Hepatocyte homeostasis for chromosome ... - Wiley Online Library

Hepatocyte Homeostasis for Chromosome Ploidization and Liver Function Is Regulated by Ssu72 Protein Phosphatase Se-Hyuk Kim,1 Yoon Jeon,1,2 Hyun-Soo Kim,1 Jin-Kwan Lee,3 Han Jeong Lim,2 Donglim Kang,1 Hyeseong Cho,4 Cheol-Keun Park,5 Ho Lee,2 and Chang-Woo Lee1,3 Hepatocyte chromosome polyploidization is an important feature of liver development and seems to be required for response to liver stress and injury signals. However, the question of how polyploidization can be tightly regulated in liver growth remains to be answered. Using a conditional knockout mouse model, liver-specific depletion of Ssu72 protein phosphatase was found to result in impairment in regulation of polyploidization. Interestingly, the aberrant polyploidization in Ssu72-depleted mice was associated with impaired liver damage response and increased markers of liver injury and seemed to mimic the phenotypic features of liver diseases such as fibrosis, steatosis, and steatohepatitis. In addition, depletion of Ssu72 caused deregulation of cell cycle progression by overriding the restriction point of the cell cycle and aberrantly promoting DNA endoreplication through G2/M arrest. Conclusion: Ssu72 plays a substantial role in the maintenance of hepatic chromosome homeostasis and would allow monitoring of liver function. (HEPATOLOGY 2016;63:247-259)

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hromosome polyploidy is an important aspect of postnatal liver development and results in the formation of highly differentiated hepatocytes.14 Polyploidization may have functions in the regulation of liver size and organization, increase in the number of genes dedicated to liver-specific functions, and adaptation to injury and stress signals.3,5,6 Thus, both liver development and regeneration are highly associated with a pronounced increase in chromosome polyploidy. At weaning, most hepatocytes are normal diploids, but >60% of hepatocytes in mice and 30%-40% in humans display polyploidy by adulthood, indicating that normal hepatocytes become polyploid in adults. During postnatal development, diploid hepatocytes (mononucleated 2c) can engage in the normal cell cycle to generate two diploid hepatocytes or, in an alternative cell cycle with incomplete cytokinesis, a tetraploid hepatocyte (binucleated 2 3 2c). Further polyploidization results in

the generation of tetraploid and octoploid hepatocytes containing one nucleus or two nuclei. Hepatocyte polyploidization is not an irreversible process. In liver development, polyploid hepatocytes can produce a highly diverse population of daughter cells with both numerical chromosomal abnormalities and uniparental chromosome sets.7 Furthermore, in response to injury or stress signals, the process of chromosome polyploidization is highly prone to triggering of autoregulatory cell death and susceptible to augmentation of apoptotic cell death. Thus, polyploidization should be tightly linked to cell division controls, such as DNA endoreplication, sister chromatid segregation, and cytokinesis.8,9 Also, polyploidy hepatocytes have a coping mechanism in response to energy demands for hepatocyte proliferation and regeneration and to stress and damage signals.5,8-10 Nevertheless, both the exact physiological significance of hepatocyte polyploidization and

Abbreviations: ALT, alanine aminotransferase; a-SMA, a-smooth muscle actin; AST, aspartate aminotransferase; CldU, chlorodeoxyuridine; HCC, hepatocellular carcinoma; H&E, hematoxylin and eosin; IL, interleukin; MEF, mouse embryonic fibroblast; PCNA, proliferating cell nuclear antigen; PCR, polymerase chain reaction; Rb, retinoblastoma; TNF-a, tumor necrosis factor a; TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling; WT, wild type. From the 1Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, Korea; 2Graduate School of Cancer Science and Policy, Research Institute, National Cancer Center, Goyang, Korea; 3Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul, Korea; 4 Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, Suwon, Korea; 5Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea Received April 23, 2015; accepted October 7, 2015. Additional Supporting Information may be found at onlinelibrary.wiley.com/doi/10.1002/hep.28281/suppinfo. 247

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the mechanism by which hepatic chromosome polyploidization can be adapted during postnatal development remain obscure. Ssu72 is known to have phosphatase activity on Ser5P and Ser7-P of the C-terminal domain of RNA polymerase II, and hypophosphorylation of the C-terminal domain of RNA polymerase II by Ssu72 appears to play an important role in regulating its ability to recruit transcription factors at appropriate points in the transcription cycle.11,12 We recently reported that Ssu72 also functions as a cohesin-binding phosphatase for regulation of sister chromatid cohesion and separation.13,14 Depletion or mutational inactivation of Ssu72 phosphatase activity led to premature dissociation of sister chromatid cohesion, indicating that Ssu72 plays a critical role in maintaining the integrity of duplicated sister chromatid. However, the physiological relevance of Ssu72 loss of function remains unknown. In this study, we unexpectedly found that Ssu72 protein phosphatase is essential for the maintenance of hepatic chromosome polyploidization and monitoring of liver function.

