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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The liver is characterized by sexually dimorphic gene expression translating into sex-specific differences in lipid, drug, steroid hormone, and xenobiotic metabolism, with distinct responses of males and females to environmental challenges. Here, we investigated the role of the Krüppel-associated box (KRAB)-associated protein 1 (KAP1) epigenetic regulator in this process. Liver-specific KAP1 knockout (KO) led to strikingly sexually dimorphic phenotypic disturbances, including male-predominant steatosis and hepatic tumors with up-regulation of protein kinase B and extracellular signal-related kinases 1/2 mitogen-activated protein kinase signaling. This correlated with the sex-specific transcriptional dysregulation of a wide range of metabolic genes, notably those involved in retinol and sex hormone processing as well as in detoxification. Furthermore, chromatin immunoprecipitation followed by deep sequencing indicated that a number of dysregulated genes are direct targets of the KRAB/KAP1 repression system. Those genes include sexually dimorphic cytochrome P 450 Cyp2d9, glutathione S-transferase π, Cyp2a, Cyp2b, and Cyp3a gene clusters. Additionally, we identified a male-restricted KAP1-binding site in the fat-specific protein 27 gene, correlating with its male-predominant up-regulation upon Kap1 deletion, suggesting that the latter might be an important trigger in the development of male-specific hepatosteatosis and secondary tumorigenesis. Conclusion: This work reveals KRAB/KAP1-mediated transcriptional regulation as a central event in metabolic control hormones, drugs, and xenobiotics in the liver and further links disturbances in these processes with hepatic carcinogenesis. (HEPATOLOGY 2012)

Metabolism is orchestrated by complex gene-regulatory networks, and the liver is central to this process. This organ is characterized by sexually dimorphic gene expression, with numerous genes transcribed in a sex-dependent manner.1 In mice, examples of such genes include the male-predominant cytochrome P450 Cyp2d9, which encodes for a testosterone-16-α-hydroxylase inactivating the main male sex hormone and various drugs, and the female-predominant Cyp2a and Cyp2b genes involved in xenobiotic and drug metabolism.2 The sex-specific expression of these and other classes of enzymes results in the differential response of males and females to environmental challenges, with males displaying a higher predisposition than females for liver inflammation, cirrhosis, and tumors subsequent to infections or nonalcoholic steatohepatitis.3, 4

Sexually dimorphic gene expression in the liver is established by the sex-specific action of liver transcription factors.1 One of those, regulator of sex limitation (Rsl), belongs to the Krüppel-associated box zinc finger proteins (KRAB-ZFPs) family of tetrapod-specific transcription repressors, which counts between 300 and 400 members in mice and humans.5 KRAB-ZFPs are characterized by the presence of an N-terminal KRAB domain required for repression and a C-terminal array of DNA-binding zinc fingers (ZFs). Few have been assigned a physiological role in vivo, yet enough data are available to indicate that these proteins display a wide range of biological activities, from the control of imprinting to regulation of metabolic processes in neurons.6, 7

Whereas the role and gene targets of the vast majority of KRAB-ZFPs remain ill defined, how they repress transcription is comparatively well understood. Many KRAB-ZFPs likely bind DNA in a sequence-specific manner through their ZFs and recruit KRAB-associated protein 1 (KAP1; also known as TIF1β, TRIM28, and KRIP-1), which serves as their universal cofactor. KAP1, in turn, acts as a scaffold for a chromatin-remodeling complex comprising the histone methyltransferase SET domain bifurcated 1 (SETDB1), which catalyzes histone 3 lysine 9 trimethylation (H3K9me3), histone deacetylases (HDACs), nuclear remodeling factors, as well as heterochromatin protein 1. The formation of heterochromatin ensues, leading to epigenetic silencing.8

In the mouse, constitutive Kap1 knockout (KO) is early embryonic lethal, correlating with an absence of gastrulation.9 Deleting Kap1 in embryonic stem cells (ESCs) leads to loss of pluripotentiality, imprinting defects, and failure to silence endogenous and some exogenous retroviruses.6, 8, 10 In adult tissues, KAP1 regulates spermatogenesis and affects the management of behavioral stress.8, 11 Furthermore, inbred mice heterozygous for a null mutation in Kap1 were found to exhibit greater variance in body weight than wild-type (WT) littermates and, for some animals, liver steatosis, adipocyte hypertrophy, and impaired glucose tolerance.12

Here, we explored the role of KRAB/KAP1 in liver function. The analysis of the hepato-specific conditional Kap1 KO mouse revealed a sexually dimorphic metabolic syndrome, with male-predominant liver steatosis and tumorigenesis. This correlated with the sex-specific transcriptional dysregulation of genes involved in retinol, xenobiotic, and drug metabolism. The identification of KAP1-binding sites in the mouse liver further revealed that a number of the affected genes are direct KAP1 targets. This study thus unveils the important contribution of KRAB/KAP1-mediated regulation to liver function and metabolism.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Mouse Generation and Phenotyping.

