Francisco M. Rausa

Elevated Levels of Hepatocyte Nuclear Factor 3β in Mouse Hepatocytes Influence Expression of Genes Involved in Bile Acid and Glucose Homeostasis

ABSTRACT The winged helix transcription factor, hepatocyte nuclear factor-3β (HNF-3β), mediates the hepatocyte-specific transcription of numerous genes important for liver function. However, the in vivo role of HNF-3β in regulating these genes remains unknown because homozygous null HNF3β mouse embryos die in utero prior to liver formation. In order to examine the regulatory function of HNF-3β, we created transgenic mice in which the −3-kb transthyretin promoter functions to increase hepatocyte expression of the rat HNF-3β 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-3β on the hepatic expression of the endogenous mouse HNF-3α,-3β, -3γ, 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-3β transcriptional network regulates expression of hepatocyte-specific genes required for bile acid and glucose homeostasis, as well as postnatal growth.

The nuclear receptor fetoprotein transcription factor is coexpressed with its target gene HNF-3β in the developing murine liver intestine and pancreas

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.

Association between Hepatocyte Nuclear Factor 6 (HNF-6) and FoxA2 DNA Binding Domains Stimulates FoxA2 Transcriptional Activity but Inhibits HNF-6 DNA Binding

ABSTRACT 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 3β [HNF-3β]), 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.

Stability of the Hepatocyte Nuclear Factor 6 Transcription Factor Requires Acetylation by the CREB-binding Protein Coactivator

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 Foxa2·HNF6 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 HNF6·Foxa2 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 Foxa2·HNF6 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.

C/EBPα and HNF6 protein complex formation stimulates HNF6‐dependent transcription by CBP coactivator recruitment in HepG2 cells

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 α (C/EBPα), but not by the related C/EBPβ or C/EBPδ proteins. Formation of the C/EBPα–HNF6 protein complex required the HNF6 cut domain and the C/EBPα activation domain (AD) 1/AD2 sequences. This C/EBPα–HNF6 transcriptional synergy required both the N‐terminal HNF6 polyhistidine and serine/threonine/proline box sequences, as well as the C/EBPα 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/EBPα–HNF6 transcriptional synergy in cotransfection assays. Co‐immunoprecipitation assays with liver protein extracts demonstrate an association between the HNF6 and C/EBPα transcription factors and the CBP coactivator protein in vivo. Furthermore, chromatin immunoprecipitation assays with hepatoma cells demonstrated that increased levels of both C/EBPα 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/EBPα–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.)