Materials and Methods Animal Experiments. All animal experiments were approved by the Institutional Animal Care and Use Committee (15-6) of Sungkyunkwan University School of Medicine, which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International and abides by the guidelines issued by the Institute of Laboratory Animal Resources. All mice were bred into the C57BL/6 background for at least seven generations. Primary Hepatocyte Culture. Primary hepatocytes were isolated from 3-week-old, 5-week-old, and 10week-old Ssu72f/f (wild type [WT]) and Alb-Cre;Ssu72f/f (Ssu72Dhep) mice by liver perfusion with collagenase IV (C5138; Sigma-Aldrich, St. Louis, MO). After isolation, cells were seeded in M199 medium (M4530; Sigma-Aldrich) supplemented with 10% fetal bovine serum (10082-147; Gibco BRL Life Technologies, Grand Island, NY), 1% antibiotic antimycotic solution (LS203-01; Welgene, Daegu, South Korea), 23 mM 4(2-hydroxyethyl)-1-piperazine ethanesulfonic acid

HEPATOLOGY, January 2016

(H0887; Sigma-Aldrich), and 10 nM dexamethasone (D4902; Sigma-Aldrich). Cells were maintained in a 37 C incubator with a 5% CO2 atmosphere. Statistical Analysis. All data are presented as means 6 standard errors of means. Statistical analyses were performed using the Student t test, and statistical significance was accepted for P < 0.05. Additional Methods. For additional details, please refer to the Supporting Information.

Results Aberrant Chromosome Polyploidy by Loss of Ssu72 Function During Postnatal Liver Development. To examine the effect of Ssu72 loss of function in vivo, a conditional Ssu72 allele was generated by a gene targeting mouse embryonic stem cells. The targeting strategy was to flank Ssu72 exons 1 and 2 with loxP sites and then allow Cre-mediated partial deletion of exon 1, including the main ATG codon. To accomplish this, Ssu72f/f mice were crossed with transgenic mice expressing Cre recombinase under the control of the hepatocyte-specific albumin promoter (Alb-Cre) to generate Alb-Cre;Ssu72f/f mice (Supporting Fig. S1A-F). To compare the cell cycle profiles of the WT and Ssu72depleted hepatocytes, primary hepatocytes isolated from Ssu72f/f (WT) and Alb-Cre;Ssu72f/f (Ssu72Dhep) mice at 3 weeks (before weaning) and 10 weeks (after weaning) were stained with propidium iodide (Fig. 1A; Supporting Figs. S2 and S3). Flow-cytometric analyses revealed that the hepatocytes isolated from both WT and Ssu72Dhep mice were composed of predominantly diploid (2c), with some tetraploid (4c), hepatocytes before weaning. As expected, the WT hepatocytes contained higher proportions of tetraploid and octoploid (8c) hepatocytes and lower proportions of diploid hepatocytes after weaning. Surprisingly, however, the Ssu72depleted hepatocytes showed markedly higher proportions of octoploid hepatocytes but almost no diploid hepatocytes after weaning (Fig. 1A). Upon comparison of the hepatic polyploidy of 3-week-old to 10-week-old mice, it was found that the proportion of octoploid hepatocytes in the Ssu72Dhep mice gradually increased with age to reach almost 60% at 10 weeks, whereas

Address reprint requests to: Chang-Woo Lee, Ph.D., Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 16419, Korea. E-mail: [email protected]. or to: Ho Lee, Ph.D., Graduate School of Cancer Science and Policy, National Cancer Center, Goyang 10408, Korea. E-mail: [email protected]. C 2015 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.28281 Potential conflict of interest: Nothing to report.

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Fig. 1. Ssu72 is involved in regulation of hepatic chromosome polyploidization. (A) The genome ploidies of hepatocytes were characterized by propidium iodide staining, followed by fluorescence-activated cell sorting. Hepatocytes were isolated from WT and Ssu72Dhep mice at 3 weeks and 10 weeks of age. (B) Quantitation of DNA contents isolated from hepatocytes of 3-week-old, 5-week-old, or 10-week-old WT and Ssu72Dhep mice (n 5 4 per genotype [3 weeks], n 5 6 per genotype [5 and 10 weeks]). (C) Representative images of immunofluorescence staining for bcatenin (plasma membrane, green) and nuclei (blue) in liver sections of 3-week-old and 10-week-old WT and Ssu72Dhep mice. Original magnification 3400; scale bars 5 10 lm. (D) Quantification of nuclear diameters and nuclear sizes of mononucleated hepatocytes from liver sections of 3-week-old and 10-week-old WT and Ssu72Dhep mice (n 5 4 mice per genotype). (E) Quantification of binucleated hepatocytes in liver sections of 3-week-old, 5-week-old, and 10-week-old WT and Ssu72Dhep mice. Results are presented as means 6 standard error of the means (n 5 4 mice per genotype). *P < 0.05, **P < 0.01, ***P < 0.001 versus WT. Abbreviations: FSC, forward scatter; NS, not significant.

fewer than 20% of octoploid hepatocytes were observed in WT mice at the same age (Fig. 1B). Although polyploidy is a general physiological process in adult mice,

deletion of Ssu72 resulted in marked increases of the hepatic polyploidy population. Regulation of hepatic polyploidization by Ssu72 appeared to occur in a tissue-

