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Activation of Inactive Hepatocytes through Histone Acetylation

A Mechanism for Functional Compensation after Massive Loss of Hepatocytes
  • Yujun Shi
    Affiliations
    Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, China
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  • Huaiqiang Sun
    Affiliations
    Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, China
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  • Ji Bao
    Affiliations
    Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, China
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  • Ping Zhou
    Affiliations
    Department of Pathology, West China Hospital, Sichuan University, Chengdu, China
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  • Jie Zhang
    Affiliations
    Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, China
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  • Li Li
    Affiliations
    Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, China
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  • Hong Bu
    Correspondence
    Address reprint requests to Hong Bu, M.D., Ph.D., Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University. 37 Guoxue Rd., Chengdu, Sichuan, China, 610041
    Affiliations
    Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, China

    Department of Pathology, West China Hospital, Sichuan University, Chengdu, China
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Open ArchivePublished:July 15, 2011DOI:https://doi.org/10.1016/j.ajpath.2011.05.029
      The mechanisms by which hepatic function is maintained after extensive parenchymal loss are unclear. In this study, we propose a novel concept of “functional heterogeneity” of hepatocytes based on their different expression of acetylated histones, the markers of active gene transcription, to explain the powerful compensatory capability of the liver. In the healthy human liver, only a fraction of the hepatocytes were marked by acetylated histones (ac-H2AK5, ac-H2BK5, ac-H3K9, ac-H3K14, ac-H3K27, and ac-H3K9/14). With the progression of cirrhosis, the ratio of the positive cells was gradually elevated, accompanied by the gradual exhaustion of the negative cells. By examining the global transcriptome of the mouse hepatocytes, we observed that the primed genes in the positive cells were much more numerous than those in negative cells. In a 70% hepatectomized mouse, the remnant hepatocytes were extensively activated, and the liver function was well maintained even when regeneration was severely inhibited. The functional compensation was absolutely dependent on the elevated expression of acetyl-histones. Additionally, when liver regeneration was blocked, the metabolism-related genes seemed to be preferentially transcribed. In conclusion, we demonstrate that normally, part of the active hepatocytes are competent for routine physiological requirements. The inactive hepatocytes, delicately regulated by acetyl-histones, act as a functional reservoir for future activation to restore the liver function after massive parenchymal loss.
      The liver has a unique regenerative capacity that restores its mass and function after diverse injuries, including a 70% to 95% partial hepatectomy (PH) in rodents and humans, by the proliferation of differentiated hepatocytes.
      • Fausto N.
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      Marginal hepatectomy in the rat: from anatomy to surgery.
      Liver regeneration is commonly considered to be the adaptive process by which liver structure and function are recovered. However, in some cases, rodents can survive for an extended period of time after a 70% PH even when liver regeneration is chemically disrupted.
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      Cyclin D1 is up-regulated in hepatocytes in vivo following cell-cycle block induced by retrorsine.
      Similarly, despite the on-going decrement of hepatocytes, patients suffering from chronic cirrhosis can keep their liver function normal for many years.
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      Comparison and improvement of MELD and Child-Pugh score accuracies for the prediction of 6-month mortality in cirrhotic patients.
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      Noninvasive quantitative testing of liver function using ultrasonography in patients with cirrhosis.
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      Natural history of decompensated hepatitis C virus-related cirrhosis A study of 200 patients.
      This evidence suggests that the remnant liver can compensate for the initial loss of hepatic function independent of cell mass recovery. The powerful compensatory capability of the liver raises an interesting question: why does the body need so many hepatocytes when a minority of the liver mass seems competent? We hypothesize that only a subset of hepatocytes is functionally active at any given time, or each individual hepatocyte is only partially active. The subset of the inactive or the incompletely activated hepatocytes, therefore, acts as a functional reservoir that can be invoked for functional compensation. Molecular studies of gene cascades have provided profound insights into the signaling pathways that are activated in the regenerating liver.
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      A common set of immediate-early response genes in liver regeneration and hyperplasia.
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      Liver regeneration: from myth to mechanism.
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      RNA interference against hepatic epidermal growth factor receptor has suppressive effects on liver regeneration in rats.
      • Yang X.
      • Guo M.
      • Wan Y.J.
      Deregulation of growth factor, circadian clock, and cell cycle signaling in regenerating hepatocyte RXRalpha-deficient mouse livers.
      However, just how the liver maintains its function during the process of liver regeneration remains unclear.
      • Taub R.
      Liver regeneration: from myth to mechanism.
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      • Ciliberto G.
      • Taub R.
      Interleukin-6-induced Stat3 and AP-1 amplify hepatocyte nuclear factor 1-mediated transactivation of hepatic genes, an adaptive response to liver injury.
      Histone acetylation serves as a key modulator of chromatin structure and gene transcription.
      • Lo W.S.
      • Henry K.W.
      • Schwartz M.F.
      • Berger S.L.
      Histone modification patterns during gene activation.
      • Choi J.K.
      • Howe L.J.
      Histone acetylation: truth of consequences?.
      Histone acetylation, catalyzed by the histone acetyltransferases (HATs) to add an acetyl group to the lysine residuals (Lys or K), increases the accessibility of transcription factors to their target genes to prime transcription and elongation.
      • Racey L.A.
      • Byvoet P.
      Histone acetyltransferase in chromatin Evidence for in vitro enzymatic transfer of acetate from acetyl-coenzyme A to histones.
      • Khan S.N.
      • Khan A.U.
      Role of histone acetylation in cell physiology and diseases: an update.
      Conversely, histone deacetylation, catalyzed by the histone deacetylases (HDACs) to remove the acetyl group from the lysine residuals, leads to chromatin compaction and results in transcriptional repression.
      • Lo W.S.
      • Henry K.W.
      • Schwartz M.F.
      • Berger S.L.
      Histone modification patterns during gene activation.
      HATs and HDACs coordinate to regulate gene expression and control numerous pathophysiological processes.
      • Wang Z.
      • Zang C.
      • Cui K.
      • Schones D.E.
      • Barski A.
      • Peng W.
      • Zhao K.
      Genome-wide mapping of HATs and HDACs reveals distinct functions in active and inactive genes.
      • Glozak M.A.
      • Seto E.
      Acetylation/deacetylation modulates the stability of DNA replication licensing factor Cdt1.
      • Johnsson A.E.
      • Wright A.P.
      The role of specific HAT-HDAC interactions in transcriptional elongation.
      In the regenerating liver, histone acetylation is correlated with RNA synthesis and DNA replication.
      • Pogo B.G.
      • Pogo A.O.
      • Allfrey V.G.
      • Mirsky A.E.
      Changing patterns of histone acetylation and RNA synthesis in regeneration of the liver.
      • Pogo B.G.
      • Pogo A.O.
      • Allfrey V.G.
      Histone acetylation and RNA synthesis in rat liver regeneration.
      Enhanced histone acetylation promotes the transcription of many genes, whose products subsequently stimulate mitosis of the remnant hepatocytes.
      • Latasa M.U.
      • Boukaba A.
      • García-Trevijano E.R.
      • Torres L.
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      • Lu S.C.
      • López-Rodas G.
      • Franco L.
      • Mato J.M.
      • Avila M.A.
      Hepatocyte growth factor induces MAT2A expression and histone acetylation in rat hepatocytes: role in liver regeneration.
      • Weiss G.
      • Puschendorf B.
      The maximum of the histone acetyltransferase activity precedes DNA-synthesis in regenerating rat liver.
      The role of histone acetylation in liver regeneration has been extensively investigated; however, its role in maintaining and compensating the liver function is still unclear.
      As a marker of active transcription,
      • Lo W.S.
      • Henry K.W.
      • Schwartz M.F.
      • Berger S.L.
      Histone modification patterns during gene activation.
      the presence of acetylated histones might represent the functional status of hepatocytes. In this report, we examined the different expression levels of acetyl-histones in liver specimens from healthy humans and patients suffering from early to end-stage cirrhosis, to investigate the levels of histone acetylation during normal, compensatory, or decompensatory stages. We established a 70% PH mouse model, and the liver regeneration was severely inhibited by retrorsine pretreatment,
      • Laconi S.
      • Doratiotto S.
      • Montisci S.
      • Pani P.
      • Laconi E.
      Repopulation by endogenous hepatocytes does not reconstitute liver mass in rats treated with retrorsine.
      • Pitzalis S.
      • Doratiotto S.
      • Greco M.
      • Montisci S.
      • Pasciu D.
      • Porcu G.
      • Pani P.
      • Laconi S.
      • Laconi E.
      Cyclin D1 is up-regulated in hepatocytes in vivo following cell-cycle block induced by retrorsine.
      • Laconi S.
      • Curreli F.
      • Diana S.
      • Pasciu D.
      • De Filippo G.
      • Sarma D.S.
      • Pani P.
      • Laconi E.
      Liver regeneration in response to partial hepatectomy in rats treated with retrorsine: a kinetic study.
      • Gordon G.J.
      • Coleman W.B.
      • Hixson D.C.
      • Grisham J.W.
      Liver regeneration in rats with retrorsine-induced hepatocellular injury proceeds through a novel cellular response.
      • Picard C.
      • Lambotte L.
      • Starkel P.
      • Sempoux C.
      • Saliez A.
      • Van Den Berge V.
      • de Saeger C.
      • Horsmans Y.
      Retrorsine: a kinetic study of its influence on rat liver regeneration in the portal branch ligation model.
      • Avril A.
      • Pichard V.
      • Bralet M.P.
      • Ferry N.
      Mature hepatocytes are the source of small hepatocyte-like progenitor cells in the retrorsine model of liver injury.
      to investigate the compensatory mechanisms of existing hepatocytes in the absence of proliferation.
      We demonstrate for the first time that in healthy liver, only part of hepatocytes are positively stained by acetyl-histones; however, in developing cirrhotic liver, the ratio of the positive hepatocytes is gradually elevated, accompanied by the gradual exhaustion of the negative cells. In intact mouse liver, the hepatocytes could also be divided into the acetyl-histone–positive and -negative subgroups. The positive cells possess much more active transcription than the negative cells. Therefore, our data indicate a novel concept of “functional heterogeneity” of the hepatocytes based on their different expression of acetyl-histones. As an active marker, histone acetylation also participates in the regulation of functional compensation after massive parenchymal lose.