Generation and genotyping of mice with a floxable Kap1 allele (Kap1fl/fl) and the albumin/Cre recombinase (Alb-Cre) mouse strain have been previously described.9, 13 Animals were housed in standard conditions with water and chow or high-fat diet (HFD) provided ad libitum (for details, see Supporting Materials). All animal experiments were approved by the local veterinary office and were carried out in accord with the European Community Council Directive (86/609/EEC) for care and use of laboratory animals. Phenotyping was performed on at least 8 males and females (for details, see Supporting Materials).

Histological Tissue Processing and Analysis.

Hematoxylin and eosine (HE) and Oil Red O (ORO) stainings were performed according to standard procedures. Histopathology evaluation, ORO scoring, and tumor occurrence analysis were done by a board-certified pathologist (F.A.). Hepatocellular lesions were classified as foci of cellular alteration, hepatocellular adenomas, and hepatocellular carcinoma (HCC) according to established criteria.14 For bromodeoxyuridine (BrdU) pulse-labeling, mice received 3 mg/mL of BrdU in drinking water for 6 days and the liver tissue was stained for BrdU (Oxford Biomedical Research, Kidlington, UK). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed with the Apoptag kit (Millipore, Bedford, Massachusetts, USA) according to the manufacturer's instructions.

Small-Scale DNA, RNA, and Protein Analysis.

Genotyping, RNA extraction and reverse-transcription, as well as protein, analysis were performed according to standard procedures as previously described, see Supplemental Methods.11

Microarray Analysis.

Mice (8-10 weeks old) were food-deprived at 07:00 a.m., and liver was isolated at 11:00 a.m. to 2:00 p.m. Total RNA was extracted with the mirVana kit (Ambion Biotechnology, Houston, TX) and treated with DNAse (Ambion), following the manufacturer's instructions. Three RNA pools from 9 mutant and WT male livers were prepared, whereas 3 individual female KO and WT livers were used separately. Microarray analysis was performed as previously described.11 Significantly deregulated genes (P < 0.05 by unpaired t test and fold-change ≥2) were used to interrogate the DAVID bioinformatic database.15

Chromatin Immunoprecipitation Sequencing and Chromatin Immunoprecipitation Polymerase Chain Reaction.

Liver chromatin was performed as previously described, with minor modifications.16 For chromatin immunoprecipitation (ChIP) sequencing, chromatin from 2 male and female Kap1fl/fl and 1 male and female Alb-Cre Kap1fl/fl control were prepared. ChIP with an affinity-purified rabbit polyclonal antibody, kindly provided by Dr. J.F. Rauscher,17 was performed as previously described.18 Details describing sequencing, read alignment, and subsequent analyses are provided in the Supporting Materials.

Statistical Analysis.

Body- and organ-weight measurements as well as plasma biochemistry were analyzed with the two-tailed Student t test or two-way analysis of variance (ANOVA), followed by Bonferroni's post-test. Contingency testing was done with Fisher's exact test. For statistical analysis of high-throughput data, see related paragraphs.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Generation of Hepatocyte-Specific Kap1 KO Mice.

We crossed Kap1fl/fl mice9 in a mixed C57/Bl6-Sv129 background with animals expressing Cre under the control of an Alb promoter.13 We confirmed the liver specificity of Kap1 deletion by locus-specific polymerase chain reaction (PCR), and determined that, in whole liver from mutant mice, KAP1 messenger RNA (mRNA) was reduced by more than 75% and protein was undetectable by western blotting, whereas immunohistochemistry confirmed that its loss was restricted to hepatocytes (Supporting Fig. 1). Histology combined with ORO staining further revealed that 8-10-week-old male Alb-Cre Kap1fl/fl mice, but not their female counterparts, suffered from mild liver steatosis (Supporting Fig. 1). These results suggested that loss of KAP1 perturbed metabolic homeostasis in the liver and pointed to possible sex-specific differences in this process.

Sex-Dependent Phenotypic Consequences of Hepatocyte-Specific Kap1 KO.

Because of this early finding and of known sex-related differences in metabolism and liver function in rodents and other mammals, including humans,1, 19 we followed a phenotyping protocol that separately examined male and female mice (see above). At 8-10 weeks of age, no differences in body weight, food intake, and plasma markers of liver function and lipid metabolism between mutant and WT mice were observed (not illustrated). However, histopathology revealed significant fat accumulation with small vacuoles in all 12 KO male livers, compared to only one third (4 of 12) of controls (Fisher's exact test; P = 0.0013; not shown), whereas females were exempt of this pathology. The steatosis noted in male KO mice was not the result of Cre, because no difference was observed between Alb-Cre Kap1wt/wt and Kap1wt/wt animals (not illustrated).