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specific manner, however, as the depletion of Ssu72 in primary mouse embryonic fibroblasts (MEFs) resulted in normal diploidy, similar to the WT MEFs (Supporting Fig. S4A-C). To determine the number of nuclei per hepatocyte, nuclear and cell membrane (b-catenin) immunofluorescence staining of liver sections was performed at 3 weeks and 10 weeks (Fig. 1C). At 3 weeks of age, WT and Ssu72Dhep livers showed significant populations of binucleated hepatocytes. However, at 10 weeks, the binucleation slightly increased in WT hepatocytes, while the number of mononucleated polyploid hepatocytes decreased. However, the depletion of Ssu72 in the livers of same-aged mice resulted in strong induction of mononucleated polyploid hepatocytes with enlarged nuclei compared with WT. Further analyses revealed that the depletion of Ssu72 caused a profound increase in both the nuclear diameters and the sizes of the mononuclear hepatocytes at 10 weeks (Fig. 1D). In addition, the depletion of Ssu72 significantly decreased the proportions of binucleated hepatocytes compared with control hepatocytes, starting at 5 weeks (Fig. 1E). Liver-Specific Depletion of Ssu72 Was Associated With Impaired Liver Damage Response and Increased Liver Injury. To determine whether Ssu72 deficiency affects liver physiology, livers from WT and Ssu72Dhep mice at 3 weeks, 5 weeks, and 10 weeks of age were stained with hematoxylin and eosin (H&E) and the expression of differentiation marker proteins, such as glutamine synthetase (a marker of pericentral expression), carbamoylphosphate synthetase I (a marker of periportal expression), cytokeratin 19 (a marker of bile duct and oval cell expression), sirius red (a marker of fibrillar collagen deposition), and a-smooth muscle actin (a-SMA, a marker of activated hepatic stellate cell), was examined. The livers were also stained by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) (Fig. 2A; Supporting Fig. S5). Interestingly, H&E staining of the liver sections revealed significant changes in the physiological phenotypes from Ssu72 loss. The Ssu72-depleted livers showed a number of apparent clinical signs of abnormality, including fat storage, necrosis, inflammatory cell infiltration, cytomegalic hepatocytes, and cytoplasmic vacuolization (Fig. 2A). In addition, fibrillar collagen observed initially around the portal veins in Ssu72deprived mice gradually enhanced to the extent that many livers were fibrotic at 5 weeks and 10 weeks of age (Fig. 2A,C). Furthermore, depletion of Ssu72 caused a significant increase in the appearance and expansion of activated hepatic stellate cells, as visualized by a-SMA staining (Fig. 2A). In contrast, only marginal differences


in the expression levels of glutamine synthetase, carbamoylphosphate synthetase, and cytokeratin 19 were observed between WT and Ssu72Dhep livers at 5 weeks and 10 weeks (Supporting Fig. S5). Also, hepatocyte apoptosis was significantly higher in the Ssu72Dhep mice, particularly at 5 weeks (Fig. 2A,B). Next, serum accumulations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the WT and Ssu72Dhep mice were measured before and after weaning (Fig. 2D,E). The enzymes ALT and AST localized in liver cells leak out into the general circulation when the liver cells become injured. Interestingly, the levels of these two enzymes were highly elevated in Ssu72Dhep mice after weaning (at age 5 weeks and 10 weeks) compared with WT mice, whereas the levels of both enzymes did not significantly differ before weaning (at 3 weeks of age). Moreover, the levels of serum ALT and AST in the Ssu72Dhep mice were higher at 5 weeks than at 10 weeks. However, the levels of both enzymes in the WT mice remained nearly constant at the ages analyzed. In a separate experiment, carbon tetrachloride, which directly causes severe liver necrosis, was intraperitoneally injected into WT and Ssu72Dhep mice to induce acute liver damage (Supporting Fig. S6A). After carbon tetrachloride injection, the Ssu72-deficient mice exhibited severe necrosis and fibrosis with significantly higher serum ALT, AST, and TUNEL levels than the control mice (Supporting Fig. S6B-E). Depletion of Ssu72 also resulted in elevation of intrahepatic fibrogenic cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor a (TNF-a) (Supporting Fig. S6F). Interestingly, we also observed that Ssu72Dhep mice with mostly polyploid hepatocytes are capable of regeneration after partial hepatectomy compared to WT mice (Supporting Fig. S7), indicating that hepatic polyploidy is associated with liver regeneration. However, the physiological relevance of highly polyploid hepatocytes developed in Ssu72Dhep mice to liver regeneration remains to be further evaluated. Together, these results indicate that loss of Ssu72 directly affects susceptibility to liver injury and augments the liver damage response. Depletion of Ssu72 Leads to a Higher Incidence of Chronic Liver Diseases. To investigate whether the Ssu72-depleted mice showed progressive pathological signs of liver diseases with age, the livers of 12month-old WT and Ssu72Dhep mice were stained with H&E, oil red O (a marker for neutral triglycerides and lipid), sirius red, a-SMA, and F4/80 (Fig. 3A). H&E staining of the liver sections of Ssu72Dhep mice revealed significant signs of liver disease, including fatty change, necrosis, and inflammatory cell infiltration, compared to those of the WT mice. The Ssu72-depleted livers also