      Materials and Methods

      Immunochemistry of Human Livers

      Human liver tissues were obtained from three healthy living liver transplant donors (two males and one female; the median age was 36 years and ranged from 29 to 44 years), whose livers were partial hepatectomized for living donor liver transplantation, and the tissue specimens were immediately subjected to quality examination, 10 patients (seven males andthree females; the median age was 56 years and ranged from 33 to 72 years) suffering from early-stage cirrhosis (as determined by ultrasonography and biopsy) with a Child-Pugh score of A, and 10 patients (nine males and one female; the median age was 53 years and ranged from 28 to 66 years) with end-stage cirrhosis with a Child-Pugh score of C, whose livers were removed before liver transplantation. The liver sections were stained with antibodies against ac-H2AK5 (acetylation of the fifth lysine residual at the N terminal) (ab45152), ac-H2BK5 (ab1759), and ac-H3K27 (ab4729), from Abcam, Cambridge, UK; ac-H3K9 (#1328-1) and ac-H3K14 (#1921-1), from Epitomics, Burlingame, CA, and ac-H3K9/14 (#06-599) from Millipore, Billerica, MA. Three pathologists independently reviewed all of the slides. For each of the patients, 10 high-power fields (×200) were randomly selected; the positive and negative hepatocytes were counted, respectively, and their ratio was calculated. All of the patients were treated at the West China Hospital, Sichuan University, and their medical records were reviewed to determine their clinical presentation.

