Title: Cited2, a coactivator of HNF4α, is essential for liver development
Abstract: Article11 October 2007free access Cited2, a coactivator of HNF4α, is essential for liver development Xiaoling Qu Xiaoling Qu Department of Pharmacology and Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA Search for more papers by this author Eric Lam Eric Lam Department of Pharmacology and Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA Search for more papers by this author Yong-Qiu Doughman Yong-Qiu Doughman Department of Pediatrics, Rainbow Babies and Children's Hospital, Case Western Reserve University School of Medicine, Cleveland, OH, USA Search for more papers by this author Yu Chen Yu Chen Department of Pharmacology and Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA Search for more papers by this author Yu-Ting Chou Yu-Ting Chou Department of Pharmacology and Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA Search for more papers by this author Minh Lam Minh Lam Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Mona Turakhia Mona Turakhia Department of Pharmacology and Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA Search for more papers by this author Sally L Dunwoodie Sally L Dunwoodie Developmental Biology Program, the Victor Chang Cardiac Research Institute, and Faculties of Science and Medicine, University of New South Wales, Sydney, Australia Search for more papers by this author Michiko Watanabe Michiko Watanabe Department of Pediatrics, Rainbow Babies and Children's Hospital, Case Western Reserve University School of Medicine, Cleveland, OH, USA Search for more papers by this author Bing Xu Bing Xu Department of Pharmacology and Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA Search for more papers by this author Stephen A Duncan Stephen A Duncan Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI, USA Search for more papers by this author Yu-Chung Yang Corresponding Author Yu-Chung Yang Department of Pharmacology and Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Xiaoling Qu Xiaoling Qu Department of Pharmacology and Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA Search for more papers by this author Eric Lam Eric Lam Department of Pharmacology and Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA Search for more papers by this author Yong-Qiu Doughman Yong-Qiu Doughman Department of Pediatrics, Rainbow Babies and Children's Hospital, Case Western Reserve University School of Medicine, Cleveland, OH, USA Search for more papers by this author Yu Chen Yu Chen Department of Pharmacology and Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA Search for more papers by this author Yu-Ting Chou Yu-Ting Chou Department of Pharmacology and Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA Search for more papers by this author Minh Lam Minh Lam Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Mona Turakhia Mona Turakhia Department of Pharmacology and Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA Search for more papers by this author Sally L Dunwoodie Sally L Dunwoodie Developmental Biology Program, the Victor Chang Cardiac Research Institute, and Faculties of Science and Medicine, University of New South Wales, Sydney, Australia Search for more papers by this author Michiko Watanabe Michiko Watanabe Department of Pediatrics, Rainbow Babies and Children's Hospital, Case Western Reserve University School of Medicine, Cleveland, OH, USA Search for more papers by this author Bing Xu Bing Xu Department of Pharmacology and Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA Search for more papers by this author Stephen A Duncan Stephen A Duncan Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI, USA Search for more papers by this author Yu-Chung Yang Corresponding Author Yu-Chung Yang Department of Pharmacology and Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Author Information Xiaoling Qu1, Eric Lam1, Yong-Qiu Doughman2, Yu Chen1, Yu-Ting Chou1, Minh Lam3, Mona Turakhia1, Sally L Dunwoodie4, Michiko Watanabe2, Bing Xu1, Stephen A Duncan5 and Yu-Chung Yang 1,3 1Department of Pharmacology and Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA 2Department of Pediatrics, Rainbow Babies and Children's Hospital, Case Western Reserve University School of Medicine, Cleveland, OH, USA 3Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, USA 4Developmental Biology Program, the Victor Chang Cardiac Research Institute, and Faculties of Science and Medicine, University of New South Wales, Sydney, Australia 5Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI, USA *Corresponding author. Department of Pharmacology, Case Western Reserve University School of Medicine, 2109 Adelbert Road, W353, Cleveland, OH 44106-4965, USA. Tel.: +1 216 368 6931; Fax: +1 216 368 3395; E-mail: [email protected] The EMBO Journal (2007)26:4445-4456https://doi.org/10.1038/sj.emboj.7601883 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The transcriptional modulator Cited2 is induced by various biological stimuli including hypoxia, cytokines, growth factors, lipopolysaccharide (LPS) and flow shear. In this study, we report that Cited2 is required for mouse fetal liver development. Cited2−/− fetal liver displays hypoplasia with higher incidence of cell apoptosis, and exhibits disrupted cell-cell contact, disorganized sinusoidal architecture, as well as impaired lipid metabolism and hepatic gluconeogenesis. Furthermore, we demonstrated the physical and functional interaction of Cited2 with liver-enriched transcription factor HNF4α. Chromatin immunoprecipitation (ChIP) assays further confirmed the recruitment of Cited2 onto the HNF4α-responsive promoters and the reduced HNF4α binding to its target gene promoters in the absence of Cited2. Taken together, this study suggests that fetal liver defects in mice lacking Cited2 result, at least in part, from its defective coactivation function for HNF4α. Introduction Hepatogenesis proceeds through multiple developmental stages. During embryonic development, the induction of hepatic fate depends on the reciprocal interactions between ventral foregut endoderm and adjacent mesenchymal tissues (Zaret, 2002). At around embryonic day 8 (E8) in the mouse, the ventral wall of foregut endoderm initiates its development toward a hepatic fate in response to the inductive growth factor signaling, including fibroblast growth factor (FGF) signaling from the adjacent cardiogenic mesoderm and bone morphogenic proteins (BMPs) signaling from nearby septum transversum (st) mesenchyme (Jung et al, 1999; Rossi et al, 2001; Duncan, 2003). Then cells within the foregut endoderm start to proliferate and differentiate, leading to the formation of the liver bud at E9. Once the liver bud is generated and the hepatoblasts delaminate from the foregut and migrate as cords into the septum transversum mesenchyme, the cells within the embryonic liver environment must reorganize to generate the complex hepatic architecture that is crucial for normal liver function (Zhao and Duncan, 2005). This requires extensive differentiation of the hepatocytes, organization of the extracellular matrix, development of the biliary tract, maturation of sinusoidal capillaries and hepatic vasculature and formation of a polarized epithelium (Lemaigre and Zaret, 2004). While the embryonic liver lacks most of the liver functions in the adult as the center of metabolism, the liver acquires those functions during the perinatal and postnatal stages. Over the last decade, analyses of transcriptional regulation of liver development have identified several liver-enriched transcription factors that are involved in liver development, differentiation and function, including C/EBP family, HNF1α and β, HNF3α, β and γ, HNF4α and HNF6 (Duncan, 2000; Costa et al, 2003). The cumulative data also strongly suggest that HNF4α acts as a central regulator of hepatogenesis, through the activation of a cascade of transcription factors that ultimately define the gene expression profile of the mature hepatocytes (Watt et al, 2003). Genetic studies in mice have also revealed that numerous signaling molecules are required for continued fetal liver growth. Although the combined use of molecular genetics, molecular biology and embryology has allowed us to realize the proceedings in unraveling of the mechanisms that control hepatogenesis, the blueprint regarding hepatic development remains largely unknown. Cited2 is a founding member of a new family of non-DNA-binding transcriptional coregulators (Shioda et al, 1997; Dunwoodie et al, 1998). Members of the Cited family share a well-conserved C-terminal acidic domain (the conserved region 2, CR2) that binds to the transcriptional coactivators CBP/p300 with high affinity (Bhattacharya et al, 1999; Leung et al, 1999). On this basis, Cited proteins are thought to target specific transcriptional responses through interactions with other transcription factors, and to modify these responses through binding with CBP/p300. Indeed, several lines of evidence supported the view that Cited2 associates with various DNA-binding transcription factors and acts as a transcriptional cofactor to regulate gene expression mediated by these proteins, including the LIM domain-containing DNA-binding protein Lhx2 (Glenn and Maurer, 1999), the transcription factor AP-2 (TFAP-2) (Braganca et al, 2003), the nuclear receptors peroxisome proliferator-activated receptors (PPAR) α and γ (Tien et al, 2004), and the TGFβ-signaling mediators Smad2/3 (Chou et al, 2006). An additional role has been proposed for Cited2 as a negative regulator of DNA-binding protein hypoxia-inducible factor-1α (HIF-1α) (Bhattacharya et al, 1999; Freedman et al, 2003). Collectively, these initial in vitro studies underscore the potential roles of Cited2 in different biological processes. Cited2 plays an essential role in mouse embryonic development based on knockout studies (Bamforth et al, 2001, 2004; Martinez-Barbera et al, 2002; Yin et al, 2002; Weninger et al, 2005; Withington et al, 2006). During early mouse embryogenesis, Cited2 expression is clearly evident in the cardiac mesenchyme and septum transversum mesenchyme, which gives rise to aspects of the mature liver (Dunwoodie et al, 1998), suggesting its potential role in hepatogenesis. In this study, we characterized fetal liver defects of Cited2−/− embryos, which are represented by hepatic hypoplasia and dysfunction of hepatocytes. These phenotypes are accompanied by the downregulation of a subset of genes involved in hepatogenesis. Moreover, we demonstrated that Cited2 interacts with HNF4α and coactivates HNF4α-mediated transcription. Therefore, we propose that fetal liver defects observed in Cited2−/− embryos result, at least in part, from its defective function as an HNF4α coactivator. Results Expression of Cited2 in the developing mouse liver During early mouse embryogenesis (8- to 12-somite stage), Cited2 transcripts are localized to the myocardium and septum transversum mesenchyme (Dunwoodie et al, 1998). By E9.25, we found Cited2 continued to express in specified hepatic endodermal cells within the liver bud as well as in the surrounding septum transversum mesenchyme, as demonstrated by the coexpression of hepatocyte-specific marker albumin and β-galactosidase from the Cited2-lacZ allele in heterozygous embryos (Figure 1A). At E10.5, the lacZ- and albumin-double-positive cells could be found in the hepatic primordia, as well as in the mesenchymal portion of the developing liver (Figure 1A). At E12.5, the lacZ-positive staining was detected in hepatocytes (HNF4α-positive cells) as well as in non-hepatocytes (Figure 1A). The Northern blot analysis of developing fetal liver (E12.5–E18.5), new-born (NB) liver and adult liver (8-week-old) further showed that Cited2 transcripts are highly expressed in fetal livers from E12.5 through E16.5, compared with the low expression levels in E17.5 and E18.5 livers and intermediate expression levels in newborn and adult livers (Figure 1B). The RNA in situ hybridization analysis also confirmed the high expression of Cited2 in E14.5 liver (Supplementary Figure 1). Taken together, the expression pattern of Cited2 in developing mouse liver supports the idea that Cited2 may play an important role in liver development and function. Figure 1.Cited2 expression in developing liver. (A) Cited2 expression in liver (E9.25, E10.5 and E12.5) by lacZ staining. Arrows indicate that lacZ-positive cells are also positive for albumin or HNF4α. The lacZ staining (blue) was pseudocolorized to red for the ease in visualization. st, septum transversum mesenchyme. (B) Northern blot analysis of Cited2 expression in developing liver. Cited2−/− fetal liver (KO) was used as a negative control. NB, newborn. Download figure Download PowerPoint Liver hypoplasia in Cited2-deficient embryos To test whether Cited2 plays a role in liver development, we examined the gross morphology of fetal livers from wild-type and Cited2−/− embryos. Compared with wild-type littermate controls, Cited2−/− embryos could be recognized by their growth retardation and hypoplastic liver starting from E13.5. Although smaller in size, liver from Cited2−/− embryos had the correct number of lobes and appeared red, suggesting that the initial steps of liver development are intact in Cited2−/− embryos (Figure 2A). However, liver weight/body weight ratio was significantly decreased in Cited2-null embryos from E13.5 to E17.5 when compared with wild-type controls (Figure 2B). Analysis of hematoxylin and eosin (H&E)-stained liver sections from both E14.5 wild-type and Cited2-null littermates revealed the distorted liver architecture with reduced cellularity, increased sinusoidal space and dissociated parenchymal cells in Cited2−/− fetal liver (Figure 2C). Three possibilities could account for the observed hypocellularity and smaller sizes of Cited2-null livers: (i) increased cell death, (ii) reduced cell proliferation and (iii) disrupted cell–cell interaction. We examined apoptosis by the TUNEL assay and cell proliferation by the BrdU incorporation assay for E14.5 wild-type and Cited2-null liver sections, followed by immunostaining of cell type-specific markers such as vimentin for mesenchymal cells and albumin or HNF4α