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Fig. 2. Loss of Ssu72 promotes hepatic damage and injury. (A) Representative images of H&E-stained livers, as well as immunohistochemical analyses for TUNEL, sirius red, and aSMA in the livers of WT and Ssu72Dhep mice at 3 weeks, 5 weeks, and 10 weeks of age. Inset shows macroscopic images of the corresponding tissue samples. Arrow indicates inflammatory infiltration, while arrowheads indicate TUNEL-positive hepatocytes. Original magnification 3100; inset and TUNEL staining magnification 3200; scale bars 5 20 lm. (B) Quantification of TUNEL-positive hepatocytes in the liver sections of WT and Ssu72Dhep mice (n 5 5 mice per genotype). (C) Quantification of sirius red– positive areas in the liver sections of WT and Ssu72Dhep mice (n 5 5 mice per genotype). Bars represent percentages of sirius red–positive areas versus total areas. (D,E) Serum ALT and AST levels, measured in WT and Ssu72Dhep mice at indicated ages (n 5 7 mice per genotype). Results are presented as means 6 standard error of the means. *P < 0.05, **P < 0.01, ***P < 0.001 versus WT. Abbreviation: NS, not significant.

showed extensive signs of steatosis with increased lipid deposition in the hepatocytes and hepatocyte ballooning, both characteristic pathological features in nonalcoholic steatohepatitis (Fig. 3A).15 In addition, depletion of Ssu72 greatly increased the fibrillary collagen deposition in the livers, as well as expansions of activated hepatic stellate cells and liver macrophages (Fig. 3A), indicating that loss of Ssu72 leads to the development of a high incidence of liver diseases, including fibrosis, stea-

tosis, and steatohepatitis, but not hepatocellular carcinoma (HCC) (Fig. 3B). Next, the serum accumulation of ALT was monitored in mice at 3 weeks, 5 weeks, 10 weeks, 20 weeks, and 52 weeks of age (Fig. 3C). At 5 weeks of age (after weaning), Ssu72Dhep mice showed highly elevated levels of ALT compared with WT mice. Moreover, the levels of ALT enzyme were persistently elevated in Ssu72Dhep mice at all ages, whereas significant differences in the

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Fig. 3. Depletion of Ssu72 leads to high incidence of chronic liver diseases. (A) Microscopic liver morphology, revealed by H&E staining and immunohistochemical analyses for oil red O, sirius red, a-SMA, and F4/80 in the livers of WT and Ssu72Dhep mice at the age of 52 weeks. H&E staining magnification 3100; oil red O, sirius red, a-SMA, and F4/80 staining magnification 3200; scale bars 5 20 lm. (B) Relative incidence of liver diseases, including fibrosis, steatosis, and steatohepatitis, in WT and Ssu72Dhep mice. Asterisk indicates mild steatosis. (C) Comparison of serum ALT levels between WT and Ssu72Dhep mice at different time points (n 5 7 mice per genotype). (D) Messenger RNA levels of the intracellular cytokines IL-6, TNF-a, and IL-1b, as measured by quantitative PCR using liver extracts from WT and Ssu72Dhep mice (n 5 5 mice per genotype, 50-52 weeks old). (E) Relative messenger RNA levels of genes involved in lipid synthesis in liver extracts isolated from WT and Ssu72Dhep mice at 12 months of age (n 5 5 mice per genotype). Results are presented as means 6 standard error of the means. *P < 0.05, **P < 0.01, ***P < 0.001 versus WT. Abbreviations: mRNA, messenger RNA; NS, not significant.

ALT levels of WT mice were not observed between those ages, indicating that the loss of Ssu72 function leads to chronic liver injury. Evaluation of the IL-1b, IL-6, and TNF-a expression levels revealed that depletion of

Ssu72 consistently also led to the elevation of intrahepatic IL-1b, IL-6, and TNF-a levels compared to WT (Fig. 3D). Furthermore, the levels of lipogenic markers, including chREBP, SREBP1c, SREBP2, FAS, and ACC,


