Title: The SWI/SNF chromatin-remodeling complex subunit SNF5 is essential for hepatocyte differentiation
Abstract: Article1 September 2005free access The SWI/SNF chromatin-remodeling complex subunit SNF5 is essential for hepatocyte differentiation Lionel Gresh Lionel Gresh Unité Expression Génétique et Maladies—CNRS FRE 2850, Département de Biologie du Développement, Institut Pasteur, Paris, France Search for more papers by this author Brigitte Bourachot Brigitte Bourachot Unité Expression Génétique et Maladies—CNRS FRE 2850, Département de Biologie du Développement, Institut Pasteur, Paris, France Search for more papers by this author Andreas Reimann Andreas Reimann Unité Expression Génétique et Maladies—CNRS FRE 2850, Département de Biologie du Développement, Institut Pasteur, Paris, France Search for more papers by this author Bruno Guigas Bruno Guigas Hormone and Metabolic Research Unit, Christian de Duve Institute of Cellular Pathology, UCL, Brussels, Belgium Search for more papers by this author Laurence Fiette Laurence Fiette Unité de Recherche et d'Expertise en Histotechnologie et Pathologie, Institut Pasteur, Paris, France Search for more papers by this author Serge Garbay Serge Garbay Unité Expression Génétique et Maladies—CNRS FRE 2850, Département de Biologie du Développement, Institut Pasteur, Paris, France Search for more papers by this author Christian Muchardt Christian Muchardt Unité Expression Génétique et Maladies—CNRS FRE 2850, Département de Biologie du Développement, Institut Pasteur, Paris, France Search for more papers by this author Louis Hue Louis Hue Hormone and Metabolic Research Unit, Christian de Duve Institute of Cellular Pathology, UCL, Brussels, Belgium Search for more papers by this author Marco Pontoglio Marco Pontoglio Unité Expression Génétique et Maladies—CNRS FRE 2850, Département de Biologie du Développement, Institut Pasteur, Paris, France Search for more papers by this author Moshe Yaniv Corresponding Author Moshe Yaniv Unité Expression Génétique et Maladies—CNRS FRE 2850, Département de Biologie du Développement, Institut Pasteur, Paris, France Search for more papers by this author Agnès Klochendler-Yeivin Agnès Klochendler-Yeivin Department of Cellular Biochemistry and Human Genetics, The Hebrew University-Hadassah Medical School, Jerusalem, Israel Search for more papers by this author Lionel Gresh Lionel Gresh Unité Expression Génétique et Maladies—CNRS FRE 2850, Département de Biologie du Développement, Institut Pasteur, Paris, France Search for more papers by this author Brigitte Bourachot Brigitte Bourachot Unité Expression Génétique et Maladies—CNRS FRE 2850, Département de Biologie du Développement, Institut Pasteur, Paris, France Search for more papers by this author Andreas Reimann Andreas Reimann Unité Expression Génétique et Maladies—CNRS FRE 2850, Département de Biologie du Développement, Institut Pasteur, Paris, France Search for more papers by this author Bruno Guigas Bruno Guigas Hormone and Metabolic Research Unit, Christian de Duve Institute of Cellular Pathology, UCL, Brussels, Belgium Search for more papers by this author Laurence Fiette Laurence Fiette Unité de Recherche et d'Expertise en Histotechnologie et Pathologie, Institut Pasteur, Paris, France Search for more papers by this author Serge Garbay Serge Garbay Unité Expression Génétique et Maladies—CNRS FRE 2850, Département de Biologie du Développement, Institut Pasteur, Paris, France Search for more papers by this author Christian Muchardt Christian Muchardt Unité Expression Génétique et Maladies—CNRS FRE 2850, Département de Biologie du Développement, Institut Pasteur, Paris, France Search for more papers by this author Louis Hue Louis Hue Hormone and Metabolic Research Unit, Christian de Duve Institute of Cellular Pathology, UCL, Brussels, Belgium Search for more papers by this author Marco Pontoglio Marco Pontoglio Unité Expression Génétique et Maladies—CNRS FRE 2850, Département de Biologie du Développement, Institut Pasteur, Paris, France Search for more papers by this author Moshe Yaniv Corresponding Author Moshe Yaniv Unité Expression Génétique et Maladies—CNRS FRE 2850, Département de Biologie du Développement, Institut