Decreased pulmonary expression of Forkhead Box f1 (Foxf1) transcription factor was associated with lethal alveolar hemorrhage in 55% of the Foxf1 +/- newborn mice. The severity of the pulmonary abnormalities correlates with the levels of Foxf1 mRNA. Defects in alveolarization and vasculogenesis were observed in subsets of the Foxf1 +/- mice with relatively low levels of expression from the normal Foxf1 allele. Lung hemorrhage was coincident with disruption of the mesenchymal-epithelial cell interfaces in the alveolar and bronchiolar regions of the lung parenchyma and was associated with increased apoptosis and reduced surfactant protein B (SP-B) expression. Finally, the lung defect associated with the Foxf1 +/- mutation was accompanied by reduced expression of vascular endothelial growth factor (VEGF), the VEGF receptor 2 (Flk-1), bone morphogenetic protein 4 (Bmp-4), and the transcription factors of the Brachyury T-Box family (Tbx2-Tbx5) and Lung Kruppel-like Factor. Reduction in the level of Foxf1 caused neonatal pulmonary hemorrhage and abnormalities in alveologenesis, implicating this transcription factor in the regulation of mesenchyme-epithelial interaction critical for lung morphogenesis.

Murine hepatocyte nuclear factor-3 beta (HNF-3 beta) protein is a member of a large family of developmentally regulated transcription factors that share homology in the winged helix/fork head DNA binding domain and that participate in embryonic pattern formation. HNF-3 beta also mediates cell-specific transcription of genes important for the function of hepatocytes, intestinal and bronchiolar epithelial, and pancreatic acinar cells. We have previously identified a liver-enriched transcription factor, HNF-6, which is required for HNF-3 beta promoter activity and also recognizes the regulatory region of numerous hepatocyte-specific genes. In this study we used the yeast one-hybrid system to isolate the HNF-6 cDNA, which encodes a cut-homeodomain-containing transcription factor that binds with the same specificity as the liver HNF-6 protein. Cotransfection assays demonstrate that HNF-6 activates expression of a reporter gene driven by the HNF-6 binding site from either the HNF-3 beta or transthyretin (TTR) promoter regions. We used interspecific backcross analysis to determine that murine Hnf6 gene is located in the middle of mouse chromosome 9. In situ hybridization studies of staged specific embryos demonstrate that HNF-6 and its potential target gene, HNF-3 beta, are coexpressed in the pancreatic and hepatic diverticulum. More detailed analysis of HNF-6 and HNF-3 beta's developmental expression patterns provides evidence of colocalization in hepatocytes, intestinal epithelial, and in the pancreatic ductal epithelial and exocrine acinar cells. The expression patterns of these two transcription factors do not overlap in other endoderm-derived tissues or the neurotube. We also found that HNF-6 is also abundantly expressed in the dorsal root ganglia, the marginal layer, and the midbrain. At day 18 of gestation and in the adult pancreas, HNF-6 and HNF-3 beta transcripts colocalize in the exocrine acinar cells, but their expression patterns diverge in other pancreatic epithelium. HNF-6, but not HNF-3 beta, expression continues in the pancreatic ductal epithelium, whereas only HNF-3 beta becomes restricted to the endocrine cells of the islets of Langerhans. We discuss these expression patterns with respect to specification of hepatocytes and differentiation of the endocrine and exocrine pancreas.

The winged helix transcription factor, hepatocyte nuclear factor-3␤ (HNF-3␤), mediates the hepatocytespecific transcription of numerous genes important for liver function. However, the in vivo role of HNF-3␤ in regulating these genes remains unknown because homozygous null HNF3␤ mouse embryos die in utero prior to liver formation. In order to examine the regulatory function of HNF-3␤, we created transgenic mice in which the ؊3-kb transthyretin promoter functions to increase hepatocyte expression of the rat HNF-3␤ protein.Postnatal transgenic mice exhibit growth retardation, depletion of hepatocyte glycogen storage, and elevated levels of bile acids in serum. The retarded growth phenotype is likely due to a 20-fold increase in hepatic expression of insulin-like growth factor binding protein 1 (IGFBP-1), which results in elevated levels in serum of IGFBP-1 and limits the biological availability of IGFs required for postnatal growth. The defects in glycogen storage and serum bile acids coincide with diminished postnatal expression of hepatocyte genes involved in gluconeogenesis (phosphoenolpyruvate carboxykinase and glycogen synthase) and sinusoidal bile acid uptake (Ntcp), respectively. These changes in gene transcription may result from the disruptive effect of HNF-3␤ on the hepatic expression of the endogenous mouse HNF-3␣,-3␤, -3␥, and -6 transcription factors. Furthermore, adult transgenic livers lack expression of the canalicular phospholipid transporter, mdr2, which is consistent with ultrastructure evidence of damage to transgenic hepatocytes and bile canaliculi. These transgenic studies represent the first in vivo demonstration that the HNF-3␤ transcriptional network regulates expression of hepatocyte-specific genes required for bile acid and glucose homeostasis, as well as postnatal growth.The liver performs essential functions in the body by uniquely expressing both hepatocyte-specific genes encoding plasma proteins and enzymes involved in the detoxification and in the homeostasis of glucose, cholesterol, and bile salts (4). Functional analysis of numerous hepatocyte-specific promoter and enhancer regions reveals that they are composed of multiple cis-acting DNA sequences that bind different families of hepatocyte nuclear factors (HNF) (reviewed in reference 4). These include the HNF-1, HNF-3, HNF-4, CCAAT/enhancer binding protein (C/EBP), HNF-6, and fetoprotein transcription factor families (4,29,15,52,53,57). Although none of these transcriptional regulatory proteins is entirely liver specific, the requirement for combinatorial protein interactions among them in order to achieve abundant transcriptional activity plays an important role in maintaining hepatocyte-specific gene expression.The HNF-3 proteins are members of an extensive family of transcription factors that share homology in the winged helix DNA binding domain and use a modified helix-turn-helix motif to bind DNA as a monomer (8, 37). To date, the winged helix family consists of over 50 members, which play important roles in the differentiat...