which are involved in the development of hepatic steatosis, were also significantly elevated in the Ssu72Dhep mice compared to the WT mice (Fig. 3E). Aberrant Up-Regulation of Genes Involved in Cell Cycle Control and DNA Replication After Loss of Ssu72 Expression. To elucidate the functional correlation between loss of Ssu72 expression and aberrant hepatic chromosome polyploidization, complementary DNA microarray analysis was performed on 5-week-old WT and Ssu72-depleted hepatocytes (Fig. 4A). The analysis showed that three to four times more transcripts were up-regulated than down-regulated in primary hepatocytes isolated from the Ssu72Dhep mice compared with those from the WT mice. Functional annotation of the genes up-regulated in the Ssu72-depleted hepatocytes revealed a bias toward cell cycle–related gene products known to be involved in cell cycle control (e.g., Ccna2, Ccne2, Cdc20, and Bub1), DNA replication (e.g., Mcm5, Mcm6, Cdc6, and proliferating cell nuclear antigen [PCNA]), cell proliferation (e.g., Hgf and Ccnd1), and DNA repair (e.g., H2afx and Esco2) (Fig. 4A-C; Supporting Table S1). Interestingly, a number of cell cycle transcripts were upregulated exclusively at 5 weeks, but not at 3 weeks (data not shown), of which a predominant subset was involved in the cell cycle stages of DNA replication and chromosome duplication. Further reverse-transcription polymerase chain reaction (PCR) and immunoblotting analyses confirmed the altered expression of these genes in the 5week-old Ssu72-depleted hepatocytes (Fig. 4D-F). In particular, the levels of Ccna2 (Cyclin A2), Ccne1 (Cyclin E1), PCNA, Mcm6, and Cdc6 were highly elevated in the 5-week-old, but not in the 3-week-old, Ssu72-depleted hepatocytes (data not shown). Importantly, several genes among the up-regulated transcripts, including Cyclin A2,16 Cyclin B1,17 Cyclin E1,18 Mcm5,19 Mcm6,19 and Cdc6,17 are directly controlled by the retinoblastoma protein (Rb)-E2F signaling pathway. However, most of the down-regulated transcripts did not appear to be directly involved in the Rb-E2F pathway. Ssu72 Binds to and Dephosphorylates Rb. The amino acid sequence of the Ssu72 protein includes a putative LxCxE motif, a highly conserved domain referred to as the “pocket” that interacts with the retinoblastoma gene product Rb (Supporting Fig. S8A).20 To determine whether the Ssu72 phosphatase regulates the phosphorylation status of Rb, and thus activates RbE2F signaling, the interaction between Ssu72 and Rb was examined. A series of pull-down and immunoprecipitation assays clearly showed that Ssu72 and Rb proteins were present in a complex in vitro and in vivo (Fig. 5A,B). Next, to assess the effect of Ssu72 expression on

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the phosphorylation status of Rb, two-dimensional isoelectric focusing gel electrophoresis was performed using lysates of 5-week-old WT and Ssu72-depleted hepatocytes (Fig. 5C). Interestingly, the appearance of a remarkable shift in Rb polypeptides was found for the primary hepatocytes isolated from the Ssu72Dhep mice, indicating that depletion of Ssu72 causes hyperphosphorylation of the Rb protein. The phosphorylation status of Rb is associated with entry into the cell cycle and proliferation. Particularly, phosphorylation at residues Ser773 (Ser780 in humans) and Ser800/804 (Ser807/811 in humans) plays a key role in the interaction of Rb with E2F1 and in the activation of transcriptional regulation of E2F1 signaling and provides a selective growth advantage for the cells in the quiescence stage.21,22 Figure 5D shows the immunohistochemical staining of representative liver sections from WT and Ssu72Dhep mice using two phosphospecific Rb antibodies (Ser773 and Ser800/804). Interestingly, significant enhancement of the phosphorylation levels at residue Ser800/804 in Ssu72Dhep mice was observed compared to WT mice, whereas phosphorylation at residue Ser773 was consistently observed in both WT and Ssu72Dhep mice. The observations above were further supported by gene-set enrichment analysis, which showed that the set of up-regulated genes in Ssu72-depleted hepatocytes was also significantly enriched in Rb-null MEFs (Supporting Fig. S8B). Overall, these data support the idea that Ssu72 targets Rb for dephosphorylation particularly of Ser800/804 in mice. Loss of Ssu72 Expression Overrode the R-Point and Aberrantly Activated DNA Replication. Because Rb is frequently inactivated during pathogenesis causing release of E2F1, which in turn can activate downstream genes that control cell cycle progression,22 we next examined whether Ssu72 affects the transcriptional activity of E2F1. Using the promoter taken from the dihydrofolate reductase gene (DHFR), which contains an E2F1-responsive gene element, transcription of the luciferase reporter gene was efficiently induced by introduction of E2F1 into Huh7 HCC cells and further augmented by cointroduction of E2F1 with its heterodimeric partner DP1 (Fig. 5E). The transcriptional activity of the promoterless control reporter gene was unaffected by expression of E2F1 or E2F1 together with DP1 (E2F1/DP1). Importantly, higher levels of Ssu72 caused a concomitant decline in the transcriptional activities of E2F1 and E2F1/DP1 (Fig. 5E). Quantitative reverse-transcription PCR analysis using hepatocytes isolated from WT and Ssu72Dhep mice further showed that the expression of several target genes of the Rb-E2F pathway, including E2F1-3, Mcm2, Mcm7,