for hepatocytes. The TUNEL assay showed increased apoptosis in Cited2−/− livers (12.8±1.2%), whereas the wild-type littermate controls had 3.9±1.5% apoptotic cells (Figure 2C). The immunostaining showed that majority of the apoptotic cells were mesenchymal cells, although some were hepatocytes (Figure 2C). The BrdU incorporation assay showed that 37.8±1.8 and 36.9±1.5% of cells in normal and Cited2−/− livers were BrdU positive, respectively, suggesting that wild-type and Cited2-knockout embryos were not significantly different in the proportion of liver cells that are undergoing proliferation (Figure 2C). The proliferating cells were both hepatocytes (albumin positive) and non-hepatocytes (Figure 2C). Figure 2.Liver hypoplasia in Cited2-deficient embryos. (A) Hypoplasia of Cited2−/− embryonic liver compared with a wild-type littermate control. (B) Liver weight/body weight ratio of wild-type versus Cited2−/− embryos from E13.5 to E17.5. The data represent the mean±s.e.m.; n=4–6 mice for each group. *P-value from Student's t-test <0.05 when comparing wild-type with Cited2-null mice. (C) H&E-stained E14.5 sagittal liver sections showed that Cited2−/− embryonic liver has a loosened liver structure with enlarged sinusoidal space and dissociated parenchymal cells (asterisks). TUNEL staining indicated an elevated number of apoptotic cells in E14.5 Cited2−/− liver. Majority of the apoptotic cells (brown) are mesenchymal cells (vimentin positive, arrows), although a few of apoptotic cells are hepatocytes (HNF4α positive, arrowheads). The HNF4α staining (green) was pseudocolorized to blue. The percentage of the apoptotic cells was evaluated from 3–5 sections (5000 cells/section) obtained from three each of wild-type and Cited2−/− embryos. BrdU incorporation assay indicated normal hepatic proliferation in E14.5 Cited2−/− fetal liver. Some of the BrdU-positive cells are hepatocytes (albumin positive, arrows) while some are non-hepatocytes (arrowheads). BrdU staining (brown) was pseudocolorized to blue. The percentage of the BrdU-positive cells was evaluated from 3–5 sections (2000 cells/section) obtained from three each of wild-type and Cited2−/− embryos. Download figure Download PowerPoint To address whether hypoplastic fetal liver phenotype in Cited2−/− embryos results from the impaired cell–cell contact, we investigated cell–cell interaction by examining the localization and expression levels of cell junction markers, E-cadherin and connexin 32, in E14.5 livers. Immunostaining showed that E-cadherin and connexin 32 were primarily membranous in wild-type liver, but the staining was much weaker or even absent from a substantial proportion of cells in Cited2−/− liver, which is suggestive of defective cell–cell interaction (Figure 3A). Furthermore, Western blot analysis of E-cadherin and connexin 32 expression levels confirmed the above observation (Supplementary Figure 2). These preliminary results suggested that mutant hepatocytes might have altered adhesive properties. We therefore cultured E14.5 hepatocytes from normal and mutant livers to directly test cell adhesion. Hematoxylin staining showed that wild-type hepatocytes were able to adhere and form good epithelial sheets on the extracellular matrix, collagen, whereas Cited2−/− hepatocytes failed to adhere to the substrate (Figure 3B). Unlike cells from wild type, most cells from Cited2−/− liver were small and round and adhered loosely to the few cells that attached to the substrate (Figure 3B). Viability of these cells was monitored using trypan blue staining and the results indicated that the small round-shaped cells were not dead but were non-adherent (data not shown). Rhodamine–phalloidin staining showed that normal hepatocytes primarily expressed cortical actin bundles, further confirming the epithelial nature of these cells (Figure 3B). However, Cited2−/− hepatocytes contained multiple stress fibers (Figure 3B). E-cadherin was detected at the site of juxtaposed plasma membrane of wild-type hepatocytes, but the expression was much weaker in Cited2−/− hepatocytes (Figure 3B). Furthermore, adenovirus-mediated exogenous Cited2 expression could rescue the ability of Cited2−/− hepatocytes to adhere to the substrate (Supplementary Figure 3). Figure 3.Disruption of cell–cell interaction in Cited2−/− fetal liver. (A) Immunostaining showed that the membranous expression of E-cadherin and connexin 32 was intense in E14.5 wild-type liver but much weaker or even absent in a substantial proportion of cells in Cited2−/− littermate liver. (B) Perturbation of cellular adhesion in E14.5 Cited2−/− hepatocytes. Hematoxylin staining, rhodamine–phalloidin staining and E-cadherin immunostaining indicated defective adhesion of Cited2−/− hepatocytes. Download