HNF1 Protein Research Articles

NR2F1 disrupts synergistic activation of the MTTP gene transcription by HNF-4α and HNF-1α

Regulation of microsomal triglyceride transfer protein (MTP) expression mainly occurs at the transcriptional level. We have previously shown that MTTP gene expression was repressed in nondifferentiated intestinal cells by nuclear receptor 2 family 1 (NR2F1). However, mechanisms involved in the repression of MTP by NR2F1 were not elucidated. Here, we show that MTP expression requires hepatic nuclear factor (HNF)-4α transcription factor. Different HNF-1 proteins synergistically enhance MTP promoter activity along with HNF-4α by binding to different cis elements. NR2F1 does not alter individual effects of HNF-4α and HNF-1 proteins on the MTTP gene promoter. However, NR2F1 suppresses synergistic activation of the MTP promoter by HNF-4α/HNF-1α by binding to a direct repeat 1 (DR1) element. This suppression is further enhanced in the presence of nuclear receptor corepressor 1. In short, these studies identified a novel mechanism of MTP repression that involves binding of NR2F1 to the DR1 element and recruitment of corepressors. In this mechanism, NR2F1 does not affect activities of individual transcription factors; instead, it abrogates synergistic activation by HNF-4α and HNF-1 proteins.

Transcription factors in liver development, differentiation, and regeneration.

Summary and Future Directions. The liver-en-riched transcription factors (C/EBP, HNF1, HNF3,HNF4, and HNF6) bind to multiple promoter/enhancersites and synergistically interact with each other to stim-ulate hepatocyte-specific gene transcription. Liver devel-opment requires retention of numerous proliferation-specific transcription factors that required proliferation,migration, and survival of hepatic progenitor cells. TheHNF6 and HNF1 proteins are critical for gallbladderand bile duct development, while the mesodermal Foxf1transcription factor is essential for gallbladder develop-ment. The HNF1 , HNF4 , Foxa2, and Foxa3 tran-scription factors regulate expression of genes critical forhepatocyte differentiation during embryonic and postna-talliverdevelopment.ThenuclearorphanreceptorsLXR,FXR, and PPAR are also known to play critical roles inregulating expression of hepatic genes involved in lipid,cholesterol, and bile acid metabolism. These nuclear re-ceptor knockout mouse studies have been summarized in

Hyperphenylalaninemia and Impaired Glucose Tolerance in Mice Lacking the Bifunctional DCoH Gene

The bifunctional protein DCoH (Dimerizing Cofactor for HNF1) acts as an enzyme in intermediary metabolism and as a binding partner of the HNF1 family of transcriptional activators. HNF1 proteins direct the expression of a variety of genes in the liver, kidney, pancreas, and gut and are critical to the regulation of glucose homeostasis. Mutations of the HNF1alpha gene underlie maturity onset diabetes of the young (MODY3) in humans. DCoH acts as a cofactor for HNF1 that stabilizes the dimeric HNF1 complex. DCoH also catalyzes the recycling of tetrahydrobiopterin, a cofactor of aromatic amino acid hydroxylases. To examine the roles of DCoH, a targeted deletion allele of the murine DCoH gene was created. Mice lacking DCoH are viable and fertile but display hyperphenylalaninemia and a predisposition to cataract formation. Surprisingly, HNF1 function in DCoH null mice is only slightly impaired, and mice are mildly glucose-intolerant in contrast to HNF1alpha null mice, which are diabetic. DCoH function as it pertains to HNF1 activity appears to be partially complemented by a newly identified homolog, DCoH2.

Pyridoxal 5′-phosphate inhibits DNA binding of HNF1

An efficient Escherichia coli expression system was constructed for the production of a variant form of HNF1 protein having the additional five amino acid residues (Asp-Arg-Trp-Gly-Ser) at the NH 2-terminal. The cDNA encoding HNF1 was ligated to 6×His tag and inserted into an inducible bacterial expression vector pRSET A. After expression in E. coli, the recombinant product was purified by Ni-NTA affinity column chromatography. The purified product showed expected NH 2-terminal sequence and specific binding to the HNF1 site. The effect of pyridoxal 5′-phosphate and its analogues on the binding activity of recombinant HNF1 was examined and found that only pyridoxal 5′-phosphate effectively inhibited the DNA binding. The concentration of pyridoxal 5′-phosphate that inhibited 50% of DNA binding was around 100 μM. Furthermore, we identified Lys197 of HNF1 molecule as the essential residue of DNA binding. These observations suggest that pyridoxal 5′-phosphate directly interacts with tissue-specific transcription factor HNF1 and modulates the binding to DNA.

High-resolution structure of the HNF-1alpha dimerization domain.