      Animals and Treatments

      Six-week-old C57BL/6 male mice were used for this study. The mice were maintained in alternating 12-hour light/dark cycles with regular chow. Animal procedures and care were conducted in accordance with the institutional guidelines in compliance with national and international laws and policies.
      The mice were randomized into retrorsine-treated (Sigma Chemical Co., St. Louis, MO) and vehicle-treated groups at the onset of the experiment. The mice received two treatments of retrorsine [30 mg/kg intraperitoneal injection (i.p.)] 2 weeks apart, as previously described.
      • Gordon G.J.
      • Coleman W.B.
      • Hixson D.C.
      • Grisham J.W.
      Liver regeneration in rats with retrorsine-induced hepatocellular injury proceeds through a novel cellular response.
      Five weeks after the second injection, the mice were randomized to receive hepatectomy, anacardic acid
      • Kim M.K.
      • Shin J.M.
      • Eun H.C.
      • Chung J.H.
      The role of p300 histone acetyltransferase in UV-induced histone modifications and MMP-1 gene transcription.
      • Eliseeva E.D.
      • Valkov V.
      • Jung M.
      • Jung M.O.
      Characterization of novel inhibitors of histone acetyltransferases.
      [AA, a product of Merck (Whitehouse Station, NJ), diluted in glycerol at 1 mg/mL, 10 mg/kg i.p.] injection, or both hepatectomy and acid injection (n = 13 in each group). The mice were fasted overnight but allowed free access to water before treatment. Administration of isoflurane inhalation anesthesia and surgical PH (70%) were performed as previously described.
      • Boyce S.
      • Harrison D.
      A detailed methodology of partial hepatectomy in the mouse.
      Three mice in each group were sacrificed to harvest liver tissue and to isolate hepatocytes 48 hours after treatment. A single dose of 5-bromo-2′-deoxyuridine (BrdU) (Sigma) was administered (50 mg/kg i.p.) 30 minutes before sacrifice. The body weight and liver weight were recorded. The liver tissues were subjected to DNA quantification, paraffin-embedded sections for pathological examination, and BrdU immunohistochemistry. The serum was used for alanine transaminase (ALT) and glutamic-oxaloacetic transaminase (AST) measurement. Hepatocytes were isolated by a two-step perfusion method as previously described.
      • Seglen P.
      Preparation of isolated rat liver cells.
      Another three mice in each group were subjected to the indocyanine green (ICG) 15-minute retention (R15) test at 48 hours. Briefly, ICG (0.5 mg/kg) was administered via the femoral vein. Samples of venous blood (0.1 mL) were taken before and 15 minutes after injection via the contralateral femoral vein. Each sample was diluted to one-fourth of the concentration with saline, and the specimens were analyzed for ICG concentration in a spectrophotometer at 805 nm.
      • Lin K.J.
      • Liao C.H.
      • Hsiao I.T.
      • Yen T.C.
      • Chen T.C.
      • Jan Y.Y.
      • Chen M.F.
      • Yeh T.S.
      Improved hepatocyte function of future liver remnant of cirrhotic rats after portal vein ligation: a bonus other than volume shifting.
      The remaining seven mice in each group were analyzed for survival rates, which were expressed using Kaplan-Meier curves. In addition, all of the surviving animals were subjected to the ICG-R15 test at the end of experiment.

      Hepatocyte Sorting

      Hepatocytes isolated from mice were suspended in phosphate buffered saline (PBS) and fixed by the addition of paraformaldehyde at a final concentration of 0.5% for 30 minutes. The cells were then washed and resuspended in PBS with 0.1% Triton X-100 (Sigma) and incubated for 3 minutes. The cell suspension was then centrifuged and resuspended in 0.2 mL of PBS with 2 μg of anti–ac-H2AK5 antibody, and the mixture was incubated for 1 hour at room temperature. Following incubation, the cells were centrifuged, resuspended in 0.2 mL of PBS with 0.1% Triton X-100 and 2 μg of fluorescein isothiocyanate–conjugated goat anti-rabbit IgG, incubated for 30 minutes in the dark, and again washed in 1 mL of 0.1% Triton X-100 in PBS. Immediately before flow cytometer analysis, the cell suspensions were filtered using a 37-μm nylon monofilament mesh filter. The fluorescein isothiocyanate–positive and -negative cells were sorted using a Beckman flow cytometer (Beckman Coulter, Brea, CA).
      • Bauer K.D.
      • Clevenger C.V.
      • Endow R.K.
      • Murad T.
      • Epstein A.L.
      • Scarpelli D.G.
      Simultaneous nuclear antigen and DNA content quantitation using paraffin-embedded colonic tissue and multiparameter flow cytometry.
      • Kurki P.
      • Ogata K.
      • Tan E.M.
      Monoclonal antibodies to proliferating cell nuclear antigen (PCNA)/cyclin as probes for proliferating cells by immunofluorescence microscopy and flow cytometry.

      Immunofluorescence

      The isolated hepatocytes were fixed with a methanol/acetic acid (3:1) mixture and were dropped onto poly(l-lysine)-coated slides for immunofluorescence staining. Antibodies against ac-H2AK5, ac-H2BK5, ac-H3K9, ac-H3K14, ac-H3K27, and ac-H3K9/14 were applied.

      Immunoblot Analysis

      Hepatocytic nuclear extract was prepared as previously described.
      • Choudhury M.
      • Shukla S.D.
      Surrogate alcohols and their metabolites modify histone H3 acetylation: involvement of histone acetyl transferase and histone deacetylase.
      The nuclear proteins were analyzed by SDS-polyacrylamide gel electrophoresis with antibodies specific to ac-H2AK5, ac-H2BK5, ac-H3K9, ac-H3K14, ac-H3K27, and ac-H3K9/14. The amount of total H3 (sc-10809; Santa Cruz Biotechnology, Santa Cruz, CA) was used as the loading control. Protein bands were detected by an enhanced chemiluminescent reaction.

      Measurement of HAT Activity

      HAT activity in the nuclear extracts was measured using a HAT activity assay kit (Millipore) according to the manufacturer's protocol and as previously described.
      • Park P.H.
      • Lim R.W.
      • Shukla S.D.
      Involvement of histone acetyltransferase (HAT) in ethanol-induced acetylation of histone H3 in hepatocytes: potential mechanism for gene expression.