We then asked whether a metabolic challenge would accentuate the phenotype of male Kap1 KO mice. For this, we fed 10-week-old male and female Alb-Cre Kap1fl/fl and Kap1fl/fl littermates with HFD (60% calories from fat) or chow diet (CD) for 19 weeks. CD-fed Kap1 KO males displayed progressive hepatosteatosis characterized by increased liver weight and fatty change of mild to moderate degree at 6 months of age (Fig. 1B,C; Table 1). In contrast, control littermates exhibited minimal lipid accumulation in the liver, as revealed by ORO staining (Fig. 1B,C). These changes were not accompanied by weight gain and increased food intake and only by a trend to up-regulation of plasma markers of liver function (Fig. 1A,D; Table 1; and data not shown). Interestingly, Kap1 KO males displayed significantly lower plasma free fatty acid (FFA) concentration than littermate controls, suggesting an increased rate of hepatocytic FFA uptake after Kap1 deletion (Table 1).

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Figure 1. Phenotypic analyses of Kap1 KO mice. (A) Body weight of male (n = 9-12; top) and female (n = 10; bottom) mice fed indicated diets. *P < 0.05, as assessed by two-way ANOVA, followed by Bonferroni's post-test. (B) HE staining of liver sample from CD-fed 29-week-old indicated mice. Inlet: ORO staining. Scale bar: 50 μm. (C) Semiquantitative ORO staining score of male (top) and female (bottom) mice fed indicated diets ***P < 0.001, as assessed by two-way ANOVA, followed by Bonferroni's post-test. (D and E) Plasma levels of ALT and lactate dehydrogenase (LDH) (n = 7-9; D) and liver protein levels of proinflammatory cytokines IL-6 and TNF-α (n = 5; E) in 29-week-old female (left) and male (right) mice. Black bars: CD; gray bars: HFD. *P < 0.05, as assessed by two-way ANOVA, followed by Bonferroni's post-test. Error bars represent standard error of the mean.

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Table 1. Plasma Markers of Lipid Metabolism and Liver Function in 29-Week-Old Mice
ParameterFemale KO CD (n = 7)Female WT CD (n = 8)Female KO HFD (n = 8)Female WT HFD (n = 8)Male KO CD (n = 10)Male WT CD (n = 7)Male KO HFD (n = 10)Male WT HFD (n = 9)
  • WT mice and animals bearing liver-specific KAP1 KO were fed chow diet (CD) or HFD for 19 weeks and fasted for 4 hours prior to experiment.

  • Abbreviations: BW, body weight; PG, perigonadal; ND, not determined; WAT, white adipose tissue; HDL, high-density lipoprotein; LDL, low-density lipoprotein; LDH, lactate dehydrogenase; γGT, gamma-glutamyl transpeptidase; ALP, alkaline phosphatise.

  • *

    P < 0.05.

  • P < 0.01.

  • P < 0.001, as compared to control KAP1 WT of the same sex and diet by two-way ANOVA, followed by Bonferroni's post-test.

BW (g)31.4 ± 1.430.0 ± 1.446.2 ± 2.241.0 ± 2.638.0 ± 1.435.9 ± 1.551.5 ± 2.252.4 ± 1.2
Body fat (%)27.6 ± 3.026.1 ± 1.848.2 ± 1.441.6 ± 2.223.0 ± 1.623.3 ± 3.039.4 ± 0.638.6 ± 1.1
Liver (% BW)4.1 ± 0.23.8 ± 0.24.0 ± 0.33.0 ± 0.24.8 ± 0.24.0 ± 0.26.3 ± 0.4*5.3 ± 0.3
PG-WAT (% BW)6.3 ± 0.96.4 ± 0.610.9 ± 0.310.3 ± 0.64.7 ± 0.24.4 ± 0.44.5 ± 0.33.8 ± 0.2
Ovaries/testes (% BW)0.33 ± 0.030.34 ± 0.030.23 ± 0.020.25 ± 0.040.91 ± 0.030.93 ± 0.030.62 ± 0.02*0.72 ± 0.04
Testosterone (ng/mL)NDNDNDND3.8 ± 1.42.2 ± 0.51.3 ± 0.64.5 ± 2.4
Cholesterol (mM)4.8 ± 0.44.5 ± 0.36.0 ± 0.6*4.1 ± 0.55.7 ± 0.24.5 ± 0.38.2 ± 0.47.9 ± 0.4
HDL cholesterol (mM)1.34 ± 0.25*1.67 ± 0.172.11 ± 0.101.98 ± 0. 172.19 ± 0.092.33 ± 0.112.11 ± 0.102.95 ± 0.08
LDL cholesterol (mM)1.25 ± 0.55*0.86 ± 0.330.73 ± 0.180.38 ± 0.060.44 ± 0.040.24 ± 0.031.10 ± 0.090.83 ± 0.06
Triglycerides (mM)1.46 ± 0.131.20 ± 0.091.15 ± 0.05*0.86 ± 0.031.56 ± 0.151.38 ± 0.161.10 ± 0.080.92 ± 0.09
Total protein (g/L)63.4 ± 1.263.1 ± 0.768.2 ± 1.4*63.5 ± 1.863.9 ± 0.763.1 ± 1.368.6 ± 1.467.5 ± 0.7
FFAs (mM)1.60 ± 0.401.70 ± 0.211.66 ± 0.141.28 ± 0.091.06 ± 0.11*1.75 ± 0.401.50 ± 0.141.77 ± 0.13
AST (U/L)252 ± 79173 ± 50342 ± 82123 ± 18118 ± 19173 ± 67373 ± 58239 ± 72
γGT (U/L)10.3 ± 2.16.4 ± 1.43.7 ± 0.44.6 ± 0.57.3 ± 0.66.3 ± 0.66.4 ± 0.54.9 ± 0.6
IL-1β (ng/mL/mg protein)4,355 ± 106*3,881 ± 914,380 ± 1404,257 ± 1804,234 ± 1953,614 ± 1644,021 ± 2414,485 ± 184
ALP (U/L)79.8 ± 5.886.8 ± 4.31,04.3 ± 20.0*66.8 ± 5.872.5 ± 3.867.1 ± 4.81,14.5 ± 14.7*82.1 ± 5.1
Total bilirubin (μM/L)2.7 ± 0.62.2 ± 0.42.7 ± 0.51.5 ± 0.31.6 ± 0.22.1 ± 0.52.4 ± 0.41.3 ± 0.1
Glucose (mM)15.8 ± 0.816.1 ± 0.917.0 ± 1.416.4 ± 1.317.5 ± 1.118.7 ± 1.217.9 ± 0.719.1 ± 1.0