figure Download PowerPoint Disorganized sinusoidal architecture, impaired lipid homeostasis and hepatic gluconeogenesis in Cited2−/− fetal liver Since well-developed sinusoidal architecture is necessary for liver function, we examined if the sinusoidal architecture is normal in Cited2−/− liver. PECAM-1 immunostaining for E16.5 liver sections showed that wild-type liver had very organized sinusoidal architecture, with PECAM-1-positive endothelial cells surrounding large vessels as well as sinusoids, which were distributed throughout the parenchyma (Figure 4A). In contrast, the sinusoidal structure of Cited2−/− liver was greatly disrupted, exhibiting disorganized sinusoidal capillaries (Figure 4A). Figure 4.Disorganized sinusoidal architecture, impaired lipid homeostasis and hepatic gluconeogenesis in Cited2−/− fetal liver. (A) Disorganized sinusoidal architecture was identified by PECAM-1 immunostaining (brown) in E16.5 Cited2−/− fetal liver. (B) Oil red O staining showed that abundant fine- to medium-sized lipid storage droplets accumulated diffusely in E17.5 Cited2−/− liver, whereas lipid storage droplets were rarely observed in wild-type fetal liver. Ultrastructural analysis showed abundant lipid droplets accumulated in the sinusoidal space in E17.5 Cited2−/− hepatocytes (asterisks). Original magnification: × 6000. (C) PAS staining revealed impaired glycogen storage in E17.5 Cited2−/− fetal liver, compared with wild type. Download figure Download PowerPoint Proper formation of the intricate sinusoidal vascular network provides all hepatocytes direct access to the blood plasma and efficient lipoprotein metabolism (Postic et al, 2004). Therefore, the disrupted sinusoidal architecture observed in Cited2−/− liver could affect the ability of the liver to properly function in lipid homeostasis. Oil red O staining showed abundant fine- to medium-sized lipid storage droplets accumulated diffusely in Cited2−/− liver, whereas lipid storage droplets were rarely observed in wild-type liver (Figure 4B). Electron microscopic analysis further showed numerous lipid droplets being accumulated in the sinusoidal space in E17.5 Cited2−/− liver, whereas normal liver contained less lipid droplets (Figure 4B). In addition, periodic acid–Schiff (PAS) histochemistry revealed that E17.5 wild-type liver exhibited robust levels of glycogen accumulation. In contrast, accumulation of intracellular glycogen was greatly reduced in E17.5 Cited2−/− liver, which displayed only sparse and weaker glycogen staining than wild-type liver (Figure 4C). Gene expression profiling in Cited2−/− fetal liver To determine whether loss of Cited2 alters the gene expression profile in developing liver, we performed Affymetrix mouse oligonucleotide gene array analysis, which identified 160 and 238 genes whose expression was downregulated and upregulated⩾2-fold (P⩽0.05) in E14.5 Cited2-null liver when compared with wild-type liver, respectively. To validate microarray data, we compared steady-state mRNA levels of differentially expressed genes identified by microarray in sorted wild-type and Cited2-null hepatocytes by real-time RT–PCR. We isolated the hepatocyte populations (albumin+CD45−Ter119−) from wild-type and Cited2−/− fetal livers (E14.5 and E17.5) by flow cytometry. The percentage of hepatocytes (albumin+CD45−Ter119−) in Cited2-null embryos (16.66% in E14.5 liver and 20.03% in E17.5 liver) was slightly lower than that in wild-type (18.10% in E14.5 liver and 21.07% in E17.5) littermate controls. The RNA samples extracted from sorted wild-type and Cited2-null hepatocytes were used for real-time RT–PCR. The downregulated genes were categorized based on their biological functions. Figure 5A shows that in the absence of Cited2, the steady-state mRNA levels of a subset of genes which are involved in cell–cell contact assembly (E-cadherin (E-cad), gap junction protein β1 (Gjb1, also known as connexin 32), claudin 1 (Cldn1), occludin (Ocln), F11 receptor (F11r), crumb 3 (Crb3)), hepatic carbohydrate homeostasis (amylase 2 (AMY2), arginase 1 (ARG1), PPARγ coactivator-1α (PGC-1α), glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), glucokinase (GK)) and several well-characterized hepatic differentiation markers (transferrin (TFN), transthyretin (TTR), tyrosine amino transferase (TAT), inter-α-trypsin inhibitor-4 (Itih-4), haptoglobin (HP)) were downregulated. However, the mRNA expression levels of several hepatic regulators such as HNF4α, HNF1α, HNF1β, HNF3 and HNF6 did not show significant difference between sorted wild-type and Cited2−/− hepatocytes (Figure 5A). Notably, most of these genes with altered expression profiles are putative HNF4α target genes when compared with the array data of fetal liver-specific HNF4α deficient mouse (Battle et al, 2006; Supplementary Table 1). It is also worth noting that this downregulation of HNF4α target genes by Cited2 deficiency does not appear to be due to decreased expression of HNF4α mRNA, since real-time RT–PCR did not show statistical difference (<2-fold) in HNF4α expression levels between wild-type and Cited2-null livers (Figure 5A). The immunostaining (Figure 5B) and Western blot analysis (Figure 5C) of HNF4α protein levels in wild-type and Cited2−/− liver also did not show significant difference. Rather, these results are consistent with the observation that Cited2 coactivates HNF4α through direct physical association and augmentation of its transcriptional activity (see below). Figure 5.Gene expression profiling in Cited2−/− fetal liver. (A) Compared with wild type, a subset of genes involved in cell junction assembly (E-cadherin, Gjb1, Cldn1, Ocln, F11r, Crb3), hepatic carbohydrate metabolism (AMY2, ARG1, PGC-1, G6Pase, PEPCK, GK), as well as several well-characterized hepatic differentiation markers (TF, TTR, TAT, Itih-4, HP) were downregulated in Cited2−/− hepatocytes. The steady-state mRNA levels of HNF4α, HNF1α, HNF1β, HNF3 and HNF6 in wild-type and Cited2-null hepatocytes did not show significant difference (<2-fold). Bars represent the mean±s.e.m.; n=3–5 RNA samples from different mice for each group. The immunofluorescence (B) and western blot analysis (C) of HNF4α proteins in wild-type and Cited2−/− liver did not show significant difference. The immunofluorescence of HNF4α was presented as grayscale. Download figure Download PowerPoint Cited2 acts as a coactivator for HNF4α Cited2 is a transcriptional cofactor that in vitro can act as either a positive or a negative regulator of transcription (Bhattacharya et al, 1999; Glenn and Maurer, 1999; Braganca et al, 2003; Tien et al, 2004; Chou et al, 2006). Therefore, one mechanism that may underlie the abnormalities observed in Cited2−/− fetal liver is defective coactivation of a transcription factor that is critical for liver development and function. Because Cited2-deficient fetal liver displays overlapping phenotypes with those of fetal liver-specific HNF4α knockout, we hypothesized that Cited2 could serve as a coactivator for HNF4α and regulate hepatic gene expression during liver development and differentiation. To test this possibility, we first examined subcellular localization of Cited2 and HNF4α proteins by confocal microscopic analysis, which showed that these two proteins colocalized in the nuclei (Supplementary Figure 4). The in vitro GST pull-down assay showed that GST-Cited2, but not GST, efficiently pulled down Myc-HNF4α protein (Figure 6A), indicating the interaction of Cited2 with HNF4α in vitro. Mammalian two-hybrid analysis further showed that HNF4α failed to interact with the Cited2 mutant missing its C-terminal region, but was still able to bind to the mutant without the N-terminal amino acids (Figure 6B), indicating that Cited2 C-terminal region is essential for interacting with HNF4α. Figure 6.Physical and functional interaction of Cited2 with HNF4α. (A) Interaction of Cited2 with HNF4α in vitro. GST or GST-Cited2 was immobilized on glutathione-conjugated Sepharose beads and incubated with lysates from cells transfected with a plasmid expressing Myc-HNF4α protein or vector alone (mock). After extensive washing, coprecipitated Cited2 was detected by anti-Myc western blotting (WB). (B) Cited2 C-terminal region is essential for interacting with HNF4α. All the luciferase reporter assays in (C–E) were performed as described in Materials and methods. (C) Cited2 enhances HNF4α-mediated apoCIII transcription in a dose-dependent manner. (D) Coactivator p300 synergizes with Cited2 to enhance HNF4α transcriptional activity in a dose-dependent manner. The data represent the mean±s.e.m.; n=3–5 samples for each group in panels B–D. *P-value from Student's t-test <0.05 when comparing the two indicated bars. (E) Loss of Cited2 abrogates HNF4α-mediated transactivation activity. HNF4α and various amounts of apoCIII promoter/reporter plasmids were cotransfected into wild-type and Cited2-null cells, followed by luciferase reporter assays. The data represent the mean±s.e.m.; n=3–5 samples for each group. *P-value from Student's t-test <0.05 when comparing wild-type with Cited2-null cells. Download figure Download PowerPoint The functional significance of the identified interaction between Cited2 and HNF4α was further assessed in the luciferase reporter assay. HepG2 cells (with endogenous HNF4α expression), or HeLa and HEK293 cells (without endogenous HNF4α expression) were cotransfected with the plasmid expressing HNF4α, apoCIII promoter/luciferase reporter plasmid harboring HNF4α-responsive elements, together w