulated. A majority of genes showing sex differential expression in adult liver did not display sex differences prior to puberty, at which time extensive changes in sex-specific gene expression were seen, primarily in males. Thus, in male liver, 76% of male-specific genes were up regulated and 47% of female-specific genes were down regulated from 3 to 8 wk of age, whereas in female liver 67% of sex-specific genes showed no significant change in expression. In both sexes, genes up regulated from 3 to 8 wk were significantly enriched (p < E-76) in the set of genes positively regulated by the liver transcription factor HNF4α, as determined in a liver-specific HNF4α knockout mouse model, while genes down regulated during this developmental period showed significant enrichment (p < E-65) for negative regulation by HNF4α. Significant enrichment of the developmentally regulated genes in the set of genes subject to positive and negative regulation by pituitary hormone was also observed. Five sex-specific transcriptional regulators showed sex-specific expression at 4 wk (male-specific Ihh; female-specific Cdx4, Cux2, Tox, and Trim24) and may contribute to the developmental changes that lead to global acquisition of liver sex-specificity by 8 wk of age.ConclusionsOverall, the observed changes in gene expression during postnatal liver development reflect the deceleration of liver growth and the induction of specialized liver functions, with widespread changes in sex-specific gene expression primarily occurring in male liver.

Hepatocyte-enriched nuclear factor (HNF)6 and CUX2 are GH and STAT5-regulated homeobox transcription factors. CUX2 shows female-specific expression and contributes to liver sex differences by repressing many male-biased genes and inducing many female-biased genes, whereas HNF6 is expressed at similar levels in male and female liver. In cell-based transfection studies, CUX2 inhibited HNF6 transcriptional regulation of the sex-specific gene promoters CYP2C11 and CYP2C12, blocking HNF6 repression of CYP2C11 and HNF6 activation of CYP2C12. These inhibitory actions of CUX2 can be explained by competition for HNF6 DNA binding, as demonstrated by in vitro EMSA analysis and validated in vivo by global analysis of the HNF6 cistrome. Approximately 40 000 HNF6-binding sites were identified in mouse liver chromatin, including several thousand sites showing significant sex differences in HNF6 binding. These sex-biased HNF6-binding sites showed strong enrichment for correspondingly sex-biased DNase hypersensitive sites and for proximity to genes showing local sex-biased chromatin marks and a corresponding sex-biased expression. Further, approximately 90% of the genome-wide binding sites for CUX2 were also bound by HNF6. These HNF6/CUX2 common binding sites were enriched for genomic regions more accessible in male than in female mouse liver chromatin and showed strongest enrichment for male-biased genes, suggesting CUX2 displacement of HNF6 as a mechanism to explain the observed CUX2 repression of male-biased genes in female liver. HNF6 binding was sex independent at a majority of its binding sites, and HNF6 peaks were frequently associated with cobinding by multiple other liver transcription factors, consistent with HNF6 playing a global regulatory role in both male and female liver.