Liver-specific ablation of Krüppel-associated box-associated protein 1 in mice leads to male-predominant hepatosteatosis and development of liver adenoma

Abstract

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.

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 post-test. CD - chow diet, HFD - high fat diet. B. HE staining of liver sample from chow diet-fed 29-week-old indicated mice. Inlet: ORO staining. Scale bar: 50 μm. C. Semi-quantitate 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 post-test. D. & E. Plasma levels of alanine transaminase (ALT) and lactate dehydrogenase (LDH) (n=7-9; D.) and liver protein levels of pro-inflammatory cytokines IL6 and TNFα (n=5; E.) in 29-week-old female (left) and male (right) mice. Black bars: CD, grey bars: HFD. *: p<0.05 as assessed by two-way ANOVA followed by Bonferroni post-test. Error bars represent S.E.M.

Figure 2. Liver tumorigenesis in KAP1 KO mice.

A. Left, liver adenoma with a well demarcated nodular proliferation and a clear expansile growth; right, hepatocellular carcinoma with poorly demarcated irregular borders suggestive of infiltrative growth (black solid line), increased anisocytosis and anisokaryosis within the tumor, when compared to the adjacent normal 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 blot of indicated proteins in Kap1 KO tumor tissue and normal 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 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 S.E.M.

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 vs WT mice. Less than 50% of deregulated genes are shared between sexes. B. Functional annotation chart of significantly deregulated (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 SEM. E. Dysregulation of mRNA levels of selected direct KAP1-target genes; n=3-4. Error bars represent SEM.

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 transcriptional start sites (TSS). For distance of -200, 0 and 200 bp relative to TSS KAP1 is significantly enriched (P<0.05) as assessed by the Wilcoxon sum rank 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 downregulated (left) or upregulated (right) in Kap1 mutant livers. Frequency is defined at the % of peaks normalized for the interval size.

Figure 5. Selected KAP1 putative binding sites in mouse liver.

Screen-shots from the UCSC Genome Browser illustrating the KAP1 ChIP-seq data from female and male Kap1 KO (n=1) and WT (n=2) hepatocytes for indicated loci. Direction of transcription is indicated by arrow. Regions targeted by primers used for the validation by qPCR are shown.

Figure 6. Chromatin studies on KAP1-regulated genes.

A. qPCR validation of KAP1 binding in female and male livers (n=4). Data are represented as an enrichment of wild type 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 S.E.M. B. & C. H3K9me3 (B.) and H3 acetylation (C.) ChIP-qPCR in female and male nuclei (n=3) of wild type and KAP1-depleted liver. Data are represented as an enrichment of wild type over KO sample relative to an average of three control loci (GAPDH, RPS9 and β-actin). Error bar is S.E.M.