Pasteur, Paris, France Search for more papers by this author Agnès Klochendler-Yeivin Agnès Klochendler-Yeivin Department of Cellular Biochemistry and Human Genetics, The Hebrew University-Hadassah Medical School, Jerusalem, Israel Search for more papers by this author Author Information Lionel Gresh1, Brigitte Bourachot1, Andreas Reimann1, Bruno Guigas2, Laurence Fiette3, Serge Garbay1, Christian Muchardt1, Louis Hue2, Marco Pontoglio1, Moshe Yaniv 1 and Agnès Klochendler-Yeivin4 1Unité Expression Génétique et Maladies—CNRS FRE 2850, Département de Biologie du Développement, Institut Pasteur, Paris, France 2Hormone and Metabolic Research Unit, Christian de Duve Institute of Cellular Pathology, UCL, Brussels, Belgium 3Unité de Recherche et d'Expertise en Histotechnologie et Pathologie, Institut Pasteur, Paris, France 4Department of Cellular Biochemistry and Human Genetics, The Hebrew University-Hadassah Medical School, Jerusalem, Israel *Corresponding author. Unité Expression Génétique et Maladies—CNRS FRE 2850, Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France. Tel.: +33 1 4568 8512; Fax: +33 1 4061 3033; E-mail: [email protected] The EMBO Journal (2005)24:3313-3324https://doi.org/10.1038/sj.emboj.7600802 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Regulation of gene expression underlies cell differentiation and organogenesis. Both transcription factors and chromatin modifiers are crucial for this process. To study the role of the ATP-dependent SWI/SNF chromatin-remodeling complex in cell differentiation, we inactivated the gene encoding the core complex subunit SNF5/INI1 in the developing liver. Hepatic SNF5 deletion caused neonatal death due to severe hypoglycemia; mutant animals fail to store glycogen and have impaired energetic metabolism. The formation of a hepatic epithelium is also affected in SNF5-deficient livers. Transcriptome analyses showed that SNF5 inactivation is accompanied by defective transcriptional activation of 70% of the genes that are normally upregulated during liver development. These include genes involved in glycogen synthesis, gluconeogenesis and cell–cell adhesion. A fraction of hepatic developmentally activated genes were normally expressed, suggesting that cell differentiation was not completely blocked. Moreover, SNF5-deleted cells showed increased proliferation and we identified several misexpressed genes that may contribute to cell cycle deregulation in these cells. Our results emphasize the role of chromatin remodeling in the activation of cell-type-specific genetic programs and driving cell differentiation. Introduction During the past decade, it has become clear that modulation of the chromatin state plays a major role in the regulation of gene expression in eukaryotes (see Khorasanizadeh, 2004). The chromatin state is affected by covalent modification of histone tails which are associated with either activated or repressed transcription (see Fischle et al, 2003). Such modifications can influence both the accessibility of nucleosomal DNA and the recruitment of regulatory proteins. Chromatin structure can also be affected by large remodeling complexes that are well conserved between eukaryotes, such as the SWI/SNF (switching defective/sucrose nonfermenting) complex (see Becker and Horz, 2002; Martens and Winston, 2003). First described in the yeast Saccharomyces cerevisiae, the 2 MDa SWI/SNF complex includes a DNA-dependent ATPase subunit, SNF2, that functions as the motor of the complex. Yeast contains a single SNF2 gene, whereas two homologous genes have been characterized in mammals, Brm/SNF2α and Brg1/SNF2β. These two subunits are mutually exclusive in mammalian complexes. In addition to the catalytic subunit, two other proteins form the core complex: SNF5/INI1/BAF47 and SWI3 (BAF170 or BAF155 in mammals) (Phelan et al, 1999). These core subunits are thought to be present in all SWI/SNF complexes that contain in addition roughly 10 distinct subunits. The SWI/SNF complex plays an important role in transcriptional regulation in the yeast S. cerevisiae. Whole-genome transcription analysis revealed that roughly 6% of the genes are SWI/SNF-dependent (Holstege et al, 1998; Sudarsanam et al, 