The hepatocyte nuclear factor (HNF)4alpha, a member of the nuclear receptor superfamily, regulates genes that play a critical role in embryogenesis and metabolism. Recent studies have shown that mutations in the human HNF4alpha gene cause a rare form of type 2 diabetes, maturity onset diabetes of the young (MODY1). To investigate the properties of these naturally occurring HNF4alpha mutations we analysed five MODY1 mutations (R154X, R127W, V255M, Q268X and E276Q) and one other mutation (D69A), which we found in HepG2 hepatoma cells. Activation of reporter genes in transfection assays and DNA binding studies showed that the MODY1-associated mutations result in a variable reduction in function, whereas the D69A mutation showed an increased activity on some promoters. None of the MODY mutants acted in a dominant negative manner, thus excluding inactivation of the wild-type factor as a critical event in MODY development. A MODY3-associated mutation in the HNF1alpha gene, a well-known target gene of HNF4alpha, results in a dramatic loss of the HNF4 binding site in the promoter, indicating that mutations in the HNF4alpha gene might cause MODY through impaired HNF1alpha gene function. Based on these data we propose a two-hit model for MODY development.

The gene encoding the tissue specific transcription factor HNF1α is expressed in vertebrates in tissues of endodermal origin such as the liver and the gut as well as in the kidney, a mesoderm derived organ. Using a 6 kb HNF1α promoter fragment linked to GFP we observed green fluorescence in transgenic embryos restricted to the liver and gut as well as to the pronephros, the embryonic kidney. By deletion and mutation analysis of the HNF1α promoter we succeeded in dissecting the HNF1α promoter into two entities that are either active in the endoderm or the mesoderm. In conclusion, our data establish that the generation of transgenic Xenopus allows the functional dissection of promoters in the context of the entire organism.

BACKGROUND:Human renal cell carcinogenesis is associated with loss of expression of tissue-specific genes and loss of function of tissue-specific transcription factors such as HNF(hepatic nuclear factor)1 alpha.MATERIALS AND METHODS:In this study HNF1 alpha DNA-binding activities and protein amounts were determined by gel retardation assay and Western blot analysis, respectively, in 42 non-metastasized renal cell carcinomas and paired normal tissues.RESULTS:36 tumors out of 42 (86%) showed diminished binding activity of HNF1 alpha. In most cases (26 out of 42) this appeared to be due to decreased amounts of HNF1 alpha protein, but 10 tumors contained equal or even higher amounts of HNF1 alpha, in spite of reduced binding to DNA. Only 6 tumors out of 42 had unaltered HNF1 alpha binding activity. A clinical follow-up was obtained for 40 patients. Over an average follow-up period of 39 months no significant differences in the survival rate were observed between patients having lost or retained HNF1 alpha function. However, since most of the patients with retained function are still alive, long-term follow-up might be warranted.CONCLUSIONS:The very high incidence of loss of HNF1 alpha function indicates the important biological role of this change in renal cell carcinoma.

The gene encoding the tissue speci®c transcription factor HNF1a is expressed in vertebrates in tissues of endodermal origin such as the liver and the gut as well as in the kidney, a mesoderm derived organ. Using a 6 kb HNF1a promoter fragment linked to GFP we observed green ̄uorescence in transgenic embryos restricted to the liver and gut as well as to the pronephros, the embryonic kidney. By deletion and mutation analysis of the HNF1a promoter we succeeded in dissecting the HNF1a promoter into two entities that are either active in the endoderm or the mesoderm. In conclusion, our data establish that the generation of transgenic Xenopus allows the functional dissection of promoters in the context of the entire organism. q 2000 Elsevier Science Ireland Ltd. All rights reserved.

Human renal cell carcinoma (RCC) is a common malignant disease of the kidney characterized by dedifferentiation of renal epithelial cells. Our previous experiments showed that most RCCs have a loss of function of the tissue-specific transcription factor hepatocyte nuclear factor (HNF) 1 alpha. Detailed analyses of the 10 exons encoding HNF1 alpha in 32 human RCCs by single-strand conformation polymorphism analysis and direct DNA sequencing revealed no tumor-associated mutation, whereas with the same probes we frequently found mutations in the von Hippel-Lindau tumor suppressor gene. No mutation leading to loss of HNF1 alpha function was detected by analyzing the integrity of the HNF1 alpha transcripts in the RNA derived from RCCs by the protein truncation test. Investigating human RCC cell lines by western blotting and gel retardation assays showed a dramatic loss in the expression of the tissue-specific transcription factor HNF1 alpha in eight of 10 cell lines. As the HNF1 alpha-related transcription factor HNF1 beta was expressed in all these tumor cell lines, the loss of HNF1 alpha expression was a specific event and was maintained in RCC cell lines. The loss of HNF1 alpha expression in RCC cell lines on the RNA level was confirmed by reverse transcription polymerase chain reaction. We propose that tumor-associated mutations in the HNF1 alpha gene do not occur in human RCC and that the loss of function is partially due to a transcriptional inactivation of the HNF1 alpha gene. (C) 1999 Wiley-Liss, Inc.

The transcription factor hepatocyte nuclear factor 4 alpha (HNF4 alpha) is a tissue specific transcription factor mainly expressed in the liver, kidney, intestine and the endocrine pancreas, but is also an essential regulator for early embryonic events, Based on its protein structure HNF4 alpha. is classified as an orphan member of the nuclear receptor superfamily, Comparing HNF4 alpha transcription factors in the differentiated and dedifferentiated murine hepatocyte cell line MHSV-12 we identified in dedifferentiated cells the novel splice variant HNF4 alpha 7, This variant is characterized by an alternative first exon and has a lower transactivation potential in transient transfection assays using HNF4 dependent reporter genes, HNF4 alpha 7 mRNA and the corresponding protein are expressed in the undifferentiated pluripotent embryonal carcinoma cell line F9, whereas HNF4 alpha 1 only appears after differentiation of F9 cells to visceral endoderm, HNF4 alpha 7 mRNA is also found in totipotent embryonic stem cells, However, the function of HNF4 alpha 7 seems not to be restricted to embryonic cells as the HNF4 alpha 7 mRNA is also present in adult tissues, most notably the stomach, All these features suggest that the presence of distinct splice variants of HNF4 alpha modulates the activity of HNF4 alpha in a cell type specific way.