      ChIP-on-Chip and RT-PCR

      To facilitate the antibody binding to nuclear antigens for flow cytometric sorting, cells were fixed with paraformaldehyde, and the cell membrane was perforated with Triton X-100. The mRNA purified from the sorted cells was severely degraded and would be no longer suitable for subsequent microarray analysis. Chromatin immunoprecipitation (ChIP)-on-chip analysis offers a rational strategy to reveal the global transcriptional activity because the specimen for the ChIP reaction is DNA. The binding of RNA polymerase II (Pol II) to the DNA sequence of target genes always indicates the priming and elongation of transcription.
      • Baugh L.R.
      • Demodena J.
      • Sternberg P.W.
      RNA Pol II accumulates at promoters of growth genes during developmental arrest.
      • Muse G.W.
      • Gilchrist D.A.
      • Nechaev S.
      • Shah R.
      • Parker J.S.
      • Grissom S.F.
      • Zeitlinger J.
      • Adelman K.
      RNA polymerase is poised for activation across the genome.
      • Zeitlinger J.
      • Stark A.
      • Kellis M.
      • Hong J.W.
      • Nechaev S.
      • Adelman K.
      • Levine M.
      • Young R.A.
      RNA polymerase stalling at developmental control genes in the Drosophila melanogaster embryo.
      Using ChIP, the Pol II–bound genes are coprecipitated with anti-Pol II antibody. The precipitated genes could be further analyzed by microarray (chip), thus to obtain the global transcriptome profile of the cells.
      • Tummala P.
      • Mali R.S.
      • Guzman E.
      • Zhang X.
      • Mitton K.P.
      Temporal ChIP-on-Chip of RNA-Polymerase-II to detect novel gene activation events during photoreceptor maturation.
      • Sandoval J.
      • Rodríguez J.L.
      • Tur G.
      • Serviddio G.
      • Pereda J.
      • Boukaba A.
      • Sastre J.
      • Torres L.
      • Franco L.
      • López-Rodas G.
      RNAPol-ChIP: a novel application of chromatin immunoprecipitation to the analysis of real-time gene transcription.
      The sorted cells from three mice were mixed, and DNA was purified and immunoprecipitated with Pol II antibody (Millipore). ChIP assay was performed using an EZ-ChIP Kit (Millipore). The ChIP DNA was amplified and then hybridized to a Roche-NimbleGen MM8 RefSeq mouse promoter chip (C4222-00-01), and all of the hybridizations were repeated two times. The hybridized chips were scanned, and the results were analyzed with GenePix Pro 6.0 software (Molecular Devices, Sunnyvale, CA); genes with a mean peak false discovery rate of ≤0.2 were regarded as being bound by Pol II.
      • Ji H.
      • Jiang H.
      • Ma W.
      • Johnson D.S.
      • Myers R.M.
      • Wong W.H.
      An integrated software system for analyzing ChIP-chip and ChIP-seq data.
      We then performed reverse transcription–polymerase chain reaction (RT-PCR) to partially verify the ChIP-on-chip results. Seven genes important in metabolic process were selected for analysis (see Table 1). Total RNA was subjected to RT-PCR using a PrimeScript One Step RT-PCR Kit (Takara, Dalian, China); the PCR products were electrophoresed in agarose gel containing ethidium bromide for visualization.
      Table 1The Selected Metabolism-Related Genes and the Primers Used in RT-PCR
      GeneFull namePrimersProduct size
      Acsm1Acyl-CoA synthetase medium-chain family memberSense5′-CAGGATGGATTCTGGCTAC-3′141 bp
      NM_054094Antisense5′-CAAGGCACTGAGTGATGG-3′
      Atp5lATP synthase, H+ transporting, mitochondrial F0Sense5′-CCCTGCTGAAATCCCTAC-3′186 bp
      NM_013795Antisense5′-CAAACATCATAGCCAACAAT-3′
      SerhlSerine hydrolase proteinSense5′-TTACATGGCTATGGATTTCG-3′100 bp
      NM_023475Antisense5′-CACCCTTCGGACCTCACT-3′
      Hk2Hexokinase 2Sense5′-GGGACAGTCTTGCGAATA-3′160 bp
      NM_013820Antisense5′-CGTTCCATACTGCTCCTC-3′
      SdhdSuccinate dehydrogenase complex, subunit DSense5′-TGCTGGGCTTTGCTACTT-3′134 bp
      NM_025848Antisense5′-AGGTGAACGGCATTGGTA-3′
      Gykl1Glycerol kinase-like 1Sense5′-GTCTATTATGCCCTGGAA-3′147 bp
      NM_010293Antisense5′-TGGGACGAAGTAACAACC-3′
      Ptdss1Phosphatidylserine synthase 1Sense5′-TACACGAGAAGCGGACAT-3′141 bp
      NM_008959Antisense5′-CCATAACTACGGATCAACAA-3′
      GAPDHGlyceraldehyde-3-phosphate dehydrogenaseSense5′-AAAATGGTGAAGGTCGGTGTGAACG-3′257 bp
       BC_145810Antisense5′-CATTTGATGTTAGTGGGGTCTCG-3′

      Statistical Analysis

      Data were expressed as the mean ± SD. Statistical significance between the groups was determined via one-way analysis of variance using the SPSS 13.0 software (Armonk, New York). The significance of differences in proportions was analyzed using the χ2 test. A P value <0.05 was considered statistically significant.