Upon HFD challenge, both Alb-Cre Kap1fl/fl and Kap1fl/fl males developed severe hepatic steatosis (Fig. 1C), but the liver was heavier in Kap1 KO animals, which also presented with a bigger epididymal fat pad and a decreased testis weight (Table 1). Although this latter trait suggested an impaired sex hormone balance, a condition also observed in humans in cases of severe liver steatosis,4 total plasma testosterone levels were not different between KO and WT littermates (Table 1).

In contrast to males, CD-fed females did not present significant liver lipid accumulation, although Kap1 KO animals showed plasma dyslipidemia and up-regulation of liver enzymes (Fig. 1A-D; Table 1). When subjected to HFD, Kap1 mutant females displayed a higher body weight than littermate controls, probably as a result of increased body fat content, as assessed by echo magnetic resonance imaging (Fig. 1A; Table 1). Furthermore, these mice were characterized by (1) a moderate degree of liver steatosis (Fig. 1C), (2) mildly elevated plasma cholesterol and triglycerides, and (3) significant up-regulation of alanine aminotransferase (ALT), aspartate aminotransferase (AST), as well as a clear trend in the up-regulation of other plasma markers of hepatocytic dysfunction (Fig. 1D; Table 1). Nevertheless, Kap1 KO females were comparable to their littermates for food intake, physical activity, and energy expenditure (not illustrated).

Development of Hepatic Adenomas in Kap1 KO Males.

Anatomo-pathological examination indicated that male Alb-Cre Kap1fl/fl mice on HFD had a tendency to develop liver tumors that occasionally reached large size and formed nodular masses distorting the lobular contour (Fig. 2A,B). Hepatic adenomas were observed in 4 of 11 HFD-fed and 1 of 12 CD-fed males at 29 weeks, whereas no tumors were found in Kap1fl/fl littermates (n = 9 and n = 10 on CD and HFD, respectively). In 1 of the HFD-fed Kap1 KO males, a 2-mm diameter hepatocellular proliferative focus with features of solid carcinoma was observed. Consistent with the previously reported protective effect of female hormones,3 Kap1 KO female mice appeared largely to escape HFD-induced tumorigenesis, because only 1 of 10 HFD-fed KO females had an adenoma and no tumor was observed in WT (n = 10 on CD and n = 10 on HFD) or in CD-fed female KO mice (n = 10). To confirm these results, we analyzed the livers of CD-fed mice at ages of 53-72 weeks (Table 2). Macroscopic examination revealed that Kap1 KO mice presented with hepatomegaly (Table 2), and 9 of 15 (64%) male Alb-Cre Kap1fl/fl mice developed tumors, compared to 2 of 13 (15%; P < 0.024) WT controls, whereas females appeared partially protected against this process, with only 4 of 15 (27%) KO and 1 of 15 (7%) WT controls showing hepatocellular tumors (Table 2; Fig. 2C). Histopathological examination performed on a subset of animals (13-15 in each subgroup) confirmed that all tumors observed in Kap1-deleted mice were adenomas, whereas focal cellular alterations were detected in both control and KO mice. In addition, minimal infiltration by mixed inflammatory cells was noted in approximately one quarter of the animals, but no significant association with Kap1 status was found (not illustrated). We verified that similar results were obtained in a C57/Bl6 background (Supporting Fig. 2).

