Inhibitor of the Tissue-Specific Transcription Factor HNF4, a Potential Regulator in Early Xenopus Development

Abstract

Hepatocyte nuclear factor 4␣ (HNF4␣) is an orphan receptor of the nuclear receptor superfamily and expressed in vertebrates as a tissue-specific transcription factor in liver, kidney, intestine, stomach, and pancreas. It also plays a crucial role in early embryonic development and has been identified as a maternal component in the Xenopus egg. We now report on an activity present in Xenopus embryos that inhibits the DNA binding of HNF4. This HNF4 inhibitor copurifies with a 25-kDa protein under nondenaturing conditions but can be separated from this protein by sodium dodecyl sulfate treatment. Protease treatment of the inhibitor results in a core fragment of about 5 kDa that retains full inhibitory activity. The activity of the HNF4 inhibitor can also be monitored in the absence of DNA, as it alters the mobility of the HNF4 protein in native polyacrylamide gels and the accessibility of antibodies. Comparing the activity of the HNF4 inhibitor with acyl coenzyme A's, recently proposed to be ligands of HNF4, we observe a more stringent specificity for the HNF4 inhibitor activity. Using deletion constructs of the HNF4 protein, we could show that the potential ligandbinding domain of HNF4 is not required, and thus the HNF4 inhibitor does not represent a classical ligand as defined for the nuclear receptor superfamily. Based on our previous finding that maternal HNF4 is abundantly present in Xenopus embryos but the target gene HNF1␣ is only marginally expressed, we propose that the HNF4 inhibitor functions in the embryo to restrict the activity of the maternal HNF4 proteins.Hepatocyte nuclear factor 4 (HNF4) constitutes transcription factor subfamily 2A (28), whose first member, HNF4␣ (NR2A1), has been identified as a factor interacting with promoter elements mediating liver-specific transcription (35). Based on the zinc finger motif of the DNA-binding domain and on a potential ligand-binding domain, HNF4 is classified as a member of the nuclear orphan receptor superfamily (33). Recently, it has been reported that acyl coenzyme A's (acylCoAs) are potential ligands of HNF4␣: acyl-CoAs containing fatty acids with 16 C residues or shorter act as agonists by increasing the DNA-binding potential of HNF4␣, whereas acyl-CoAs with 18 C residues or longer have antagonistic properties and inhibit DNA binding of HNF4␣ (11). HNF4␣ turned out to be present as well in nonhepatic cells such as kidney, intestine, stomach, and pancreas (23,38,45). The importance of HNF4␣ in gene control in tissues distinct from the liver has been documented by the fact that an inherited human disease is based on the expression of a mutated HNF4␣ gene in the ␤ cells of the endocrine pancreas, leading to maturity-onset diabetes of the young (MODY1 [42]). Most interestingly, another MODY gene identified in humans represents the tissuespecific transcription factor HNF1␣ (43), known to be tightly regulated by HNF4 (17,39,44).In addition to its role as a tissue-specific transcription factor, HNF4␣ is also a maternal component in the egg of Drosophila melano...

Mutations in the human genes encoding the transcription factors of the hepatocyte nuclear factor (HNF)1 and HNF4 families: functional and pathological consequences

“…A series of coactivators have been identified that interact with defined regions of the HNF4 protein (listed in Sladek & Seidel 2001). No definitive ligand for HNF4 has been identified as the recent claim that fatty acylcoenzyme A thioesters might be ligands (Hertz et al 1998) has been challenged by the finding that these compounds exert only a minor effect and have a limited specificity (Peiler et al 2000, Sladek & Seidel 2001. Thus HNF4 remains an orphan receptor, but the identification of potential ligands is still an important issue as such compounds might be important in influencing HNF4 activity in the organism.…”

“…Thus HNF4 remains an orphan receptor, but the identification of potential ligands is still an important issue as such compounds might be important in influencing HNF4 activity in the organism. Recently, a low molecular compound has been identified in embryonic extracts of Xenopus that inhibits quite specifically the DNA binding properties of HNF4 (Peiler et al 2000). However, this inhibitor is distinct from the usual ligands of the nuclear hormone receptor superfamily, as it does not require the ligand binding domain of HNF4 to exert its inhibitory function.…”

“…However, this effect was significantly lower as compared with an artificial HNF4 deletion construct (amino acids 1 to 358) lacking the AF-2 module (Laine et al 2000). As R154X is known to bind to DNA albeit less efficiently (Lausen et al 2000, Peiler et al 2000, the reduction of the activity of the cotransfected wild-type protein probably reflects some dilution of the strong wild-type activity and should not be considered as a dominant negative effect. Most similar data were obtained for the nonsense mutant, Q268X, by showing that this mutant lacks DNA binding, has some altered localization in the cell compartment and has lost its transactivation potential.…”

“…The nonsense mutant, R154X, retained some reduced DNA binding activity (Lausen et al 2000, Peiler et al 2000 and showed some residual transactivation potential in transfection experiments depending on the cell type used (Laine et al 2000, Lausen et al 2000. One report found an inhibitory effect of the mutated HNF4 protein as assayed in transfection experiments by adding increasing amounts of the R154X mutant to wild-type protein.…”

Hepatocyte Nuclear Factor (HNF)‐4α Induces Expression of Endothelial Fas Ligand (FasL) to Prevent Cancer Cell Transmigration: A Novel Defense Mechanism of Endothelium against Cancer Metastasis

“…an HNF-4α inhibitor is abundantly present in Xenopus embryos, in which the HNF-1 gene is only marginally expressed, 42) we propose that similar kinds of inhibitors might be present in RLE cells to restrict the activity of the HNF-4α.…”