Sex differences in pituitary growth hormone (GH) secretion (pulsatile in males vs near continuous/persistent in females) impart sex-dependent expression to hundreds of genes in adult mouse liver. Signal transducer and activator of transcription (STAT) 5, a GH-activated transcription factor that is essential for liver sexual dimorphism, is dynamically activated in direct response to each male plasma GH pulse. However, the impact of GH-induced STAT5 pulses on liver chromatin accessibility and downstream transcriptional events is unknown. In this study, we investigated the impact of a single pulse of GH given to hypophysectomized mice on local liver chromatin accessibility (DNase hypersensitive site analysis), transcription rates (heterogeneous nuclear RNA analysis), and gene expression (quantitative polymerase chain reaction and RNA sequencing) determined 30, 90, or 240 minutes later. The STAT5-dependent but sex-independent early GH response genes Igf1 and Cish showed rapid, GH pulse-induced increases in chromatin accessibility and gene transcription, reversing the effects of hypophysectomy. Rapid increases in liver chromatin accessibility and transcriptional activity were also induced in hypophysectomized male mice for some (Ces2b, Ugt2b38) but not for other liver STAT5-dependent male-biased genes (Cyp7b1). Moreover, in pituitary-intact male mice, Igf1, Cish, Ces2b, and Ugt2b38 all showed remarkable cycles of chromatin opening and closing, as well as associated cycles of induced gene transcription, which closely followed each endogenous pulse of liver STAT5 activity. Thus, the endogenous rhythms of male plasma GH pulsation dynamically open and then close liver chromatin at discrete, localized regulatory sites in temporal association with transcriptional activation of Igf1, Cish, and a subset of STAT5-dependent male-biased genes.

Sex-dependent pituitary growth hormone (GH) secretory profiles—pulsatile in males and persistent in females—regulate the sex-biased, STAT5-dependent expression of hundreds of genes in mouse liver, imparting sex differences in hepatic drug/lipid metabolism and disease risk. Here, we examine transcriptional and epigenetic changes induced by continuous GH infusion (cGH) in male mice, which rapidly feminizes the temporal profile of liver STAT5 activity. cGH repressed 86% of male-biased genes and induced 68% of female-biased genes within 4 days; however, several highly female-specific genes showed weak or no feminization, even after 14 days of cGH treatment. Female-biased genes already in an active chromatin state in male liver generally showed early cGH responses; genes in an inactive chromatin state often responded late. Early cGH-responsive genes included those encoding two GH/STAT5-regulated transcriptional repressors: male-biased BCL6, which was repressed, and female-specific CUX2, which was induced. Male-biased genes activated by STAT5 and/or repressed by CUX2 were enriched for early cGH repression. Female-biased BCL6 targets were enriched for early cGH derepression. Changes in sex-specific chromatin accessibility and histone modifications accompanied these cGH-induced sex-biased gene expression changes. Thus, the temporal, sex-biased gene responses to persistent GH stimulation are dictated by GH/STAT5-regulated transcription factors arranged in a hierarchical network and by the dynamics of changes in sex-biased epigenetic states.

Sex differences in the incidence and progression of many liver diseases, including liver fibrosis and hepatocellular carcinoma, are associated with sex-biased hepatic expression of hundreds of genes. This sexual dimorphism is largely determined by the sex-specific pattern of pituitary growth hormone secretion, which controls a transcriptional regulatory network operative in the context of sex-biased and growth hormone-regulated chromatin states. Histone H3K27-trimethylation yields a major sex-biased repressive chromatin mark deposited at many strongly female-biased genes in male mouse liver, but not at male-biased genes in female liver, and is catalyzed by polycomb repressive complex-2 through its homologous catalytic subunits, Ezh1 and Ezh2. Here, we used Ezh1-knockout mice with a hepatocytespecific knockout of Ezh2 to investigate the sex bias of liver H3K27-trimethylation and its functional role in regulating sex-differences in the liver. Combined hepatic Ezh1/Ezh2 deficiency led to a significant loss of sex-biased gene expression, particularly in male liver, where many female-biased genes were increased in expression while male-biased genes showed decreased expression. The associated loss of H3K27me3 marks, and increases in the active enhancer marks H3K27ac and H3K4me1, were also more pronounced in male liver. Further, Ezh1/Ezh2 deficiency in male liver, and to a lesser extent in female liver, led to up regulation of many genes linked to liver fibrosis and liver cancer, which may contribute to the observed liver pathologies and the increased sensitivity of these mice to hepatotoxin exposure. Thus, Ezh1/Ezh2-catalyzed H3K27-trimethyation regulates sex-dependent genetic programs in liver metabolism and liver fibrosis through its sex-dependent effects on the epigenome, and may thereby determine the sex-bias in liver disease susceptibility.