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Figure 2. Liver tumorigenesis in KAP1 KO mice. (A) Left: liver adenoma with a well-demarcated nodular proliferation and a clear expansile growth; right: HCC with poorly demarcated irregular borders suggestive of infiltrative growth (black solid line) and increased anisocytosis and anisokaryosis within the tumor, when compared to the adjacent healthy hepatocytes (right) in male mouse fed HFD for 19 weeks. Scale bar in (A): 2 mm. Scale bar in (B): 100 μm. (B) Macroscopic view of rostral (left) and caudal (right) face of livers from indicated CD-fed, 58-week-old male mice. (C) Gross necropsy analysis of tumor occurrence in livers of mice at age 53-70 weeks. *P < 0.024 by Fisher's exact test. (D) Western blotting of indicated proteins in Kap1 KO tumor tissue and healthy liver from WT and Kap1 KO mice (left) with quantification of band intensity normalized to actin and represented as fold change over average WT liver (right). ***P < 0.001 by one-way ANOVA. (E) Phosphorylation status and total protein content of AKT and various MAPK in indicated samples determined by enzyme-linked immunosorbent assay (ELISA) and represented as fold change over average WT liver. ***P < 0.001 by one-way ANOVA. (F) Hepatocyte proliferation in livers of 35- and 68-week-old female (n = 3-5; left) and male (n = 4-5; right) mice. Data represent mean number of BrdU-positive nuclei per field of view. Error bars in (D), (E), and (F) represent standard error of the mean.

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Table 2. Parameters Describing 53-72-Week-Old KAP1 KO Mice
ParameterFemale KO CD (n = 15)Female WT CD (n = 15)Male KO CD (n = 15)Male WT CD (n = 13)
  • WT mice and animals bearing liver-specific KAP1 KO were fed CD and fasted for 4 hours before experiment.

  • Abbreviation: BW, body weight.

  • *

    Significantly different (P < 0.05), as compared to control KAP1 WT of the same sex by Student t test.

BW40.1 ± 2.546.2 ± 2.743.9 ± 2.745.9 ± 2.8
Liver weight (% BW)5.1 ± 0.3*3.8 ± 0.16.5 ± 0.7*4.5 ± 0.3
Age (weeks)60.3 ± 3.959.3 ± 1.663.5 ± 1.662.1 ± 2.0
Liver IL-1β (ng/mL/mg protein)1,611 ± 951,606 ± 631,962 ± 32*1,674 ± 78
Liver IL-6 (ng/mL/mg protein)1,178 ± 791,258 ± 511,504 ± 87*1,255 ± 47
Liver TNF-α (ng/mL/mg protein)2,085 ± 1612,123 ± 1562,682 ± 128*2,360 ± 145

Although inflammation was not a prominent histological feature of Kap1 KO livers, we observed higher intrahepatic levels of proinflammatory cytokines interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) in this setting (Fig. 1E; Tables 1 and 2). IL-6 and TNF-α contribute to hepatoprotection and induce hepatocyte proliferation during liver injury20 and have been implicated in the development of fatty liver and tumors in other mouse models of liver tumorigenesis.3 Because cytokine signaling involves several transduction pathways previously implicated in liver oncogenic transformation,21 we analyzed the activity of the mitogen-activated protein kinase (MAPK) pathway, the phosphorylation levels of protein kinase B (AKT), as well as the expression levels of phosphatase and tensin homolog (PTEN), key regulators of the phosphoinositide 3-kinase (PI3K) pathway (Fig. 2D,E). Total levels of p38 MAPK were similar in WT and KO animals, irrespective of their genetic background. However, levels of phosphorylated p38 were increased in KO livers. As an expected corollary, there also was a significant hyperphosphorylation of extracellular signal-related kinases 1/2 (ERK1/2) MAPK and MAPK kinase 1 in KAP1-depleted tissue (Fig. 2D,E). Furthermore, tumor tissue harvested from Kap1 KO liver displayed a marked up-regulation of both total and phosphorylated ERK1/2, despite normal levels of activity of other components of the MAPK pathway. Additionally, we observed AKT hyperphosphorylation in tumor tissue, despite normal total protein levels. These results suggest that amplification of ERK1/2 and increase of AKT signaling play a prominent role in the development of adenomas in Kap1 KO livers. In contrast, there was no significant change in the phosphorylation status of Jun N-terminal kinase/stress-activated protein kinase (JNK/SAP) nor abnormalities in PTEN levels (Fig. 2D).

To assess the contribution to the observed phenotype of hepatocyte proliferation and hepatic cell death, we performed BrdU pulse in 35- and 68-week-old mice and found that hepatocyte proliferation was comparable between KO and controls (Fig. 2F). Similarly, histological examination and TUNEL staining in KAP1-depleted livers did not reveal any significant signs of cell death in Kap1 KO livers (data not shown).