The N-terminal dimerization domain of the transcriptional activator hepatocyte nuclear factor-1alpha (HNF-1alpha) is essential for DNA binding and association of the transcriptional coactivator, DCoH (dimerization cofactor of HNF-1). To investigate the basis for dimerization of HNF-1 proteins, we determined the 1.2 A resolution X-ray crystal structure of the dimerization domain of HNF-1alpha (HNF-p1). Phasing was facilitated by devising a simple synthesis for Fmoc-selenomethionine and substituting leucine residues with selenomethionine. The HNF-1 dimerization domain forms a unique, four-helix bundle that is preserved with localized conformational shifts in the DCoH complex. In three different crystal forms, HNF-p1 displays subtle shifts in the conformation of the interhelix loop and the crossing angle between the amino- and carboxyl-terminal helices. In all three crystal forms, the HNF-p1 dimers pair through an exposed hydrophobic surface that also forms the binding site for DCoH. Conserved core residues in the dimerization domain of the homologous transcriptional regulator HNF-1beta rationalize the functional heterodimerization of the HNF-1alpha and HNF-1beta proteins. Mutations in HNF-1alpha are associated with maturity-onset diabetes of the young type 3 (MODY3), and the structure of HNF-p1 provides insights into the effects of three MODY3 mutations.

The bifunctional protein DCoH/PCD, a transcription factor with a cytoplasmic enzymatic activity, is a maternal factor in the rat egg and expressed tissue specifically during embryogenesis.

The bifunctional protein DCoH/PCD is both a cytoplasmatic enzyme (PCD) involved in the tetrahydrobiopterin regeneration and a transcription coactivator (DCoH). Originally detected in liver cell nuclei, it forms a 2:2 heterotetrameric complex with the nuclear transcription factors HNF1alpha and the variant form HNF1beta and enhances their transcriptional potential. To address the role of DCoH in tissue specific and developmental gene regulation we analyzed its spatial and temporal expression pattern in the rat. DCoH might have a function in tissue specific gene expression mediated by HNF1 in the adults and in the developing embryo as it is found in the kidney and the liver, organs known to contain HNF1. In addition DCoH is a maternal factor in the rat egg lacking HNF1 transcription factors. The maternal protein enters the cell nuclei at the 8-cell stage suggesting a role in early embryonic gene regulation and excluding a cytoplasmatic enzymatic function. Evidence for a HNF1 independent function of DCoH is also given by the fact that DCoH is present in the eyes (pigmented epithelium) and the brain (ependym cells) of the rat embryos, cell types lacking HNF1 proteins. The tightly regulated expression pattern of DCoH in distinct cell types originating from endo- meso- and ectoderm is conserved between the rat and the frog indicating a fundamental role for DCoH in early gene regulation among the vertebrates.

The transactivation potential of variant hepatocyte nuclear factor 1 is modified by alternative splicing.

Two forms of the transcription factor vHNF1 (HNF1 beta or LFB3) have been previously described, derived by alternative splicing from a common premessenger RNA, and have been called vHNF1-A and vHNF1-B. vHNF1 proteins share a homologous homeo-related DNA-binding domain with the HNF1 protein, initially characterized as a liver-restricted transcription factor, and bind to a similar sequence motif. Here we demonstrate that vHNF1-A is a stronger transactivator than vHNF1-B when assayed in transient transfections using two different promoters. vHNF1-A also binds DNA with a higher affinity suggesting that a region of the protein located immediately upstream of the homeodomain can modulate the protein/DNA interaction and transactivation. Both vHNF1 transcripts were found at a constant ratio in every tissue where vHNF1 expression could be detected, using a quantitative reverse transcriptase-polymerase chain reaction.

More potent transcriptional activators or a transdominant inhibitor of the HNF1 homeoprotein family are generated by alternative RNA processing.

We report the isolation of cDNAs from human liver encoding several isoforms of the hepatocyte nuclear factor homeoproteins HNF1 and vHNF1 generated by the differential use of polyadenylation sites and by alternative splicing. In the novel isoforms intron sequences that are excised in the previously described forms are translated in the same frame as exon sequences until the first termination codon is encountered. Hence, the newly found isoforms all contain different C-terminal domains. For HNF1 it has been shown that its C-terminal region is responsible for the activation of transcription. In transient transfection assays the two novel HNF1 isoforms, HNF1-B and -C, transactivate 5-fold better than the previously described HNF1 protein (HNF1-A). The newly isolated isoform of vHNF1, designated vHNF1-C, is unable to transactivate and behaves as a transdominant repressor when cotransfected with HNF1-A, -B or -C. All of the different isoforms of HNF1 and vHNF1 can form homo- and heterodimers and their mRNAs are differentially expressed in fetal and adult human liver, kidney and intestine, suggesting distinct roles during development. Our studies show that the transactivation domain of the members of the HNF1 homeoprotein family is organized in modules which can be exchanged to generate either more potent transcriptional activators or a transdominant repressor.

Analysis of the rat hepatocyte nuclear factor (HNF) 1 gene promoter: synergistic activation by HNF4 and HNF1 proteins.

Hepatocyte nuclear factor (HNF) 1 is a key transcription factor involved in the expression of many liver-specific genes. We have isolated and characterized the promoter region of the rat HNF1 gene. Transfection experiments revealed that a short region between -118 and -8 is crucial for cell type-specific expression of the HNF1 gene in the hepatoma cell line, HepG2 cells. This region contains two positive cis-elements: site A, to which the transcription factor HNF4 protein can bind, and site B, to which the HNF1 protein can bind. Mutational analyses of these sites and cotransfection assays suggested that the HNF4 protein and HNF1 protein can transactivate the HNF1 gene.