2000). Several studies suggest that the SWI/SNF complex does not only act as a transcriptional activator, but can also be a repressor (Sif et al, 2001; Martens and Winston, 2002). In multicellular organisms, cell differentiation requires extensive reprogramming of gene transcription. In addition to cell type-specific transcription factors, chromatin modifiers are thought to be involved in this process. Nevertheless, few studies have addressed the in vivo role of chromatin-remodeling complexes in cell differentiation. The first evidence for such a role in mammalian cells came from the use of homologous recombination in mice: targeted inactivation of several core subunits of the SWI/SNF complex, namely Brg1, SNF5 and BAF155, results in embryonic lethality at the peri-implantation stage (Bultman et al, 2000; Klochendler-Yeivin et al, 2000; Roberts et al, 2000; Guidi et al, 2001; Kim et al, 2001). In all the three knockout mice, inner cell mass development is altered, and Brg1 and SNF5 inactivation also affect the trophectoderm lineage. However, the transcriptional targets of SWI/SNF activity at this early stage are unknown. Using cell-specific inactivation systems, Brg1 has recently been shown to be required for T-cell development (Chi et al, 2003; Gebuhr et al, 2003). These studies suggest that Brg1 (and therefore the SWI/SNF complex) is essential to allow gene activation in response to signaling pathways that drive successive steps of T-cell differentiation. The use of in vitro models of cell differentiation has also provided some insights into the role of SWI/SNF complex. For example, SWI/SNF activity is required for the activation of several muscle-specific genes upon MyoD induction (de la Serna et al, 2001). However, the consequences of SWI/SNF inactivation on global genome reprogramming have not been investigated in these systems. Here, we studied the role of the SWI/SNF complex in cell differentiation using the hepatoblast–hepatocyte lineage as a model. We conducted specific inactivation of the complex core subunit SNF5 in these cells and evaluated its impact at the cellular and molecular levels. We show that liver-specific SNF5 inactivation impairs glycogen storage and epithelial morphogenesis, which are characteristics of hepatocyte differentiation. Transcriptome analyses revealed that these defects are associated with defective transcriptional activation of 70% of the genes that are normally upregulated during liver development. Our results provide a link between chromatin remodeling and development and show that SWI/SNF-dependent chromatin remodeling is required for the genetic programming of differentiated cells. Results Liver-specific inactivation of SNF5 in SNF5flox/− mice results in perinatal lethality We generated a SNF5 conditional allele in which two loxP sites flanked exons 1 and 2 of the gene (SNF5flox). Cre-mediated recombination results in deletion of the two first exons of SNF5, including the initiation ATG codon, and generates a null allele. SNF5 was inactivated in the developing liver using the AlfpCre transgene. AlfpCre-driven recombination begins at the onset of liver bud formation and leads to complete recombination in liver cells of endodermal origin: hepatocytes and cholangiocytes (Kellendonk et al, 2000; Coffinier et al, 2002). Our breeding strategy (AlfpCre; SNF5+/− × SNF5flox/flox) generated embryos with deleted SNF5 in Cre-expressing cells (AlfpCre; SNF5flox/− refered to as 'mutants'), and wild-type or heterozygous embryos ((SNF5flox/+), (SNF5flox/−) and (AlfpCre; SNF5flox/+), collectively referred to as 'controls'). At postnatal day 10, no mutant mice were found (n=65), while at embryonic day (E) 18.5, mutants were present at normal Mendelian ratios (n=27). Close examination revealed that mutant mice were born normally, but died within the first 12 h after birth (n=26). Nonetheless, neither growth retardation nor macroscopic liver defects were observed at birth. PCR analysis confirmed that the predicted SNF5deleted allele was detected in livers expressing Cre recombinase (Figure 1A). SNF5flox deletion was not complete. This was expected as hematopoietic cells represent more than 50% of total liver cells during fetal