In summary, these results indicate that loss of KAP1 in the liver leads to sexually dimorphic phenotypes, with males exhibiting progressive steatosis and age-related tumorigenesis and females suffering from a less dramatic syndrome with mild metabolic defects, including obesity and steatosis, revealed only upon exposure to an environmental stress.

Sex-Specific Gene Dysregulation in Kap1 KO Liver.

We performed gene-expression profiling in livers from CD-fed 8-10-week-old WT and mutant male and female mice, choosing this young age to minimize the potential effect of compensatory changes. Loss of KAP1 led to at least 2-fold dysregulation of 170 and 149 genes in male and female mice, respectively. Interestingly, less than 50% of the deregulated genes were common to the two sexes (Fig. 3A), suggesting that the KRAB/KAP1 system targets different genes in males and females. Gene ontology (GO) analysis of 2-fold dysregulated transcripts (Supporting Table 1) indicated that loss of KAP1 affected genes encoding protein endowed with oxidoreductase activity involved in xenobiotic metabolism by Cyp450 as well as the pathways of retinol, drug, and arachidonic acid metabolism (Fig. 3B). Additionally, GO terms and Kegg pathway analyses singled out genes relevant for steroid hormone biosynthesis and metabolism, peptidase inhibitor activity, and peroxisome proliferator receptor signaling in male KO mice, contrasting with genes linked to pheromone binding in their female counterparts. Approximately 50% of genes with the highest fold-change differed between male and female mice. For example, the most highly up-regulated genes in males belong to the phenobarbital-inducible Cyp2b family, whereas in females, those include serine peptidase inhibitor, Kazal type 3 (Spink3), keratin 23 (Krt23), and Cyp2d9 (Fig. 3C). We verified that the transcriptional changes noted upon Kap1 deletion were not the result of the mixed genetic background of the mice or Cre expression (Supporting Fig. 2).

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Figure 3. Hepato-specific KAP1 deletion leads to sex-specific gene dysregulation. (A) Venn diagram of genes ≥2-fold deregulated in 10-week-old female and male KO versus WT mice. Less than 50% of deregulated genes are shared between sexes. (B) Functional annotation chart of significantly deregulated genes (fold change, ≥2) in Kap1-mutant livers. (C) Top 10 up- and down-regulated genes in male (left) and female (right) livers. (D) Expression of Rsl-regulated sexually dimorphic genes in Kap1 KO and WT male and female livers (n ≥ 5). Error bars represent standard error of the mean (SEM). (E) Dysregulation of mRNA levels of selected direct KAP1-target genes; n = 3-4. Error bars represent SEM.

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KAP1 Controls Expression of Rsl-Target Genes.

Female-restricted changes in Cyp2d9, sex-limited protein (Slp), and major urinary proteins (Mups) are hallmarks of the Rsl loss-of-function phenotype.5 Though we could document both Cyp2d9 and Slp derepression in female Kap1 KO livers (Fig. 3D), Slp appeared affected to a far lower extent than reported in Rsl-deleted female mice, although a direct comparison is difficult owing to differences in the mouse strains used in the two studies. Furthermore, we observed a down-regulation, rather than an up-regulation, of Mups in Kap1 mutant mice at both RNA and protein level (Fig. 3D and Supporting Fig. 3), suggesting that either Rsl2-mediated female-specific repression5 is not mediated by KAP1 or that Mups are indirect KAP1 or Rsl targets.

KAP1 Binding Sites and KAP1-Related Chromatin Modifications in the Mouse Liver.

To better understand the molecular mechanisms of KAP1-mediated gene-expression control in the liver, we performed ChIP followed by deep sequencing on male and female WT and KAP1-depleted nuclei, using an antibody directed against the RBCC domain of KAP1.8 False-positive peaks originating from nonhepatocytic cells present in the samples were minimized by following a protocol optimized for hepatocyte-centered ChIP experiments,16 and data obtained in Kap1-deleted hepatocytes served as a control sample for peak calling. We identified 7,158 and 5,223 KAP1 peaks in male and female livers, respectively (Fig. 4A), a number comparable to that extrapolated from genome-wide KAP1-binding studies previously performed in other tissues.22 Surprisingly, few of the identified peaks (∼10%) were common to males and females (Fig. 4A), consistent with the observed sex-specific gene dysregulations induced by the Kap1 KO (Fig. 2). KAP1 peaks were significantly enriched in the vicinity of transcriptional start sites (TSSs; Fig. 4B).

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Figure 4. KAP1-binding sites in mouse liver. (A) Number of KAP1 peaks on genome from male and female hepatocytes. (B) Distribution of KAP1 putative binding sites relative to all annotated TSSs. For distance of −200, 0, and 200 base pairs relative to TSS, KAP1 is significantly enriched (P < 0.05), as assessed by Wilcoxon's rank-sum test. (C) KAP1-responsive genes are closer to the nearest KAP1 peak than random genes. Distance to the nearest peak was calculated for all KAP1 up- or down-regulated genes and compared to the average distance for all genes probed by the Illumina chip. (D) Frequency of KAP1 peaks in different intervals from the TSS for genes down-regulated (left) or up-regulated (right) in Kap1 mutant livers. Frequency is defined at the percentage of peaks normalized for the interval size.