      Results

      The Proportion of the Acetyl-Histone–Positive Hepatocytes Gradually Increases in Developing Cirrhosis

      We examined the in situ expression levels of acetyl-histones in healthy and cirrhotic human livers. In healthy livers, less than half (43.8% ± 9.9%) of the hepatocytes were positive and irregularly distributed in the parenchyma, either at the portal area or around the central vein (Figure 1, A, B, C, and F). In early-stage cirrhosis, the proportion of positive cells was significantly increased to around three-quarters (70.8% ± 13.4%) (Figure 1, D and F), and in end-stage cirrhosis, nearly all of the nuclei in the regenerative nodules, markedly enlarged with loose chromatin, were strongly positively stained (Figure 1, E and F). Correspondingly, with the progression of cirrhosis, the proportion of the negative hepatocytes was gradually decreased, and these cells were completely exhausted in patients with a Child-Pugh score of C. All of the acetyl-histone markers had the same expression patterns (see Supplemental Figure S1 at http://ajp.amjpathol.org).
      Figure thumbnail gr1
      Figure 1Hepatocytic histone acetylation differs in healthy and cirrhotic human livers. Liver tissues of healthy humans (A), and patients suffering from early (D) or end-stage cirrhosis (E) were subjected to ac-H2AK5 immunohistochemistry (at a magnification of ×200). B and C: The fields that are boxed in (A) show the distribution of the ac-H2AK5–positive cells in the central zone and portal zone, respectively. F: The proportion of positive hepatocytes was recorded based on 10 randomly selected fields at a magnification of ×200. *P < 0.01. The other acetylation markers present a similar manner of expression; for details, see at http://ajp.amjpathol.org.

      The Acetyl-Histone–Positive Hepatocytes Possess Much More Active Transcription

      We isolated hepatocytes from the intact mouse liver and examined the expression of ac-H2AK5, ac-H2BK5, ac-H3K9, ac-H3K14, ac-H3K27, and ac-H3K9/14 using immunofluorescence. Similar to healthy human liver, only part of the hepatocytes were labeled by green fluorescence (Figure 2).
      Figure thumbnail gr2
      Figure 2Histone acetylation markers are positively expressed in part of the hepatocytes. The isolated hepatocytes from the intact mouse were subjected to immunofluorescence staining of ac-H2AK5, ac-H2BK5, ac-H3K9, ac-H3K14, ac-H3K27, and ac-H3K9/14. DAPI was used to stain the nuclei. Scale bar = 50 μm.
      To directly clarify the different transcriptional activity between the acetyl-histone–positive and -negative hepatocytes, we sorted the cells based on their different expression of ac-H2AK5 (Figure 3A). The global transcriptome profiles by ChIP-on-chip analysis showed that in ac-H2AK5–expressing hepatocytes, 479 genes were identified; however, 86 genes were identified in the negative hepatocytes (Figure 3B), and 31 genes overlapped these two groups (for a detailed gene list, see Supplemental Table S1 at http://ajp.amjpathol.org).
      Figure thumbnail gr3
      Figure 3The different transcriptional activity in hepatocytes. A: The ac-H2AK5+ and ac-H2AK5 hepatocytes were sorted by flow cytometer. B: The flow cytometric–sorted hepatocytes were subjected to ChIP-on-chip assay, and the Pol II–bound genes were identified. C: Immunoblots of the histone acetylation markers in ac-H2AK5+ and ac-H2AK5 hepatocytes are shown. The expression of total histone H3 was used as a loading control. FDR, false discovery rate; FS, forward scatter; SS, side scatter.
      It was still needed to determine whether all of the active markers were expressed in the same cells. We examined the expression of each acetyl-histone in ac-H2AK5–positive and -negative cells. As shown in Figure 3C, all of the active markers were simultaneously and robustly expressed in the ac-H2AK5–positive cells.

      Liver Function Is Completely Compensated after Partial Hepatectomy, Independent of Regeneration

      To further confirm that a small part of the liver can compensate the entire function, we pretreated the mice with retrorsine before PH. Forty-eight hours after PH, mitosis was seldom seen in the livers, suggesting that liver regeneration was severely inhibited. On the contrary, many of the hepatocytes had entered mitosis in the mice that received PH alone (Figure 4, A and B). Histologically, no remarkable necrosis was seen in either group, although hepatocytic hypertrophy was prominent in PH + retrorsine mice at day 7 (Figure 4C). Despite the strong inhibition of DNA synthesis in retrorsine-pretreated mice (Figure 4D), their remnant livers were well reconstituted at day 7, and there was no significant difference in liver/body weight ratio between the PH and PH + retrorsine groups (Figure 4, E and F). Additionally, retrorsine administration alone did not lead to cell injury since the increased levels of enzymes seemed only correlated to surgery (Figure 4G). All of the animals in both groups survived the entire experimental period, suggesting that the remaining livers were competent with or without regeneration. We then performed the ICG-R15 test to manifest the retained liver function after operation. Indeed, the ICG retention was increased 48 hours after operation; however, there was no significant difference between the two groups. At day 7, the ICG retention of both groups was decreased to normal level (Figure 4H). These results indicate that the liver function was entirely compensated independent of liver regeneration.
      Figure thumbnail gr4
      Figure 4Retrorsine (Retro) inhibits hepatocyte proliferation but does not interfere with liver reconstitution or lead to direct hepatocyte damage. A: BrdU immunochemistry of the differently treated mice at day 2. B: The average BrdU-positive cells in 10 randomly chosen visual fields per section at a magnification of ×200 at day 2. C: The histology of liver tissue at day 7. D: The genomic DNA content of the livers at day 7. E: The gross view of the reconstitution of the remnant liver at day 7. F: The ratios of liver to body weight at day 7. G: The serum ALT and AST levels at day 2. All of the error bars represent the SD from the mean of three mice assays of an individual experiment. *P < 0.05. H: ICG-R15 test was performed to manifest the liver function at days 2 and 7. *P < 0.05 versus control (Con).

      The Expression of Acetyl-Histones Is Robustly Elevated after PH

      If the acetyl-histone–negative hepatocytes act as a functional reservoir, they will be activated to compensate the initial parenchymal loss. Indeed, after PH, all of the remnant hepatocytes, with or without retrorsine pretreatment, were marked by acetyl-histones (Figure 5A, only the expression of ac-H2AK5 is presented here), and immunoblot analysis also showed that the expression of all of the active markers was robustly elevated (Figure 5B).
      Figure thumbnail gr5
      Figure 5The expression of acetyl-histones is robustly elevated after PH, especially in retrorsine (Retro)-pretreated mice, but is robustly decreased in AA-treated mice. A: The immunofluorescence of ac-H2AK5 in different groups. Scale bar = 50 μm. B: The immunoblots of acetyl-histones in different groups; the expression of total histone H3 was used as a loading control (Con).