In previous studies we used transgenic mice or recombinant adenovirus infection to increase hepatic expression of forkhead box A2 (FoxA2, previously called hepatocyte nuclear factor 3␤ [HNF-3␤]), which caused diminished hepatocyte glycogen levels and reduced expression of glucose homeostasis genes. Because this diminished expression of FoxA2 target genes was associated with reduced levels of the Cut-Homeodomain HNF-6 transcription factor, we conducted the present study to determine whether there is a functional interaction between HNF-6 and FoxA2. Human hepatoma (HepG2) cotransfection assays demonstrated that HNF-6 synergistically stimulated FoxA2 but not FoxA1 or FoxA3 transcriptional activity, and protein-binding assays showed that this protein interaction required the HNF-6 Cut-Homeodomain and FoxA2 winged-helix DNA binding domains. Furthermore, we show that the HNF-6 Cut-Homeodomain sequences were sufficient to synergistically stimulate FoxA2 transcriptional activation by recruiting the p300/CBP coactivator proteins. This was supported by the fact that FoxA2 transcriptional synergy with HNF-6 was dependent on retention of the HNF-6 Cut domain LXXLL sequence, which mediated recruitment of the p300/CBP proteins. Moreover, cotransfection and DNA binding assays demonstrated that increased FoxA2 levels caused a decrease in HNF-6 transcriptional activation of the glucose transporter 2 (Glut-2) promoter by interfering with the binding of HNF-6 to its target DNA sequence. These data suggest that at a FoxA-specific site, HNF-6 serves as a coactivator protein to enhance FoxA2 transcription, whereas at an HNF-6-specific site, FoxA2 represses HNF-6 transcription by inhibiting HNF-6 DNA binding activity. This is the first reported example of a liver-enriched transcription factor (HNF-6) functioning as a coactivator protein to potentiate the transcriptional activity of another liver factor, FoxA2.

The ␣2,6-sialyltransferase (ST) is a Golgi glycosyltransferase that adds sialic acid residues to glycoprotein N-linked oligosaccharides. Here we show that two forms of ␣2,6-sialyltransferase are expressed by the liver and are encoded by two different RNAs that differ by a single nucleotide. The ST tyr possesses a Tyr at amino acid 123, whereas the ST cys possesses a Cys at this position. The ST tyr is more catalytically active than the ST cys; however, both are functional when introduced into tissue culture cells. The proteolytic processing and turnover of the ST tyr and ST cys proteins differ dramatically. The ST cys is retained intact in COS-1 cells, whereas the ST tyr is rapidly cleaved and secreted. Analysis of the N-linked oligosaccharides of these proteins demonstrates that both proteins enter the late Golgi. However, differences in ST tyr and ST cys proteolytic processing may be related to differences in their localization, because ST tyr but not ST cys is expressed at low levels on the cell surface. The possibility that the ST tyr is cleaved in a post-Golgi compartment is supported by the observation that a 20°C temperature block, which stops protein transport in the trans Golgi network, blocks both cleavage and secretion of the ST tyr.

During organogenesis, the winged helix hepatocyte nuclear factor 3beta (HNF-3beta) protein participates in regulating gene transcription in the developing esophagus, trachea, liver, lung, pancreas, and intestine. Hepatoma cell transfection studies identified a critical HNF-3beta promoter factor, named UF2-H3beta, and here, we demonstrate that UF2-H3beta is identical to the fetoprotein transcription factor (FTF). In situ hybridization studies of mouse embryos demonstrate that FTF expression initiates in the foregut endoderm during liver and pancreatic morphogenesis (day 9) and that earlier expression of FTF is observed in the yolk sac endoderm, branchial arch and neural crest cells (day 8). Abundant FTF hybridization signals are observed throughout morphogenesis of the liver, pancreas, and intestine and its expression continues in the epithelial cells of these adult organs. In day 17 mouse embryos and adult pancreas, however, expression of FTF becomes restricted to the exocrine acinar and ductal epithelial cells.