life (Paul et al, 1969) and these cells do not express the AlfpCre recombinase. Quantitative RT–PCR carried out on total liver RNAs showed that SNF5 mRNA level was reduced by 2.2-fold in mutant livers (Figure 1B). Figure 1.Liver-specific inactivation of SNF5. (A) Detection of Cre-mediated SNF5 deletion in the liver. PCR analysis using primers that amplify the null, flox and deleted alleles. Mice genotypes are indicated on the top. The deleted allele was detectable in the livers expressing Cre recombinase both before (E17.5) and after (P0) birth. (B) Quantitative RT–PCR analysis of SNF5 expression. SNF5 mRNA level was reduced by 2.2-fold in mutant livers at E18.5. Error bars represent standard error of the mean (ncontrol=4, nmutant=4). (C) AlfpCre-mediated recombination is specific of liver parenchymal cells. X-gal staining on liver sections of control AlfpCre; SNF5flox/+; ROSA26R (left panel) and mutant AlfpCre; SNF5flox/flox; ROSA26R (right panel) at E18.5. β-gal activity is an indicator of Cre-driven recombination on the ROSA26 locus. In both controls and mutants, most parenchymal cells were β-gal-positive and hematopoietic cells were β-gal-negative. For unclear reasons, the staining intensity of control parenchymal cells was weaker than that of mutant cells. Scale bars: 200 μm. Download figure Download PowerPoint To confirm the cell specificity and efficiency of AlfpCre-driven recombination, we also generated mice that included the ROSA26R reporter allele in addition to the floxed SNF5 allele (AlfpCre; SNF5flox/flox; ROSA26R). This allele contains a lacZ gene, whose activity is induced by the Cre-driven recombination (Soriano, 1999). These mice had the same phenotype as AlfpCre; SNF5flox/− animals (data not shown). SNF5 heterozygous littermates (AlfpCre; SNF5flox/+; ROSA26R) were also obtained. X-gal-stained liver sections revealed that in both mutants and controls over 95% of the parenchymal cells were β-galactosidase (β-gal)-positive at E18.5 (Figure 1C). As expected, hematopoietic cells were β-gal-negative. These data indicate that most liver parenchymal cells underwent Cre-mediated recombination. Glucose metabolism is strongly impaired in SNF5-deleted livers To unravel the cause of the early postnatal morbidity of mutant mice, we followed the glycemia within hours of birth (P0) and found that mutant mice were strongly hypoglycemic (Table I). The average glycemia of control mice was 58 mg/dl, while all 13 mutants tested presented a glycemia lower than 10 mg/dl. This defect was not linked to a feeding problem, as mutant and control mice digestive tracts both contained milk. Hypoglycemia was also seen in E18.5 mutant mice 2 h after caesarian delivery (data not shown). As these levels of glycemia are not compatible with survival, we conclude that hypoglycemia is the likely cause of perinatal lethality of mutant mice. Table 1. Liver-specific inactivation of SNF5 leads to decreased glycemia and hepatic glucose production Hepatic glucose production (μmol min−1 g−1) Genotype Glycemia (mg dl−1) Total Gluconeogenesis Glycogenolysis Control 58±13 (n=19) 2.5±0.5 (n=5) 0.56±0.14 (n=5) 1.9±0.5 (n=5) Mutant <10 (n=13) 0.8±0.2 (n=8) 0.27±0.08 (n=8) 0.6±0.3 (n=8) Glycogen synthesized from maternal glucose in the liver during gestation is essential to support life during the first hours after birth. To determine if hypoglycemia was linked to a glycogen storage defect, we stained liver sections for glycogen using the Periodic Acid-Schiff (PAS) reaction. Glycogen-specific staining was decreased in mutants at E17.5, E18.5 and P0 (Figure 2A and data not shown). To quantify this defect, we measured glycogen levels in E18.5 and P0 control and mutant livers. Glycogen levels were strongly reduced in mutant livers both before and after birth (Figure 2B). Figure 2.Mice carrying a liver-specific inactivation of SNF5 fail to store glycogen. (A) PAS staining for the presence of glycogen in control (left panel) and mutant (right panel) liver sections at E18.5. Control livers showed extensive glycogen deposition. Staining was almost absent in mutant livers. Scale bars: 200 μm. (B) Glycogen content of mutant livers (w/w) was significantly reduced (P<0.05) when compared to