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We then searched the nearest peak for each of the genes dysregulated at least 1.5-fold upon KAP1 removal (Supporting Table 1). We observed that relatively few of them had a KAP1-binding site in the gene body (less than 20%; not shown); however, the average distance between both up- and down-regulated genes and the nearest KAP1 peak was significantly shorter than for all protein-coding genes present on our Illumina chip (Fig. 4C). Furthermore, these peaks were usually found just upstream of the TSS of deregulated genes (Fig. 4D). This suggests that KAP1 might directly control many of the genes perturbed in its absence, but that it may not always be through repression.

Surprisingly, for several genes expressed and deregulated in a sex-discriminating fashion, KAP1 binding was found in both males and females (Figs. 3 and 5). These included peaks in the promoter of the male-predominant Cyp2d9 and Slp genes, both strongly up-regulated in Kap1 KO females, and in the glutathione S-transferase π (Gstπ) cluster, a late-phase drug-responsive gene preferentially expressed in males, with strong down-regulation upon KAP1 removal. Of note, the Gstπ cluster bears a male-specific KAP1-binding site downstream of the locus that could explain sex-biased dysregulation. Moreover, we identified putative KAP1-binding sites in an intron of the gene encoding fat-specific protein 27 (FSP27), which plays a role in lipid droplet formation23 and was strongly up-regulated in male KO livers, and in the gene encoding for the cellular homolog of the viral Casitas B-lineage lymphoma proto-oncogene (c-Cbl), a regulator of various tyrosine kinase signaling pathways24 up-regulated in KO livers of both sexes (Figs. 3E, 5, and 6A). We also identified KAP1-binding sites in a cluster of phenobarbital-responsive, female-predominant Cyp2b genes that were up-regulated in males and down-regulated in females upon loss of KAP1 and in clusters of other xenobiotics-processing Cyp450 genes, such as Cyp2a and Cyp3a (Figs. 3E, 5, and 6A, and Supporting Fig. 4).

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Figure 5. Selected KAP1 putative binding sites in mouse liver. Screen shots from the University of California Santa Cruz (Santa Cruz, CA) Genome Browser illustrating the KAP1 ChIP sequencing data from female and male Kap1 KO (n = 1) and WT (n = 2) hepatocytes for indicated loci. Direction of transcription is indicated by an arrow. Regions targeted by primers used for the validation by quantitative PCR are shown.

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Figure 6. Chromatin studies on KAP1-regulated genes. (A) Quantitative PCR validation of KAP1 binding in female and male livers (n = 4). Data are represented as an enrichment of WT over KO sample relative to an average of KAP1 unbound control loci (Foxp3, RPS9, and β-actin) at sites depicted in (A) Error bar is standard error of the mean (SEM). (B and C) H3K9me3 (B) and H3 acetylation (C) ChIP/quantitative PCR in female and male nuclei (n = 3) of WT and KAP1-depleted liver. Data are represented as an enrichment of WT over KO sample relative to an average of three control loci (GAPDH, RPS9, and β-actin). Error bar is SEM.

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KAP1 recruits the SETDB1 histone methyltransferase, which induces deposition of the H3K9me3 repressive mark, and HDAC-containing complexes, which result in histone deacetylation.17 We thus compared the presence of the relevant chromatin marks in WT and Kap1 KO liver at both the nearest KAP-binding site and the promoter of a few genes suspected to represent direct KAP1 targets. Loss of KAP1 correlated with a loss of H3K9me3 at the binding site for genes up-regulated in Kap1-mutant livers, such as Cyp2d9 in females and fsp27, c-Cbl, or the Cyp2b cluster in males. However, for male down-regulated Gstπ, Cyp2d9, or Slp, no change in H3K9me3 at the KAP1-binding site was detected. Notably, gain of H3K9me3 at the male KAP1-binding site in the fsp27 locus in the female liver suggests local chromatin changes that could be the cause of mild female up-regulation of the corresponding transcript. Finally, an increase in H3Ac at the promoters of Cyp2d9 in females and Fsp27 in males coincided with gene dysregulation; however, this was not the case for all tested KAP1-target loci (Fig. 6B,C).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The present work reveals the prominent role of KRAB/KAP1-mediated regulation in the control of liver metabolism and endo- and xenobiotics-detoxifying genes. The liver-targeted KO of KAP1, the universal cofactor of KRAB-ZFPs, resulted in a markedly sexually dimorphic phenotype in mice, which included male-predominant steatosis and hepatic tumors. This correlated with sex-specific transcriptional dysregulation of a wide range of metabolic genes, notably those involved in retinol and sex hormone processing as well as in detoxification. An examination of KAP1 genomic binding sites in the liver and of chromatin marks at the promoters of some of these genes further revealed that a number of detoxifying genes, such as Cyp2d9, Gstπ, and the Cyp2a, Cyp2b, and Cyp3a gene clusters, are direct KAP1 targets. Moreover, loss of KAP1 binding at the fsp27 locus was associated with the male-predominant up-regulation of its product.