      Different Genes Are Activated in Mice Receiving PH With or Without Retrorsine Pretreatment

      DNA extracted from hepatectomized mice with or without retrorsine pretreatment was subjected to ChIP-on-chip assay with Pol II antibody. The global transcriptome profiles indicated that a great number of genes were primed in hepatocytes of both groups of mice: 802 genes in the PH-alone mice and 911 genes in the PH + retrorsine mice (Figure 6A).
      Figure thumbnail gr6
      Figure 6Different genes are activated in mice receiving PH with or without retrorsine pretreatment. A: DNA extracted from hepatectomized mice with or without retrorsine (Retro) pretreatment was subjected to ChIP-on-chip assay with Pol II antibody. The identified genes were then categorized as metabolism-related genes, cell proliferation–related genes, or uncategorized genes. B: Seven selected genes that encode key enzymes involved in metabolism were analyzed using reverse transcription–PCR; GAPDH was used as a loading control (Con).
      Because it has been proved that histone acetylation plays a crucial role in the regulation of RNA synthesis and DNA replication in regenerating liver, we then hypothesized that different genes should be activated in retrorsine-pretreated liver, in which regeneration was severely deprived. We classified the identified genes into three subgroups, the metabolism-related genes, cell proliferation–related genes, and the uncategorized genes, using the online software of Gene Ontology (http://www.geneontology.org and Ref.
      • Huang da W.
      • Sherman B.T.
      • Lempicki R.A.
      Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.
      ). In mice that received PH alone, 28 (3.5%) and 235 (29.3%) genes belonged to the proliferation-related and metabolism-related gene categories, respectively. In mice that received PH + retrorsine, the number was 18 (2.0%, P = 0.057 compared to PH mice) and 334 (36.7%, P = 0.001 compared to PH mice), respectively (Figure 6A). We then performed semiquantitative RT-PCR to verify the ChIP-on-chip results. All of the selected genes that encode key enzymes involved in metabolism were notably up-regulated in both groups, especially in the PH + retrorsine mice (Figure 6B. For a detailed gene list, see Supplemental Table S2 at http://ajp.amjpathol.org).

      Inhibition of Acetyl-Histone Expression Leads to Depressed Liver Function and Increased Animal Mortality

      We have demonstrated that the elevation of histone acetylation seems closely correlated with functional compensation; therefore, inhibition of histone acetylation would be destined to produce a bad outcome. To clarify this hypothesis, we treated the mice with AA to inhibit acetyl-histone expression. As expected, in AA-treated mice, the HAT activity was tremendously inhibited (Figure 7A); as a result, the expression of acetyl-histones was remarkably decreased (PH + Retro + AA, Figure 5), and the metabolism-related genes were notably depressed (Figure 6B). We did not find any AA-related histological damage or increased serum enzyme levels in AA-treated mice (data not shown); however, AA administration did significantly increase the ICG retention (Figure 7B). AA administration alone did not lead to animal death; however, when AA was combined with retrorsine, PH, or both, the mortality was significantly increased (Figure 7C).
      Figure thumbnail gr7
      Figure 7Administration of anacardic acid (AA) leads to HAT inhibition, function deterioration, and increased mortality. A: The relative HAT activity in different groups. B: The ICG retention rate at day 2 (2d; n = 3) and day 7 (7d; all of the animals survived at day 7). C: The survival curve of the different groups. *P < 0.05. Con, control; Retro, retrorsine.

