ERH (enhancer of rudimentary homologue), a conserved factor identical between frog and human, is a transcriptional repressor.

Drosophila enhancer of rudimentary [e(r)] interacts genetically with the rudimentary gene, which encodes a protein possessing the first three enzymatic activities of the pyrimidine biosynthesis pathway. A regulatory or enzymatic activity of e(r) in pyrimidine biosynthesis and the cell cycle has been suggested, but nothing is known about its molecular function. The factor is evolutionarily highly conserved since homologues exist in plants and mammals. We cloned the Xenopus enhancer of rudimentary homologue (XERH) as an interaction partner of DCoH/PCD (dimerisation cofactor of HNF1/pterin-4alpha-carbinolamine dehydratase) in the yeast two-hybrid assay. DCoH/PCD is a multifunctional factor originally identified as a positive cofactor of the HNF1 homeobox transcription factors. XERH is a 104 amino acid protein that is identical to its mammalian homologues. The mRNA is expressed maternally, enriched in ectodermal derivatives during development and ubiquitously detectable in the adult. Fused to the DNA binding region of the GAL4 transcription factor domain, XERH represses the activity of a GAL4 responsive reporter in HeLa, but not in NIH3T3 cells. Furthermore, the DCoH/PCD coactivation of a HNF1 responsive reporter is inhibited by XERH. We propose that XERH is a cell type-specific transcriptional repressor, probably interfering with HNF1-dependent gene regulation via DCoH/PCD.

Dimerization co-factor of hepatocyte nuclear factor 1/pterin-4alpha-carbinolamine dehydratase is necessary for pigmentation in Xenopus and overexpressed in primary human melanoma lesions.

Dimerization co-factor of hepatocyte nuclear factor 1 (HNF1)/pterin-4alpha-carbinolamine dehydratase (DCoH/PCD) is both a positive co-factor of the HNF1 homeobox transcription factors and thus involved in gene regulation as well as an enzyme catalyzing the regeneration of tetrahydrobiopterin. Dysfunction of DCoH/PCD is associated with the human disorders hyperphenylalaninemia and vitiligo. In Xenopus, overexpression of the protein during development induces ectopic pigmentation. In this study loss of function experiments using DCoH/PCD-specific antibodies demonstrated that the protein is also absolutely necessary for pigment cell formation in Xenopus. In normal human skin DCoH/PCD protein is weakly expressed in the basal layer of the epidermis that consists of keratinocytes and melanocytes. Whereas only 4 of 25 benign nevi reacted with DCoH/PCD-specific antibodies, high protein levels were detectable in melanoma cell lines and 13 of 15 primary malignant melanoma lesions. The comparison with the commonly used melanoma markers S100 and HMB45 demonstrated that DCoH/PCD has an overlapping but distinct expression pattern in melanoma lesions. In addition to human colon cancer, this is the second report about the overexpression of DCoH/PCD in human tumor cells indicating that the protein might be involved in cancerogenesis.

Misexpression of Xsiah-2 induces a small eye phenotype in Xenopus.

Recent data demonstrate a structural and functional conservation of factors crucial for the development of the insect and the vertebrate eye. We isolated Xenopus siah-2, a protein with 67% identity to Drosophila sina (seven in absentia) and 85% identity to the mouse and human siah-2 proteins. Sina is required in Drosophila for the R7 photoreceptor cell formation during eye development, because it down regulates proteins that inhibit R7 differentiation via the ubiquitin/proteasome pathway. Nothing is known about the developmental function of the siah protein in vertebrates. We show that in Xenopus siah-2 is expressed maternally and is later restricted to the brain, spinal cord and the developing and mature eye. To demonstrate that the vertebrate factor participates in the process of eye formation we over expressed Xsiah-2 during Xenopus development and observed the formation of a small eye phenotype. The vertebrate counterpart of a C-terminal loss of function sina mutant, that causes a deficiency of the R7 photoreceptor cells in Drosophila, induces in Xenopus also smaller eyes. The small eyes are characterized by a reduced size of the lens, the retina and the pigmented epithelium. As this phenotype has been also described for flies expressing sina ectopically, the data demonstrate the functional and structural conservation of Xsiah-2 and sina in metazoan eye development.

The mutated human gene encoding hepatocyte nuclear factor 1beta inhibits kidney formation in developing Xenopus embryos.