Fig. 4. Depletion of Ssu72 causes aberrant activation of DNA replication and expression of cell cycle regulatory genes. (A) Gene expression heat map representing fold-change ratios of hepatocytes from Ssu72Dhep mice versus WT mice at 5 weeks of age. A filter was applied to categorize genes with fold-changes greater than (red) or less than (green) 1.5. (B) Schematic diagrams of up-regulated (red) and down-regulated (green) transcripts, as determined by microarray analyses of WT versus Ssu72Dhep hepatocytes at 5 weeks of age. (C) Categorization of upregulated genes (>1.5 fold) in terms of cell cycle involvement. (D) Reverse-transcription PCR assays were used to verify the expression levels of cell cycle–related genes in hepatocytes isolated from two different lines of Ssu72Dhep and age-matched WT mice at 5 weeks of age. Primer sequences are listed in Supporting Table S2 (n 5 3 mice per genotype). (E) Expression of the cell cycle–related genes indicated was determined by immunoblotting of hepatocytes from two different lines of 5-week-old WT and Ssu72Dhep mice (n 5 4 mice per genotype). Asterisk indicates a nonspecific band. (F) Expression levels of the indicated cell cycle–related genes, as confirmed by immunoblotting using liver extracts from WT and Ssu72Dhep mice at 3 weeks and 5 weeks of age (n 5 3 mice per each genotype). Results are representative of two independent experiments.


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Fig. 5. Ssu72 directly dephosphorylates Rb and affects Rb-E2F1-dependent transcription. (A) Glutathione S-transferase (lower arrow) or Glutathione S-transferase-Rb (upper arrow) was incubated with purified His-Ssu72 and subjected to immunoblotting with anti-Ssu72 and anti-Rb antibodies. (B) HepG2 cells were transfected with expression plasmids encoding the Myc-Rb and HA-Ssu72. Lysates were immunoprecipitated with anti-Myc antibody and immunoblotted with anti-Rb and anti-HA antibodies (upper panel). Lysates of HepG2 cells were immunoprecipitated with anti-Rb antibody and then immunoblotted with anti-Ssu72 antibody (lower panel). Asterisk indicates a nonspecific band. (C) Five-week-old WT and Ssu72Dhep hepatocyte extracts were analyzed by two-dimensional gel electrophoresis, followed by immunoblotting using anti-Rb antibody (arrow). Lamin was used as the loading control. Asterisk indicates a nonspecific band. (D) Representative images of immunohistochemical analyses for the phosphorylation of Rb at residue Ser773 or Ser800/804 in liver sections of WT and Ssu72Dhep mice at 5 weeks of age. Original magnification 3400; scale bars 5 20 lm. (E) Huh7 cells were transfected with expression vectors for E2F1 (0.2 lg) and/or DP1 (0.6 lg), together with an increasing amount of Ssu72 expression vector (1, 2, and/or 3 lg) in the presence of the E2F reporter pDHFR-luciferase (0.5 lg) (upper panel) or the control pT81-luciferase (0.5 lg) (lower panel), as indicated. The pCMV-b-galactosidase vector was included in each transfection as an internal control. The relative activity of luciferase to b-galactosidase is presented. (F) Expressions of several target genes of the Rb-E2F pathway in hepatocytes from WT and Ssu72Dhep mice at 5 weeks of age were measured by quantitative PCR (n 5 5 mice for each genotype). Abbreviations: CS, Coomassie brilliant blue staining; GST, glutathione S-transferase.

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Cdc6, PCNA, Kif4, and Plk1, was significantly higher in Ssu72Dhep hepatocytes compared with WT (Fig. 5F). Thus, these results suggest that Ssu72 can regulate the transcriptional activity of E2F1, where its effects are dependent upon Rb. To determine the proliferative capacity of hepatocytes conferred by Ssu72 loss, WT and Ssu72Dhep livers were immunostained with anti-bromodeoxyuridine (BrdU), anti-PCNA, and anti-Ki67 (Supporting Fig. S9A-C) antibodies. At 3 weeks, the proliferation indices (identified as BrdU-positive, PCNA-positive, and Ki67positive hepatocytes) of WT and Ssu72Dhep livers did not significantly differ. However, in 5-week-old Ssu72Dhep livers where the transcript profiles of cell cycle regulatory genes were altered, a drastic increase of the population of BrdU-positive, PCNA-positive, and Ki67-positive hepatocytes was observed compared with the same-aged WT livers. Moreover, the ratio of liver weight to body weight in Ssu72Dhep mice was slightly increased compared to the WT mice (Supporting Fig. S9D). This observation suggests that loss of Ssu72 function aberrantly activates the proliferation potential of nonproliferating hepatocytes. Depletion of Ssu72 Promotes Endoreplication Through G2/M Arrest. To further determine the molecular mechanisms that lead to the aberrant mononucleated polyploid hepatocyte in Ssu72Dhep, we compared the cell cycle profiles between WT and Ssu72Dhep livers by monitoring PCNA expression (an S phase marker), PCNA and phospho-H3S10 double expression (a G2 phase marker), and phospho-H3S10 expression (a marker of mitosis) (Fig. 6A,B). No significant differences were observed between the phospho-H3S10-positive proportions in the WT and Ssu72Dhep livers before weaning (3 weeks old), although the population of PCNA-positive nuclei was slightly higher in Ssu72Dhep livers. As expected, the overall cell cycle progression in WT livers sharply decreased after weaning (5 weeks old). However, 5-weekold Ssu72Dhep livers showed a marked recovery of cell cycle progression: the populations of PCNA-positive and PCNA plus phospho-H3S10–positive hepatocytes sharply increased, whereas these populations were attenuated in the same-aged WT livers. We further compared cell cycle progression between WT and Ssu72-depleted hepatocytes using the fluorescent ubiquitination–based cell cycle indicator system, which visualizes the cell cycle by green and red fluorescence, by tail vein injection of recombinant adenovirus mKO2cdt1 (Ad-G1) and mAG-Geminin (Ad-G2/M).23 Interestingly, Ssu72-depleted hepatocytes significantly prolonged the induction of green fluorescence, but not red fluorescence, compared to the control hepatocytes (Fig. 6C),