control livers. Error bars represent standard error of the mean (E18.5: ncontrol=5, nmutant=4; P0: ncontrol=5, nmutant=3). Download figure Download PowerPoint In order to evaluate the actual capacity of mutant livers to synthesize glucose, we measured the hepatic glucose production in newborn liver slices. We found a three-fold reduction (P<0.05) of total glucose production in mutant livers (Table I). Interestingly, this defect was due to a combined significant reduction of both glycogenolysis and gluconeogenesis rates, respectively by three- and two-fold (P<0.05) (Table I). These results suggest that mutant hypoglycemia is the consequence of reduced glycogenolysis due to lower glycogen accumulation, but also to a concomitant decreased in gluconeogenesis, indicating that both pathways supporting liver glucose production are impaired upon SNF5 inactivation. To gain more insights on the defects in glucose metabolism and on other potential functions of SNF5, we conducted a comparative transcriptome analysis of control versus mutant liver RNAs (see Materials and methods). The data obtained unravel the molecular basis of the drastic reduction in liver glycogen storage. First, expression of the liver glycogen synthase (Gys2) was downregulated by 2.1-fold. Second, a regulatory subunit of protein phosphatase 1 (PP1), Ppp1rc3 (PP1 regulatory subunit 3C, also known as PTG for protein targeting to glycogen), was also expressed at lower levels in mutant livers. This protein targets PP1 to the glycogen particle, and allows it to activate glycogen synthesis and inhibit glycogen breakdown (see Brady and Saltiel, 2001). Finally, we observed a downregulation of the glucose transporter Slc2a2 (Glut2), which should affect glucose uptake from maternal sources before birth (Figure 3). Figure 3.Impaired glucose metabolism upon loss of SNF5 in fetal livers. The scheme shows genes involved in energy metabolism that were significantly downregulated (P<0.05) more than two-fold in E18.75 mutant livers. Key enzymes of gluconeogenesis (Gpc, Fbp1, Pcx) and glycogen synthesis (Gys2) were strongly downregulated. For each gene, the mutant versus control fold change is indicated. Genes encoding enzymes are italicized. Boxed names represent genes encoding regulatory proteins. Gene products involved in gluconeogenesis, citric acid cycle and oxidative phosphorylation are represented in green, yellow and red, respectively. Download figure Download PowerPoint Hydrolysis of glucose 6-phosphate (G6P) into glucose and phosphate is a common and important step in both glycogenolysis and gluconeogenesis. It is accomplished by two proteins: the G6P ER translocator (G6pt1) and the phosphatase (G6pc) (see van Schaftingen and Gerin, 2002). Both genes were strongly downregulated in mutant livers (Figure 3). Five additional genes involved in gluconeogenesis were also downregulated. This should result in the inactivation of the two irreversible reactions that allow pyruvate to be converted back to G6P: the pyruvate carboxylase (Pcx) step and the fructose-1,6-bisphosphatase 1 (Fbp1) step (Figure 3) (see Hers and Hue, 1983). Other genes involved in energy metabolism were also affected in mutant livers (Figure 3). The downregulation of the two first enzymes involved in galactose catabolism (galactokinase (Glk) and galactose-1-phosphate uridyl transferase (Galt)) probably affects galactose usage as an energy source. Consequences of downregulation of three other genes, glucose phosphate isomerase 1 (Gpi1), 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1 (Pfkfb1) and pyruvate dehydrogenase kinase, isoenzyme 4 (Pdk4), are difficult to predict. Finally, isocitrate dehydrogenase 2 (Idh2) and ATPase, Na+/K+transporting, alpha 1 polypeptide (Atp1a1) downregulation could reduce ATP synthesis in mutant hepatocytes (Figure 3). Taken together, these data show that SNF5 is essential for glycogen synthesis, glycogenolysis and gluconeogenesis in the liver. SNF5 is essential for epithelial differentiation of hepatocytes Establishment of cell–cell junctions is one of the most important steps during terminal hepatocyte differentiation (Feracci et al, 1987). To explore this process, we performed immunodetection