BackgroundSenescent cells occur in adults with cirrhotic livers independent of the etiology. Aim: Investigate the presence rate of cellular senescence and expression of cell cycle check points in livers from children with end stage disease.Methodology/Principal FindingsLivers of five children aged three years or less undergoing liver transplantation due to tyrosinemia (n = 1), biliary atresia (n = 2), or fulminant hepatitis (n = 2) were analyzed for senescence associated β-galactosidase (SA-βgal) activity and p16INK4a, p21cip1 and p53. All livers displayed positive cellular staining for SA-βgal in the canals of Hering and interlobular biliary ducts. In the presence of cirrhosis (3/5 cases) SA-βgal was found at the cholangioles and hepatocytes surrounding the regenerative nodules. Children with fulminant hepatic failure without cirrhosis had significant ductular transformation with intense SA-βgal activity. No SA-βgal activity was evident in the fibrous septa. Staining for p53 had a similar distribution to that observed for SA-βgal. Staining for p16INK4a and p21cip1 was positive in the explanted liver of the patient with tyrosinemia, in the hepatocytes, the canals of Hering, cholangioles and interlobular bile ducts. In the livers with fulminant hepatitis, p21cip1 staining occurred in the areas of ductular transformation and in the interlobular bile ducts.Conclusions/SignificanceCellular senescence in livers of children with end stage disease is associated with damage rather than corresponding to an age dependent phenomenon. Further studies are needed to support the hypothesis that these senescence markers correlate with disease progression.

31Background: Sex differences in the incidence and progression of many liver diseases, including liver 32 fibrosis and hepatocellular carcinoma, are associated with sex-biased expression of hundreds of 33 genes in the liver. This sexual dimorphism is largely determined by the sex-specific pattern of pituitary 34 growth hormone secretion, which controls a transcriptional regulatory network operative in the context 35 of sex-biased chromatin states. Histone H3K27-trimethylation yields a major sex-biased repressive 36 chromatin mark that is specifically deposited by polycomb repressive complex-2, via its homologous 37 catalytic subunits Ezh1 and Ezh2, at many strongly female-biased genes in male mouse liver, but not 38 at male-biased genes in female liver. Results: We used Ezh1-knockout mice with a hepatocyte-39 specific knockout of Ezh2 to elucidate the sex bias of liver H3K27-trimethylation and its functional role 40 in regulating sex-differences in the liver. Combined hepatic Ezh1/Ezh2 deficiency led to a significant 41 loss of sex-biased gene expression, particularly in male liver, where many female-biased genes 42 increased in expression while male-biased genes showed decreased expression. The associated loss 43 of H3K27me3 marks, and increases in the active enhancer marks H3K27ac and H3K4me1, were also 44 more pronounced in male liver. Many genes linked to liver fibrosis and hepatocellular carcinoma were 45 induced in Ezh1/Ezh2-deficient livers, which may contribute to the increased sensitivity of these mice 46 to hepatotoxin-induced liver pathology. Conclusions: Ezh1/Ezh2-catalyzed H3K27-trimethyation is 47 thus essential for the sex-dependent epigenetic regulation of liver chromatin states controlling 48 phenotypic sex differences in liver metabolism and liver fibrosis, and may be a critical determinant of 49 the sex-bias in liver disease susceptibility. Background 52Liver disease shows marked sex differences. Hepatocellular carcinoma incidence and mortality is 53 three times higher in men than in women [1, 2], and male mice are more susceptible to chemical-54 induced hepatic carcinogenesis [3]. Males are also more susceptible to non-alcoholic fatty liver 55 disease, non-alcoholic steatohepatitis and liver fibrosis than females [4][5][6][7][8]. Underlying these sex-biased Lau-Corona et alPage 5 3/13/19 liver. Hepatic Ezh1/Ezh2 deficiency is also shown to down regulate many male-biased genes, 106presumed as a secondary response to the disruption of female-biased gene expression. Finally, we 107show that many genes associated with liver fibrosis and liver carcinogenesis are differentially 108 responsive to the loss of Ezh1/Ezh2 in male compared to female liver, which may contribute to the 109 observed sex-differences in the incidence and progression of liver cancer. 110 111 Methods 112 Animal tissues. Livers from 7-week-old male and female Ezh1-knockout mice with a hepatocyte-113 specific knockout of Ezh2 (E1/E2-KO mice, also designated Double-knockout (DKO) in the figures 114 and tables) and their age and sex...