Role of transcription factor CUX2 in post-natal developmental gene expression and sex-biased gene expression in mouse liver

Abstract

Sex differences in hepatic gene expression are primarily regulated by growth hormone, whose patterns of pituitary secretion differ between males and females beginning at puberty. Global gene expression analysis of male and female mouse livers at 3, 4 and 8 weeks of age showed that the majority of adult sex-biased genes displayed sex-specificity after 4 weeks. These pubertal changes preferentially occurred in male liver, where upregulation or down-regulation of genes led to the sex-biased expression observed in adult mouse liver. One of the genes displaying sex-biased expression by 4 weeks was cut-like homeodomain 2 (CUX2), a highly female-specific transcription factor. Overexpression of CUX2 in male liver repressed many male-biased genes and induced many female-biased genes, while knockdown of CUX2 expression in female liver showed complementary effects. Binding of CUX2 was enriched near male-specific STAT5 binding sites and at sites that were preferentially DNase I hypersensitive in male liver genomic DNA. Thus, CUX2 tends to bind in female liver to regions that are more open in male liver at sites also bound by STAT5. Genes identified near CUX2 binding sites were enriched for genes repressed by CUX2 overexpression or induced by CUX2 knockdown, indicating that CUX2 can act as a direct repressor. At 2 weeks of age, CUX2 gene expression and binding activity is high in both male and female mouse liver. Enrichment between male-biased genes repressed by CUX2 overexpression and male-biased genes up-regulated in male liver from 3-8 weeks suggests that the down-regulation of CUX2 contributes to the masculinization of these genes. De novo analysis of CUX2 binding sites identified a motif most similar to that of HNF6, a cut-containing homeodomain whose expression is enriched in liver. Transfection assays showed that CUX2 can inhibit the transcriptional activities of HNF6, consistent with DNA-binding assays showing that these two proteins and the related homeodomain proteins CUX1 and OC-2 display similar DNA-binding site specificities. The extensive overlap of CUX2 binding sites with HNF6 binding sites in liver in vivo suggests that these two factors compete for binding and that HNF6 plays a global role in modulating CUX2-regulated gene expression in female mouse liver.