Male liver-specific Kap1 KO mice displayed early-onset hepatosteatosis and age-related development of liver adenomas, whereas their female counterparts exhibited milder metabolic disturbances revealed only by an HFD challenge and had lower incidences of liver adenomas than their male counterparts. This correlates with the reported resistance of the female liver to environmental insults, which has been attributed to its greater ability to inactivate toxins and neutralize reactive oxygen species, and to the protective action of estrogens against inflammation and steatofibrosis, including during the course of chronic viral hepatitis.19, 25 Our results suggest that these mechanisms are influenced by KRAB/KAP1-mediated transcriptional control.

What specific mechanisms account for the accumulation of fat and tumor development in the Kap1-deleted male liver remains to be determined. We observed a male-predominant, Kap1 deletion-induced up-regulation of FSP27, correlating with a male-restricted strong KAP1-binding site in the body of this gene. FSP27 promotes fat droplet formation in adipocytes and its hepatocytic overexpression results in hepatosteatosis.23, 26 This phenotypic abnormality, which represents an early sign of hepatocyte suffering common to many pathological circumstances, has been linked to increased liver damage, inflammation, and development of HCC in other mouse models.3 Interestingly, KAP1 KO mice displayed an up-regulation of liver levels of proinflammatory cytokines IL-6 and TNF-α, which is a sign of response to hepatocyte damage. Aberrant activity of signaling pathways downstream of these cytokines have been linked to increased fat accumulation in hepatocytes and development of liver tumors, and our results suggest a role of the MAPK and AKT pathways in the increased incidence of tumors in Kap1 KO animals.3 The early development of hepatosteatosis resulting from derepression of fsp27 in KAP1 KO males may be one important trigger in a cascade of events leading to the development of liver steatosis and tumors in these mice.

Indirectly, our analyses suggest that xenobiotic stress, which, if prolonged, can lead to tumorigenesis,27 might contribute to the observed phenotype. In Kap1-deleted livers, we measured a dysregulation of drug- and xenobiotic-metabolizing phase I genes, such as members of the Cyp2a, Cyp2b, and Cyp3a families, of phase II and III genes, such as Gstπ and θ, and of genes coding for various UDP-glucuronosyltransferases required for the conjugation of molecules that need this modification for efficient excretion.2 We identified KAP1-binding sites in the vicinity of several of these genes, suggesting that they are direct targets of the transcriptional regulator.

The combined results of our transcriptome and chromatin analyses exclude a simple model for KAP1 action, whereby a given KRAB-ZFP would tether the cofactor to specific sequences, with secondary heterochromatin formation and gene silencing. Some sexually dimorphic genes, such as the male-predominant Cyp2d9, Gstπ, and Slp, carry KAP1 signatures inconsistent with such a model. For instance, the Cyp2d9 and Slp genes are Rsl1-targeted, male-predominant genes up-regulated upon Kap1 KO only in female mice. However, KAP1 was detected at similar sites, such as the promoter of Cyp2d9, in liver chromatin from both sexes. Also, KAP1 binds upstream of the Gstπ locus in both males and females, whereas this gene is highly expressed in the former and strongly repressed in the latter. These observations might reflect the effect of sex-specific dominant factors counteracting the repressing influence of KAP1 or sex-differential, post-translational modifications of the transcription regulator. KAP1 small ubiquitin-like modifier (SUMO)ylation appears to be required for its corepressor activity, whereas phosphorylation stimulates its chromatin-opening properties.8 In the case of Cyp2d9, it could be that phospho-KAP1 keeps the promoter accessible for transcription factors in males, whereas SUMO-KAP1 recruits SETDB1 and heterochromatin protein 1 to close it down in females.

It is generally acknowledged that KAP1 exerts its gene-repressor activity through an interaction with DNA-binding KRAB-ZFP family members. It will be important to identify the mediators of its activity in the liver; however, it remains that our data strongly suggest that KAP1 sometimes acts as a transcriptional activator, rather than as a repressor. Future studies should explore this possibility, which challenges the current view of KAP1 function.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors thank the Histology Core Facility at Ecole Polytechnique Fédérale de Lausanne (EPFL), the National Center of Competence in Research Genomics Platform at the University of Geneva, and the EPFL Transgenic Core Facility for technical assistance, Jacques Rougemont for bioinformatics support, and Juergen Ripperger, Walter Wahli, Gianpaolo Rando, and Kristina Schoonjans for their discussions. Computations were performed on the Vital-IT facility of the Swiss Institute of Bioinformatics.

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  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

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