      Discussion

      In this study, we proposed a novel concept of “functional heterogeneity” of the hepatocytes based on their different expression level of acetyl-histones and transcriptional activity. We demonstrated that normally, part of the hepatocytes are activated to satisfy the routine physiological requirement, and the inactive cells act as a functional reservoir for future activation, thus compensating for the initial massive loss of hepatocytes even without proliferation.
      It is generally accepted that liver regeneration is the major compensatory mechanism after massive parenchymal loss.
      • Michalopoulos G.K.
      Liver regeneration.
      • Taub R.
      Liver regeneration: from myth to mechanism.
      However, knowledge from clinical cases and animal experiments has confirmed that both human and rodents can survive on a small part of their liver even without regeneration.
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      Reconstitution of liver mass via cellular hypertrophy in the rat.
      • Laconi S.
      • Doratiotto S.
      • Montisci S.
      • Pani P.
      • Laconi E.
      Repopulation by endogenous hepatocytes does not reconstitute liver mass in rats treated with retrorsine.
      • Pitzalis S.
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      • Greco M.
      • Montisci S.
      • Pasciu D.
      • Porcu G.
      • Pani P.
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      • Laconi E.
      Cyclin D1 is up-regulated in hepatocytes in vivo following cell-cycle block induced by retrorsine.
      • Boursier J.
      • Cesbron E.
      • Tropet A.L.
      • Pilette C.J.
      Comparison and improvement of MELD and Child-Pugh score accuracies for the prediction of 6-month mortality in cirrhotic patients.
      • Yan G.Z.
      • Duan Y.Y.
      • Ruan L.T.
      • Cao T.S.
      • Yuan L.J.
      • Yang Y.L.
      Noninvasive quantitative testing of liver function using ultrasonography in patients with cirrhosis.
      • Planas R.
      • Ballesté B.
      • Alvarez M.A.
      • Rivera M.
      • Montoliu S.
      • Galeras J.A.
      • Santos J.
      • Coll S.
      • Morillas R.M.
      • Solà R.
      Natural history of decompensated hepatitis C virus-related cirrhosis A study of 200 patients.
      It is still unclear why the liver possesses such a powerful compensatory capability.
      Histone acetylation is commonly considered to be a marker of active transcription,
      • Lo W.S.
      • Henry K.W.
      • Schwartz M.F.
      • Berger S.L.
      Histone modification patterns during gene activation.
      • Liang G.
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      • Gonzales F.A.
      • Jones P.A.
      Distinct localization of histone H3 acetylation and H3-K4 methylation to the transcription start sites in the human genome.
      and the different expression of acetylated histones often indicates the transcriptional status of cells. By examining liver tissues, we found that only part of the hepatocytes in healthy human and mouse livers are marked by acetyl-histones. By examining the flow cytometric–sorted hepatocytes, we confirmed that all included acetyl-histone markers are simultaneously and robustly expressed in the ac-H2AK5–positive cells but are weakly expressed in the negative cells. The global transcriptome profiles directly demonstrated that the ac-H2AK5–positive hepatocytes possess active transcription, but to the negative cells, the transcription is robustly depressed. Thus, we can divide the hepatocytes into two subgroups, the active and the inactive subgroups, based on their different expression of ac-H2AK5 or other acetyl-histone markers. Therefore, our findings offer a novel manifestation of the “functional heterogeneity” of hepatocytes, which is completely different from the established concept of the functional heterogeneity of the periportal and perivenous hepatocytes.
      • Jungermann K.
      Functional heterogeneity of periportal and perivenous hepatocytes.
      The active hepatocytes are competent for routine physiological requirement; the inactive cells, then, might act as a functional reservoir. In progressing cirrhosis, the residual cells are extensively activated, accompanied by the gradual exhaustion of the inactive cells. Once the reserve cells are completely exhausted, the liver becomes destined for failure. Therefore, a biopsy to evaluate the proportion of acetyl-histone–positive hepatocytes might offer a potential strategy to reveal hepatic functional reserves. Additionally, the irregular distribution of the active cells in the parenchyma suggests that the cells might be alternately activated, thus to ensure all of the cells obtain the chance to rest and repair the damaged or mutated DNA. The sustained histone acetylation might in turn lead to transcriptional deregulation, which would partly explain why cirrhosis often results in hepatoma.
      • Bai X.
      • Wu L.
      • Liang T.
      • Liu Z.
      • Li J.
      • Li D.
      • Xie H.
      • Yin S.
      • Yu J.
      • Lin Q.
      • Zheng S.
      Overexpression of myocyte enhancer factor 2 and histone hyperacetylation in hepatocellular carcinoma.
      • Borzio M.
      • Trerè D.
      • Borzio F.
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      • Bruno S.
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      • Colloredo G.
      • Leandro G.
      • Oliveri F.
      • Derenzini M.
      Hepatocyte proliferation rate is a powerful parameter for predicting hepatocellular carcinoma development in liver cirrhosis.
      To prove directly that a small part of the liver can function as an intact one, we established a 70% PH mouse model in which liver regeneration was severely deprived by retrorsine pretreatment. Retrorsine is a well-established agent that inhibits hepatocyte proliferation through inhibition of DNA replication and cell cycle arrest.
      • Laconi S.
      • Doratiotto S.
      • Montisci S.
      • Pani P.
      • Laconi E.
      Repopulation by endogenous hepatocytes does not reconstitute liver mass in rats treated with retrorsine.
      • Pitzalis S.
      • Doratiotto S.
      • Greco M.
      • Montisci S.
      • Pasciu D.
      • Porcu G.
      • Pani P.
      • Laconi S.
      • Laconi E.
      Cyclin D1 is up-regulated in hepatocytes in vivo following cell-cycle block induced by retrorsine.
      • Laconi S.
      • Curreli F.
      • Diana S.
      • Pasciu D.
      • De Filippo G.
      • Sarma D.S.
      • Pani P.
      • Laconi E.
      Liver regeneration in response to partial hepatectomy in rats treated with retrorsine: a kinetic study.
      • Gordon G.J.
      • Coleman W.B.
      • Hixson D.C.
      • Grisham J.W.
      Liver regeneration in rats with retrorsine-induced hepatocellular injury proceeds through a novel cellular response.
      • Picard C.
      • Lambotte L.
      • Starkel P.
      • Sempoux C.
      • Saliez A.
      • Van Den Berge V.
      • de Saeger C.
      • Horsmans Y.
      Retrorsine: a kinetic study of its influence on rat liver regeneration in the portal branch ligation model.
      • Avril A.
      • Pichard V.
      • Bralet M.P.
      • Ferry N.
      Mature hepatocytes are the source of small hepatocyte-like progenitor cells in the retrorsine model of liver injury.
      With PH and retrorsine treatment, hepatocyte hypertrophy, accompanied by an enlargement of the mitochondria, Golgi apparatus, and endoplasmic reticulum, indicates that energy metabolism and protein synthesis have been notably enhanced.
      • Nagy P.
      • Teramoto T.
      • Factor V.M.
      • Sanchez A.
      • Schnur J.
      • Paku S.
      • Thorgeirsson S.S.
      Reconstitution of liver mass via cellular hypertrophy in the rat.
      A previous study in rat