of proteins that participate to these junctions. The Zonula occludens 1 (ZO-1) protein is normally localized to tight junctions (Stevenson et al, 1986), as observed in control animals (Figure 4A). In contrast, almost no ZO-1 protein was detected in mutants (Figure 4B), suggesting that tight junctions were absent. E-cadherin is also involved in cell–cell interactions and is normally localized at both adherens junctions and desmosomes (Johnson et al, 1993). The expression of this protein was also downregulated in mutant livers (Figure 4C and D). Furthermore, mutant livers presented ultrastructure abnormalities. Electron microscopy observations showed that intermembrane space between adjacent cells was larger than in controls and that cell–cell junctions were disrupted (Figure 4E and F). Mutant cells also showed abnormal cytoplasms with lower organelle and mitochondria content, again suggesting a defect in terminal hepatocyte differentiation (Figure 4F). Figure 4.SNF5 inactivation results in the disruption of epithelial architecture of liver parenchyma. (A, B) ZO-1 immunodetection (green) on liver sections in control (A) and mutant (B) mice at E18.5. Tight junctions are stained in control livers. In mutants, almost no signal is detected. (C, D) Loss of E-cadherin (red) in mutant livers (D) when compared to controls (C). Scale bars (A–D): 75 μm. (E, F) Electron micrographs of control (E) and mutant (F) livers at E18.5. Gaps in between cells were observed in mutant livers (arrowheads). Insets show details of cell–cell contacts. Download figure Download PowerPoint Transcriptome data are consistent with a defect in cell–cell junction formation in mutant livers (Table II). The gene encoding E-cadherin, Cadherin 1, was downregulated 1.8-fold in mutant livers. Furthermore, genes encoding proteins of desmosomes (Desmocollin 2 and Desmoglein 2), gap junctions (Gjb1/Connexin-32 and Gjb2/Connexin-26) and adherens junctions (Ceacam1) were also downregulated. Interestingly, Tjp1, the gene encoding ZO-1, was normally expressed at the mRNA level. Table 2. Genes involved in cell–cell adhesion that were significantly downregulated (P<0.05) in E18.75 mutant livers UniGene cluster Gene symbol Gene name Alternate name Type of junction Fold change 280547 Dsc2 Desmocollin 2 Desmosome −2.5 21198 Gjb1 Gap junction membrane channel protein beta 1 Connexin-32 Gap junction −2.0 34118 Gjb2 Gap junction membrane channel protein beta 2 Connexin-26 Gap junction −1.9 360512 Ceacam1 CEA-related cell adhesion molecule 1 Adherens junction −1.8 345891 Dsg2 Desmoglein 2 Desmosome −1.8 35605 Cdh1 Cadherin 1 E-cadherin Adherens junction −1.8 Taken together, these data show that SNF5 is essential for the assembly of all types of epithelial cell–cell junctions, namely tight junctions, adherens junctions, gap junctions and desmosomes. Moreover, this phenotype is underlied by a defective transcriptional activation of several genes encoding proteins that participate in these junctions. SNF5 is required for the expression of a specific set of developmentally regulated and liver-specific genes Our global transcriptome analysis showed that a considerable number of genes are dependent on SNF5 activity (ArrayExpress accession number: E-MEXP-241). We detected 9929 probe sets out of 36 000 present on the DNA chips (see Materials and methods). Of these, 412 (4.1%) were significantly decreased more than two-fold and 860 (8.7%) more than 1.5-fold in mutant livers (Supplementary Table I). The number of increased probe sets was smaller: only 71 (0.7%) were significantly increased more than two-fold and 259 (2.6%) more than 1.5-fold (Supplementary Table II). This broad effect could have been due to the lack or under-representation of hepatic cell types in mutant livers. However, histological examination (see Figures 2A and 4F) and lineage analysis (see Figure 1C) demonstrated that hepatic cells were indeed present. In addition, several specific markers of this lineage, including albumin 1 and alpha-fetoprotein, were normally expressed in mutant livers (data not shown). To further assess the effect of SNF5 inactivation on hepatocyte differentiation, we analyzed the transcriptome more thoroughly. We compared