Hepatocyte nuclear factor 4 (HNF4) was first identified as a DNA binding activity in rat liver nuclear extracts. Protein purification had then led to the cDNA cloning of rat HNF4, which was found to be an orphan member of the nuclear receptor superfamily. Binding sites for this factor were identified in many tissuespecifically expressed genes, and the protein was found to be essential for early embryonic development in the mouse. We have now isolated cDNAs encoding the human homolog of the rat and mouse HNF4 splice variant HNF4␣2, as well as a previously unknown splice variant of this protein, which we called HNF4␣4. More importantly, we also cloned a novel HNF4 subtype (HNF4␥) derived from a different gene and showed that the genes encoding HNF4␣ and HNF4␥ are located on human chromosomes 20 and 8, respectively. Northern (RNA) blot analysis revealed that HNF4␥ is expressed in the kidney, pancreas, small intestine, testis, and colon but not in the liver, while HNF4␣ RNA was found in all of these tissues. By cotransfection experiments in C2 and HeLa cells, we showed that HNF4␥ is significantly less active than HNF4␣2 and that the novel HNF4␣ splice variant HNF4␣4 has no detectable transactivation potential. Therefore, the differential expression of distinct HNF4 proteins may play a key role in the differential transcriptional regulation of HNF4-dependent genes.While intensive research during the last decade has established that tissue-specific gene expression is regulated to a large extent at the level of transcription, it has also become clear that most if not all of the tissue-specific transcription factors involved in this regulation are not restricted to a single tissue. Furthermore, the molecular cloning of these factors has shown that they are usually encoded by gene families with a multitude of subtypes and splice variants, and it is believed that it is the intra-and interfamily interplay of the cell-type-specific subset of these transcription factors that determines the identity of a certain tissue.Analysis of the regulatory regions of liver-specifically expressed genes, for example, has led to the identification of three gene families of transcription factors (i.e., the C/EBP, hepatocyte nuclear factor 1 [HNF1], and HNF3 families) and of another factor (HNF4) for which up to now splice variants but no subtypes derived from different genes had been identified (for reviews, see references 13, 19, and 25). All of these transcription factors originally considered to be liver specific are now known to be expressed also in other organs, but their combined expression is a unique feature of the liver (24).The factor HNF4 was originally identified in rat liver nuclear extracts as a protein binding to a DNA element of the transthyretin promoter (5). Protein purification and cDNA cloning revealed that HNF4 is an orphan member of the nuclear receptor superfamily with a zinc finger DNA binding domain and a putative ligand binding domain (20). Binding sites for HNF4 have been found in the regulatory regions of many ge...

The transcription factor hepatocyte nuclear factor 4 (HNF4) is an orphan member of the nuclear receptor superfamily expressed in mammals in liver, kidney, and the digestive tract. Recently, we isolated the Xenopus homolog of mammalian HNF4 and revealed that it is not only a tissue-specific transcription factor but also a maternal component of the Xenopus egg and distributed within an animal-to-vegetal gradient. We speculate that this gradient cooperates with the vegetally localized embryonic induction factor activin A to activate expression of HNF1␣, a tissue-specific transcription factor with an expression pattern overlapping that of HNF4. We have now identified a second Xenopus HNF4 gene, which is more distantly related to mammalian HNF4 than the previously isolated gene. This new gene was named HNF4␤ to distinguish it from the known HNF4 gene, which is now called HNF4␣. By reverse transcription-PCR, we detected within the 5 untranslated region of HNF4␤ two splice variants (HNF4␤2 and HNF4␤3) with additional exons, which seem to affect RNA stability. HNF4␤ is a functional transcription factor acting sequence specifically on HNF4 binding sites known for HNF4␣, but it seems to have a lower DNA binding activity and is a weaker transactivator than the ␣ isoform. Furthermore, the two factors differ with respect to tissue distribution in adult frogs: whereas HNF4␣ is expressed in liver and kidney, HNF4␤ is expressed in addition in stomach, intestine, lung, ovary, and testis. Both factors are maternal proteins and present at constant levels throughout embryogenesis. However, using reverse transcription-PCR, we found the RNA levels to change substantially: whereas HNF4␣ is expressed early during oogenesis and is absent in the egg, HNF4␤ is first detected in the latest stage of oogenesis, and transcripts are present in the egg and early cleavage stages. Furthermore, zygotic HNF4␣ transcripts appear in early gastrula and accumulate during further embryogenesis, whereas HNF4␤ mRNA transiently appears during gastrulation before it accumulates again at the tail bud stage. All of these distinct characteristics of the newly identified HNF4 protein imply that the ␣ and ␤ isoform have different functions in development and in adult tissues.A central event in early development is the establishment of different cell types from a single cell, the fertilized egg. This cell type is highly specialized, as it contains maternal components that define embryonic induction processes and the initiation of transcriptional cascades. This unique feature of the egg is especially evident in amphibians such as Xenopus laevis. In this species, no gene transcription occurs in early cleavage stages and all early events are regulated by preexisting transcripts and translational products made during oogenesis. Many of these maternal components are transcription factors that can most easily be analyzed in Xenopus (e.g., fos [13], B-myb [3], and Xrel [22]). Recently, we revealed that hepatocyte nuclear factor 4 (HNF4), previously known as a tissue-spe...

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