We previously demonstrated that the formation of complexes between the DNA binding domains of the hepatocyte nuclear factor 6 (HNF6) and Forkhead Box a2 (Foxa2) transcription factors resulted in synergistic transcriptional activation of a Foxa2 target promoter. This Foxa2⅐HNF6 transcriptional synergy was mediated by the recruitment of CREB-binding protein (CBP) coactivator through the HNF6 Cut-Homeodomain sequences. Although the HNF6 DNA binding domain sequences are sufficient to recruit CBP coactivator for HNF6⅐Foxa2 transcriptional synergy, paradoxically these HNF6 Cut-Homeodomain sequences were unable to stimulate the transcription of an HNF6-dependent reporter gene. Here, we investigated whether the CBP coactivator protein played a different role in regulating HNF6 transcriptional activity. We showed that acetylation of the HNF6 protein by CBP increased both HNF6 protein stability and its ability to stimulate transcription of the glucose transporter 2 promoter. Mutation of the HNF6 Cut domain lysine 339 residue to an arginine residue abrogated CBP acetylation, which is required for HNF6 protein stability. Furthermore, the HNF6 K339R mutant protein, which failed to accumulate detected protein levels, was transcriptionally inactive and could not be stabilized by inhibiting the ubiquitin proteasome pathway. Finally, increased HNF6 protein levels stabilized the Foxa2 protein, presumably through the formation of the Foxa2⅐HNF6 complex. These studies show for the first time that HNF6 protein stability is controlled by CBP acetylation and provides a novel mechanism by which the activity of the CBP coactivator may regulate steady levels of two distinct liver-enriched transcription factors.Synergistic transcriptional activation of hepatocyte-specific genes required the simultaneous binding of multiple families of liver-enriched transcription factors to the DNA regulatory regions of these cell type-specific genes (1). On the basis of homology within DNA binding domains of these regulatory hepatocyte nuclear factors (HNF), 1 the liver-enriched transcription factors were grouped into related protein families. These include the Forkhead Box (Fox) Foxa1 (HNF3␣), Foxa2 (HNF3␤), and Foxa3 (HNF3␥) proteins (2-5), the "ONECUT" (OC) HNF6 proteins (6 -9), the steroid hormone orphan receptor HNF4 proteins (10), the POU homeodomain-containing HNF1 proteins (11, 12), and the basic region leucine zipper CCAAT/ enhancer-binding protein (C/EBP) proteins (13,14). Many of these liver transcription factors recruit the p300/ CREB-binding protein (CBP) family of histone acetyltransferases to activate transcription of hepatocyte-specific genes. These include the HNF4␣ (15, 16), HNF1␣ (17, 18), HNF6 (19), and C/EBP transcription factors (20). Furthermore, stimulation of HNF4␣ transcriptional activity is mediated by CBP acetylation of a lysine residue within the nuclear localization sequence of the HNF4␣ protein, causing an increase in its nuclear retention (21). The p300/CBP coactivator proteins stimulate gene transcription by acetylating p...

We previously demonstrated that formation of complexes between the DNA-binding domains of hepatocyte nuclear factor 6 (HNF6) and forkhead box a2 (Foxa2) proteins stimulated Foxa2 transcriptional activity. Here, we used HepG2 cell cotransfection assays to demonstrate that HNF6 transcriptional activity was stimulated by CCAAT/enhancer-binding protein ␣ (C/EBP␣), but not by the related C/EBP␤ or C/EBP␦ proteins. Formation of the C/EBP␣-HNF6 protein complex required the HNF6 cut domain and the C/EBP␣ activation domain (AD) 1/AD2 sequences. This C/EBP␣-HNF6 transcriptional synergy required both the N-terminal HNF6 polyhistidine and serine/threonine/proline box sequences, as well as the C/EBP␣ AD1/AD2 sequences, the latter of which are known to recruit the CREB binding protein (CBP) transcriptional coactivator. Consistent with these findings, adenovirus E1A-mediated inhibition of p300/CBP histone acetyltransferase activity abrogated C/EBP␣-HNF6 transcriptional synergy in cotransfection assays. Co-immunoprecipitation assays with liver protein extracts demonstrate an association between the HNF6 and C/EBP␣ transcription factors and the CBP coactivator protein in vivo. Furthermore, chromatin immunoprecipitation assays with hepatoma cells demonstrated that increased levels of both C/EBP␣ and HNF6 proteins were required to stimulate association of these transcription factors and the CBP coactivator protein with the endogenous mouse Foxa2 promoter region. In conclusion, formation of the C/EBP␣-HNF6 protein complex stimulates recruitment of the CBP coactivator protein for expression of Foxa2, a transcription factor critical for regulating expression of hepatic gluconeogenic genes during fasting. (HEPATOLOGY 2006;43:276-286.) T ransfection studies in hepatoma cell lines demonstrated that synergistic transcriptional activation of hepatocyte-specific genes requires simultaneous binding of multiple hepatocyte nuclear factor to their promoter/enhancer regions. 1 However, the mechanisms by which these hepatocyte nuclear factor transcription factors functionally interact to elicit transcriptional synergy have yet to be deciphered. The CCAAT/enhancer Abbreviations: HNF6, hepatocyte nuclear factor 6; Foxa2, forkhead box a2; C/EBP␣, CCAAT/enhancer-binding protein