The transcription factor hepatocyte nuclear factor 1beta (HNF1beta) is a tissue-specific regulator that also plays an essential role in early development of vertebrates. In humans, four heterozygous mutations in the HNF1beta gene have been identified that lead to early onset of diabetes and severe primary renal defects. The degree and type of renal defects seem to depend on the specific mutation. We show that the frameshift mutant P328L329fsdelCCTCT associated with nephron agenesis retains its DNA-binding properties and acts as a gain-of-function mutation with increased transactivation potential in transfection experiments. Expression of this mutated factor in the Xenopus embryo leads to defective development and agenesis of the pronephros, the first kidney form of amphibians. Very similar defects are generated by overexpressing in Xenopus the wild-type HNF1beta, which is consistent with the gain-of-function property of the mutant. In contrast, introduction of the human HNF1beta mutant R137-K161del, which is associated with a reduced number of nephrons with hypertrophy of the remaining ones and which has an impaired DNA binding, shows only a minor effect on pronephros development in Xenopus. Thus, the overexpression of both human mutants has a different effect on renal development in Xenopus, reflecting the variation in renal phenotype seen with these mutations. We conclude that mutations in human HNF1beta can be functionally characterized in Xenopus. Our findings imply that HNF1beta not only is an early marker of kidney development but also is functionally involved in morphogenetic events, and these processes can be investigated in lower vertebrates.

Ectopic pigmentation in Xenopus in response to DCoH/PCD, the cofactor of HNF1 transcription factor/pterin-4alpha-carbinolamine dehydratase.

DCoH, the dimerization cofactor of the HNF-1 homeodomain proteins (hepatocyte nuclear factor-1alpha and beta), is involved in gene expression by associating with these transcription factors. The protein also called PCD for pterin-4alpha-carbinolamine dehydratase is a bifunctional factor as it catalyzes also the regeneration of tetrahydrobiopterin. This coenzyme is used by the enzyme phenylalanine hydroxylase, which generates tyrosine, the precursor of catecholamines and melanin. DCoH/PCD presumably cooperates with other partners, because it is expressed earlier than HNF1 and phenylalanine hydroxylase (PAH) in early vertebrate development. It is also found in cells lacking HNF1 and PAH like skin, brain and the pigmented epithelium of the eye suggesting a yet unknown function. We show that the overexpression of DCoH/PCD in Xenopus induces the formation of ectopic pigment cells in the epidermis, that are visible earlier than the endogenous pigmentation and broader distributed. This ectopic pigmentation is accompanied by an increase in tyrosinase activity and the amount of melanin. Overexpression of DCoH/PCD induces the appearance of pigment cells also in animal cap explants, that normally differentiate into atypical epidermis. DCoH/PCD mutants with impaired carbinolamine dehydratase activity retain the potential to induce pigmentation and we propose therefore that DCoH/PCD is not simply an essential enzyme for melanin biosynthesis, but also a regulator for the differentiation of pigment producing cells.

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.

Patterning the expression of a tissue-specific transcription factor in embryogenesis: HNF1 alpha gene activation during Xenopus development.

Tissue-specific transcription factors play an essential role in establishing cell identity during development. We review our knowledge of the molecular events involved in the activation of the gene encoding the tissue-specific transcription factor HNF1 alpha (LFB1). The available data suggest that the maternal factors OZ-1, HNF4 alpha and HNF4 beta act as initial activators of the HNF1 alpha promoter. We present evidence suggesting that the mesoderm-inducing factor activin A plays a critical role by acting through the HNF4 binding site of the HNF1 alpha promoter. The activity of this embryonic morphogen seems to form a gradient opposing the distribution of the maternal HNF4 proteins that are concentrated at the animal pole of the egg. After zygotic gene transcription the HNF1 alpha-related transcription factor HNF1 beta accumulates faster than HNF1 alpha itself and thus is likely to contribute to the activation of the HNF1 alpha transcription via the HNF1 binding site. The cofactor of the HNF1 proteins (DCoH) is present throughout development and thus cannot limit the activation potential of HNF1 alpha in early development. Our results provide a detailed description of setting up the expression pattern of a tissue-specific transcription factor during embryogenesis.

HNF4beta, a new gene of the HNF4 family with distinct activation and expression profiles in oogenesis and embryogenesis of Xenopus laevis.