      • Nagy P.
      • Teramoto T.
      • Factor V.M.
      • Sanchez A.
      • Schnur J.
      • Paku S.
      • Thorgeirsson S.S.
      Reconstitution of liver mass via cellular hypertrophy in the rat.
      and our experiment in mouse both proved that 7 days after operation, the liver could be well reconstituted to its original weight through hypertrophy in the absence of regeneration. However, in the aspect of gene activation, little is understood regarding the functional compensation in the residual hepatocytes. Our data manifested that all of the residual hepatocytes are transcriptionally activated as indicated by acetyl-histones staining. The transcriptome profile also indicated that a great number of genes are activated. Our results demonstrated that the extensive activation of the remnant hepatocytes is the major pathway to maintain and compensate the liver function. Previous studies revealed that liver regeneration after PH in retrorsine-exposed rats is accomplished through proliferation and differentiation of small hepatocyte-like progenitor cells
      • Avril A.
      • Pichard V.
      • Bralet M.P.
      • Ferry N.
      Mature hepatocytes are the source of small hepatocyte-like progenitor cells in the retrorsine model of liver injury.
      ; however, the immature hepatocytes only begin to appear at 3 days post-PH. Obviously these cells could not be responsible for functional execution, especially during the immediate days post-PH.
      • Best D.H.
      • Coleman W.B.
      Treatment with 2-AAF blocks the small hepatocyte-like progenitor cell response in retrorsine-exposed rats.
      Indeed, we found that a few BrdU-positive cells emerged at day 3 after operation; however, these cells might be the progenitor cells rather than the regenerating hepatocytes since they were much smaller and showed colony-like proliferation.
      In regenerating livers, the hepatocytes have to be responsible for their self-proliferation and functional compensation. It is well known that histone acetylation plays a critical role in regulation of RNA synthesis and DNA replication.
      • Pogo B.G.
      • Pogo A.O.
      • Allfrey V.G.
      • Mirsky A.E.
      Changing patterns of histone acetylation and RNA synthesis in regeneration of the liver.
      • Pogo B.G.
      • Pogo A.O.
      • Allfrey V.G.
      Histone acetylation and RNA synthesis in rat liver regeneration.
      • Latasa M.U.
      • Boukaba A.
      • García-Trevijano E.R.
      • Torres L.
      • Rodríguez J.L.
      • Caballería J.
      • Lu S.C.
      • López-Rodas G.
      • Franco L.
      • Mato J.M.
      • Avila M.A.
      Hepatocyte growth factor induces MAT2A expression and histone acetylation in rat hepatocytes: role in liver regeneration.
      To date, little has been clarified as to how the proliferating hepatocytes could maintain their function.
      • Taub R.
      Liver regeneration: from myth to mechanism.
      • Leu J.I.
      • Crissey M.A.S.
      • Leu J.P.
      • Ciliberto G.
      • Taub R.
      Interleukin-6-induced Stat3 and AP-1 amplify hepatocyte nuclear factor 1-mediated transactivation of hepatic genes, an adaptive response to liver injury.
      Our data indicate that numerous mitosis-related genes as well as metabolism-related genes are activated, suggesting that the remnant hepatocytes can keep a good balance between self-proliferation and functional execution. When liver regeneration is deprived, however, the metabolism-related genes seem to be preferentially transcribed. These results suggest that the cells possess the ability to determine which gene cluster should be preferentially activated, thus to adapt different physiopathological processes.
      To explore whether the elevation of acetyl-histones expression is necessary for functional compensation after PH, mice were treated with AA. AA is one of the most commonly used inhibitors of p300 and p300/CBP-associated factor,
      • Kim M.K.
      • Shin J.M.
      • Eun H.C.
      • Chung J.H.
      The role of p300 histone acetyltransferase in UV-induced histone modifications and MMP-1 gene transcription.
      • Eliseeva E.D.
      • Valkov V.
      • Jung M.
      • Jung M.O.
      Characterization of novel inhibitors of histone acetyltransferases.
      both of which are members of the HAT family.
      • Wang Z.
      • Zang C.
      • Cui K.
      • Schones D.E.
      • Barski A.
      • Peng W.
      • Zhao K.
      Genome-wide mapping of HATs and HDACs reveals distinct functions in active and inactive genes.
      • Yang X.J.
      The diverse superfamily of lysine acetyltransferases and their role in leukemia and other diseases.
      • Balasubramanyam K.
      • Swaminathan V.
      • Ranganathan A.
      • Kundu T.K.
      Small molecule modulators of histone acetyltransferase p300.
      It has been demonstrated that some hepatotoxic chemicals, such as hydralazine derivatives, have an impact on acetylation inhibition.
      • Murata K.
      • Hamada M.
      • Sugimoto K.
      • Nakano T.
      A novel mechanism for drug-induced liver failure: inhibition of histone acetylation by hydralazine derivatives.
      In our experiment, the repression of the acetyl-histones expression, gene transcription, and functional deterioration was subsequent to AA injection. The proper HAT activity is critical to regulate cell cycle,
      • Glozak M.A.
      • Seto E.
      Acetylation/deacetylation modulates the stability of DNA replication licensing factor Cdt1.
      thus, the severe inhibition of HAT might disturb cell proliferation. Indeed, the regeneration of hepatectomized liver was moderately inhibited by AA administration (data not shown); however, it did not directly lead to animal death. We cannot rule out the effect of the systemic toxicity of AA, because all of the emerged data concerning this chemical have been based on in vitro experiments,
      • Logrado L.P.
      • Santos C.O.
      • Romeiro L.A.
      • Costa A.M.
      • Ferreira J.R.
      • Cavalcanti B.C.
      • Manoel de Moraes O.
      • Costa-Lotufo L.V.
      • Pessoa C.
      • Dos Santos M.L.
      Synthesis and cytotoxicity screening of substituted isobenzofuranones designed from anacardic acids.
      • Schultz D.J.
      • Wickramasinghe N.S.
      • Ivanova M.M.
      • Isaacs S.M.
      • Dougherty S.M.
      • Imbert-Fernandez Y.
      • Cunningham A.R.
      • Chen C.
      • Klinge C.M.
      Anacardic acid inhibits estrogen receptor alpha-DNA binding and reduces target gene transcription and breast cancer cell proliferation.
      • Chandregowda V.
      • Kush A.
      • Reddy G.C.
      Synthesis of benzamide derivatives of anacardic acid and their cytotoxic activity.
      and its global toxicity in vivo has yet to be fully explored. Our data indicate that no remarkable histological damage or increased serum enzyme levels were observed in AA-treated mice; it is likely that the deteriorated liver function and increased mortality should be ascribed to HAT inhibition and the subsequent inhibition of histone acetylation and gene activation.
      In summary, we have raised, at least in part, a novel concept of functional heterogeneity of the hepatocytes to explain the powerful compensatory mechanism of the liver via gene transcription. Accordingly, under normal conditions, the hepatocytes lacking acetylated histones are inactive in gene transcription and act as a functional reservoir. These cell are activated to transcription after massive parenchymal loss, thus to maintain liver function, even without the restoration of cell mass.

      Acknowledgments

      We are grateful to Xiangli Yin, Ruina Yang, Jun Wan, and Lijia Cheng for their assistance with this experiment.

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