wild-type E18.75 livers with transcriptome data obtained from wild-type 3T3 immortalized fibroblasts (Gerald and Mechta, ArrayExpress accession number: E-MEXP-239) and from wild-type E14.5 livers (Chéret and Pontoglio, ArrayExpress accession number: E-MEXP-240). Using these comparisons, we defined two groups of genes according to tissue-specific expression ('liver-enriched genes' and 'ubiquitous genes'), and two others according to temporal expression pattern ('developmentally activated genes' and 'not developmentally regulated genes') (see Materials and methods). We monitored the effect of SNF5 inactivation by calculating the number of genes downregulated ('decreased genes'), upregulated ('increased genes') and unchanged ('not changed genes') upon SNF5 inactivation in these different groups of genes. Interestingly, the 'decreased' probe sets represented a large proportion of the 'liver-enriched genes' and the 'developmentally activated genes' (Figure 5A and B). In contrast, no gene was 'developmentally activated' and 'increased' upon SNF5 inactivation (Figure 5B). We also found that, out of 235 genes that were both 'liver-enriched' and 'developmentally activated', 164 (70%) were downregulated and 71 (30%) were not affected by SNF5 inactivation (Figure 5C). These 71 probe sets included genes involved in different hepatic functions, indicating that SNF5 inactivation did not completely block hepatocyte differentiation (a partial list is given in Table III). As stated above, a major fraction of genes involved in energy metabolism were downregulated, while half of the serum proteins were normally expressed. Finally, the expression of the majority of cytochrome P450 genes was reduced. Figure 5.SNF5 is required for the establishment of the genetic program of differentiated hepatocytes. Transcriptional effect of SNF5 inactivation on different groups of genes: (A) 'Liver-enriched genes' versus 'ubiquitous genes'; (B) 'developmentally activated genes' versus 'not developmentally regulated genes'; (C) intersection of 'liver-enriched genes' and 'developmentally activated genes'. (A, B) An important proportion of 'liver-enriched genes' (A) and 'developmentally activated genes' (B) were downregulated upon SNF5 inactivation. (C) Almost 70% of the genes that are both 'liver-enriched' and 'developmentally activated' were downregulated in the absence of SNF5. In all, 30% of those genes did not require SNF5 activity for their activation. Download figure Download PowerPoint Table 3. Specific subsets of genes are dependent or independent on SNF5 activity to be correctly activated during terminal hepatocyte differentiation. Examples of liver-enriched and developmentally activated genes that were decreased (left) or not changed (right) upon SNF5 inactivation Liver-enriched and developmentally activated genes Decreased genes upon SNF5 inactivation Genes not changed upon SNF5 inactivation Gene symbol Gene name Gene symbol Gene name Energy metabolism Acat1 Acetyl-coenzyme A acetyltransferase 1 Abcd2 ATP-binding cassette, subfamily D (ALD), member 2 Car5a Carbonic anhydrase 5a, mitochondrial Fabp1 Fatty acid-binding protein 1, liver Car8 Carbonic anhydrase 8 Gyk Glycerol kinase Fabp4 Fatty acid-binding protein 4, adipocyte Hao1 Hydroxyacid oxidase 1, liver Facl2 Fatty acid coenzyme A ligase, long chain 2 Itih4 Inter-alpha-trypsin inhibitor, heavy chain 4 Fbp1 Fructose bisphosphatase 1 Lcat Lecithin cholesterol acyltransferase G6pc Glucose-6-phosphatase, catalytic G6pt1 Glucose-6-phosphatase, transport protein 1 Pck1 Phosphoenolpyruvate carboxykinase 1, cytosolic Pcx Pyruvate carboxylase Pfkfb1 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 1 Ppp1r3c Protein phosphatase 1, regulatory (inhibitor) subunit 3C Serum proteins A2m Alpha-2-macroglobulin Afp Alpha fetoprotein Apoa5 Apolipoprotein A-V C4bp Complement component 4-binding protein Apoc4 Apolipoprotein C-IV Cfh Complement component factor h C4 Complement component 4 (within H-2S) Cfhl1 Complement component factor h-like 1 Crp C-reactive protein, petaxin related Cfi Complement component factor i F10 Coagulation factor X Cpb2 Carboxypeptidase B2 (plasma) F11 Coagulation factor XI F13b Coagulation factor XIII,