indicating that the depletion of Ssu72 arrests the hepatocyte cell cycle at G2 phase and/or delays the G2 to mitosis transition. We next compared the expression profiles of cell cycle marker proteins between the WT and Ssu72depleted hepatocytes (Fig. 6D). Phosphorylation of Cdk1 at tyrosine 15 residue (Cdk1Y15) decreases as the cells proceed through G2/M phases.24 The abundance of Cyclin A normally increases from S to G2 and then sharply decreases during mitosis. The level of Mcm proteins including Mcm6 decreases as the cells pass through mitosis for proliferation.24 In WT hepatocytes, the levels of phosphorylated Cdk1Y15, Cyclin A, and Mcm6 gradually increased, peaked at 48 hours, and then decreased at 60 hours poststimulation with epidermal growth factor. By contrast, the levels of these cell cycle markers in Ssu72-depleted hepatocytes were significantly maintained at even 60 hours poststimulation (Fig. 6D). Next, to determine whether Ssu72-deleted hepatocytes are able to bypass G2/M arrest and enter into a new round of the cell cycle, we performed liver tissue labeling using two thymidine analogues (chlorodeoxyuridine [CldU] and iododeoxyuridine [IdU]), as recently reported.24 Importantly, a number of Ssu72-depleted hepatocytes that have already progressed through S phase (CldU1, green) clearly entered into a new round of replicative cell cycle (CldU1 IdU1, yellow). Furthermore, we observed that the population of mononucleated polyploid hepatocytes (4c, 44.2%; 8c, 23.5%) was much greater than that of mononucleated diploid hepatocytes (2c, 32.3%) in the Ssu72Dhepmice (Fig. 6E,F). In addition, the abundance of Ki67-positive hepatocytes in the polyploid population (8c) was significantly increased in the Ssu72Dhep mice compared to the same-aged WT mice (Supporting Fig. S10). These data suggest that the aberrant polyploid hepatocytes generated by Ssu72 depletion are able to reenter the cell cycle despite G2/M arrest.

Discussion In liver development, hepatocytes have developmentally programmed polyploidy to regulate hepatic homeostasis and adapt to stress.10,25 In general, this scheduled process generates the successive appearance of binucleated polyploid hepatocytes through incomplete cytokinesis. On the other hand, the pronounced increase in the population of polyploid hepatocytes with a nucleus (mononucleated polyploid hepatocytes) is associated with hepatic damage or liver injury, as well as regeneration after partial hepatectomy.6,24 Although some correlation between polyploidization and various hepatic conditions is tightly regulated, little is known about the mechanisms


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Fig. 6. Loss of Ssu72 results in an altered cell cycle and leads to endoreplication. (A) Double immunofluorescence staining for PCNA (red) and phospho-H3S10 (green) expression and nuclei (blue) in the liver sections of WT and Ssu72Dhep mice at 3 weeks and 5 weeks of age. Insets are enlarged images of the double-positive staining. Original magnification 3400; inset magnification 3630; scale bars 5 20 lm. (B) Quantification of positively stained hepatocytes in liver sections. PCNA (red), a marker of the S phase; phospho-H3S10 (green), a marker of mitosis; PCNA and phospho-H3S10 double-positive (blue), a marker of the G2 phase. Results are presented as means 6 standard error of the means (n 5 4 mice per genotype). (C) Time-lapse imaging of fluorescent ubiquitination–based cell cycle indicator from primary hepatocytes in WT and Ssu72Dhep mice at 5 weeks of age. G1 phase cells express mKO2-Cdt1 (Ad-G1, red), S phase cells show yellow color due to coexpression with mKO2-cdt1 and mAG-Geminin, and cells in G2/M phase express only mAG-Geminin (Ad-G2/M, green). Original magnification 3200; scale bars 5 20 lm. Results are representative of three independent experiments. (D) Experimental scheme of hepatocytes with epidermal growth factor (upper panel). Expression of the S-related and G2/M-related marker proteins was determined by immunoblotting analysis using hepatocytes isolated from 5-week-old WT and Ssu72Dhep mice after epidermal growth factor stimulation as indicated (lower panel). (E) Schematic representation of liver tissue labeling experiment with thymidine analogues (upper panel). Representative images of CldU (green) and IdU (red) staining from liver sections in Ssu72Dhep mice at 5 weeks of age. Arrowheads indicate double-labeled nuclei (yellow, lower panel). Original magnification 3200; scale bars 5 20 lm. (F) Quantification of mononucleated hepatocytes with nuclei double labeling in Ssu72Dhep liver (n 5 5 mice per genotype). Abbreviations: EGF, epidermal growth factor; IdU, iododeoxyuridine.

