Impact of CUX2 on the Female Mouse Liver Transcriptome: Activation of Female-Biased Genes and Repression of Male-Biased Genes

Abstract

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...

Genome-Wide Analysis of Chromatin States Reveals Distinct Mechanisms of Sex-Dependent Gene Regulation in Male and Female Mouse Liver

“…CUX2 targets were highly enriched among male-biased genes in class M4 and, to a lesser extent, in class M1 (Fig. 5A), consistent with the proposed role for CUX2 as a repressor of many malebiased genes in female liver (34). CUX2 targets were also enriched among class F3 genes, consistent with CUX2 directly activating a subset of female-biased genes (34).…”

“…Enrichment scores were then calculated for each TF compared to a background set of all liver-expressed genes. Figure 5A and B present these data for three GH-regulated TFs: STAT5 (male-enriched, female-enriched, and sex-independent STAT5 binding sites) (30), BCL6, a male-biased repressor (30), and CUX2, a highly female-specific repressor (34). Data are also shown for FOXA1 and FOXA2, which contribute to the sex-biased incidence of hepatocellular carcinoma (44).…”

“…Raw sequence reads for the six sets of chromatin marks in both male and female mouse liver are available at the Gene Expression Omnibus (GEO) website as series GSE44571 (samples GSM1087069 to GSM1087105 and GSM1181495), and sequence reads for RNA-seq data for gene expression in male and female mouse liver are available as series GSE48109 (samples GSM1168535 to GSM1168538). Mouse liver DNase-seq data sets (39) are available as series GSE21777, and ChIP-seq data sets are available for STAT5 (GSE31578; male and female liver, STAT5 high-activity status), BCL6 (GSE31578; male liver) (30), CUX2 (GSE35985; female liver) (34), and FOXA1 and FOXA2 (44) (untreated male and female liver; E-MTAB-805) in ArrayExpress (www.ebi.ac.uk/arrayexpress/).…”

“…We identify genes in active, inactive, and poised chromatin states, and we establish the propensity of genes in each state for induction or repression by ligand-activated nuclear receptors, which mediate metabolic responses to myriad steroids, drugs, and environmental chemicals (41)(42)(43). Further, we elucidate sex-differential chromatin states at sex-biased genes and sex-biased DHS, and we integrate these chromatin state data with binding site data from chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) for the GH-regulated TFs STAT5 (30), BCL6 (30), and CUX2 (34) and for the pioneer factors FOXA1 and FOXA2 (11,12), which impart male bias to hepatocellular carcinoma by facilitating androgen-mediated tumor promotion in males and estrogen-dependent resistance to tumorigenesis in females (44). Our findings reveal the importance of sex-dependent chromatin states in the control of liver gene expression.…”

“…BCL6, a male-biased, GH-regulated transcriptional repressor, competes with STAT5 binding and antagonizes GH-stimulated STAT5 transcriptional activity (30,32). CUX2 is a female-specific, continuous GH-inducible repressor (33) that downregulates many male-biased genes and activates a subset of female-biased genes in female liver (34). Other liver-expressed TFs, including HNF4A and HNF6, are also important for hepatic sex differences, as indicated by knockout studies and reporter gene analyses and by the enrichment of their motifs at GH-dependent regulatory sites (35)(36)(37)(38)(39).…”