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Fig. 7. Schematic model for the role of Ssu72 in regulating hepatic homeostasis between chromosome ploidization and liver function. A detailed discussion of this model is provided in the text.

leading to the genesis of polyploid hepatocytes. In the present study, Ssu72 was found to play a critical role in maintaining hepatic chromosome polyploidization by deregulating the cell cycle. Depletion of Ssu72 induced the reinitiation of DNA replication by triggering exit from the quiescence stage and entry into the S phase after weaning. Therefore, Ssu72 depletion aberrantly activated the DNA replication cycle by overriding the R-point, which arrests cells in late G1 phase until cells received a growth signal (e.g., from a growth factor) to proceed to S phase. Ssu72 depletion then excessively generated mononucleated polyploid hepatocytes through endoreplication cycles (Fig. 7), contributing to the development of the pathogenic processes in the liver. Depletion of Ssu72 led to extensive liver diseases, including necrosis, fatty change, fibrosis, steatosis, and steatohepatitis, in the mice tested. However, we were unable to observe the development of HCC in adult Ssu72-depleted mice at around 1 year of age. Interestingly, increase of aberrant chromosome polyploidy in Ssu72-depleted liver was similar to polyploidization in nonalcoholic fatty liver disease24 and in regenerative liver.4 Although aberrant chromosome polyploidy may be a sufficient risk factor for the development of fibrosis, steatosis, and steatohepatitis, but not HCC, it also may be an efficient defense mechanism to cope with both physiological and pathological signals. Nevertheless, Ssu72Dhep mice showed persistent elevation of ALT levels up to 1 year of observation and high incidences of liver disease

manifestations of fibrosis, steatosis, and steatohepatitis (Fig. 3). Therefore, development of liver diseases by loss of Ssu72 function may be due to persistent liver damage and activated immune response. Supporting the above notion, persistent liver damage caused by infection with hepatitis B or C virus induces a wide range of genetic alterations, as well as chromosome instability, and ultimately causes HCC.26,27 It should be noted, therefore, that we do not exclude the possibility that the Ssu72depleted mice would eventually develop HCC at later periods, and further studies will elucidate the precise mechanism linking Ssu72 functions to liver pathology. It is noteworthy that a negative correlation exists between mitotic index in the liver and the level of hepatocyte polyploidization.9 Interestingly, our recent study revealed that the phosphatase activity of Ssu72 is maintained from the G1 phase to the G2 phase of the cell cycle but sharply declines during mitosis14 and that the Aurora B–mediated phosphorylation of Ssu72 causes drastic down-regulation of its phosphatase activity, while triggering the ubiquitin-dependent degradation of Ssu72 during late G2 phase and early mitosis.14 In relation to the above notion, Aurora B is overexpressed in a number of HCC cell lines,28 implying that deregulation of the Ssu72 signaling pathway may be a driver of hepatocarcinogenesis. In the present study, the phosphatase activity of Ssu72 was found to be required for monitoring the activation of unnecessary DNA endoreplication processes in hepatocytes and for preventing the


development of liver diseases. Therefore, the phosphatase activity of Ssu72 should be accurately regulated to maintain the normal polyploidy of hepatocytes. Ssu72 is essential for integrating hepatic chromosome ploidy; however, the regulation of hepatic polyploidy by Ssu72 seems to occur in a tissue-specific manner. Interestingly, Ssu72-depleted primary MEFs exhibited the same level of diploidy as WT MEFs. Although we have not yet determined the nature of the molecular mechanism responsible for maintaining the liver-specific regulation of chromosome polyploidization by Ssu72 phosphatase, it is possible that polyploidy facilitates the expression of many genes responsible for polyploidization and that the gene products provide tissue specificity. There also exists the possibility of tissue specificity to discriminate the substrates that can be targeted by Ssu72 phosphatase. Alternatively, potential tissue-specific modification states may lead to diverse functions of Ssu72, whereby different combinations of modifications direct the sequential requirements for Ssu72 phosphatase. To summarize, this study demonstrated that Ssu72 serves to maintain hepatic chromosome polyploidization and liver function. In addition, our findings suggest that regulation of the expression or modification of Ssu72 protein might provide a means of characterizing physiological and pathological liver development. Acknowledgment: We thank the Research Core Facility, Samsung Biomedical Research Institute, for supplying equipment and providing technical assistance. This study was supported by grants from the National Research Foundation funded by the Korean government (2014R1A2A1A10050775 and 2011-0030043) and the National Cancer Center, Republic of Korea (NCC1410673).

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Author names in bold designate shared co-first authorship.

Supporting Information Additional Supporting Information may be found at onlinelibrary.wiley.com/doi/10.1002/hep.28281/suppinfo.