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 HNF1alpha, 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 HNF4beta to distinguish it from the known HNF4 gene, which is now called HNF4alpha. By reverse transcription-PCR, we detected within the 5' untranslated region of HNF4beta two splice variants (HNF4beta2 and HNF4beta3) with additional exons, which seem to affect RNA stability. HNF4beta is a functional transcription factor acting sequence specifically on HNF4 binding sites known for HNF4alpha, but it seems to have a lower DNA binding activity and is a weaker transactivator than the alpha isoform. Furthermore, the two factors differ with respect to tissue distribution in adult frogs: whereas HNF4alpha is expressed in liver and kidney, HNF4beta 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 HNF4alpha is expressed early during oogenesis and is absent in the egg, HNF4beta is first detected in the latest stage of oogenesis, and transcripts are present in the egg and early cleavage stages. Furthermore, zygotic HNF4alpha transcripts appear in early gastrula and accumulate during further embryogenesis, whereas HNF4beta 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 alpha and beta isoform have different functions in development and in adult tissues.

Human hepatocyte nuclear factor 4 isoforms are encoded by distinct and differentially expressed genes.

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 tissue-specifically 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 alpha 2, as well as a previously unknown splice variant of this protein, which we called HNF alpha 4. More importantly, we also cloned a novel HNF4 subtype (HNF4 gamma) derived from a different gene and showed that the genes encoding HNF 4 alpha and HNF4 gamma are located on human chromosomes 20 and 8, respectively. Northern (RNA) blot analysis revealed that HNF4 GAMMA is expressed in the kidney, pancreas, small intestine, testis, and colon but not in the liver, while HNF4 alpha RNA was found in all of these tissues. By cotransfection experiments in C2 and HeLa cells, we showed that HNF4 gamma is significantly less active than HNF4 alpha 2 and that the novel HNF4 alpha splice variant HNF4 alpha 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.

The DNA binding activity of the liver transcription factors LFB1 (HNF1) and HNF4 varies coordinately in rat hepatocellular carcinoma.

As renal cell carcinomas are characterized by the disappearance of the transcription factor LFB1, which is known to be primarily involved in gene regulation in the liver, we have measured the presence of LFB1 in rat hepatocellular carcinomas induced by diethylnitrosamine. The level of LFB1 binding activity in adenoid-cystic as well as trabecular tumours shows some variation and may either be lower or higher than in the non-tumorous tissue. The amount of LFB1 binding activity correlates with the binding activity of HNF4, a transcription factor reported to stimulate LFB1 expression. As the levels of LFB1 and HNF4 binding activity differ extensively in various hepatocellular carcinomas, it is unlikely that these transcription factors play a general role in hepatocarcinogenesis. This is in contrast to renal carcinogenesis where a dramatic loss of LFB1 is a consistent feature.

Chimeric liver transcription factors LFB1 (HNF1) containing the acidic activation domain of VP16 act as positive dominant interfering mutants.

The transcription factor LFB1 (HNF1) involved in the expression of liver-specific genes is characterized by a serine/threonine-rich activation domain whose transactivation potential differs between mammals and Xenopus. Exchanging the activation domain between the Xenopus and rat LFB1, we produced chimeric transactivators whose activities are primarily determined by the origin of the activation domain. By replacing the serine/threonine-rich activation domain of LFB1 with the acidic activation domain of VP16, we generated transcription factors that act as dominant positive interfering mutants on endogenous LFB1 in differentiated hepatoma cells. As these LFB1/VP16 chimeras show no self-squelching as observed with wild-type LFB1 and increase the activity of saturating LFB1, we postulate that acidic and serine/threonine-rich activation domains use different targets of the basal transcription machinery. Stable transfection of various LFB1 derivatives, including those containing the VP16 transactivation domain, into the dedifferentiated C2 hepatoma cell resulted in cell clones stably expressing LFB1 function. However, as in none of these clones the chromosomal albumin genes are activated, we conclude that the presence of functional LFB1 may not be sufficient to reactivate liver-specific functions lost in dedifferentiated hepatoma cells.

Developmental regulation and tissue distribution of the liver transcription factor LFB1 (HNF1) in Xenopus laevis.

The transcription factor LFB1 (HNF1) was initially identified as a regulator of liver-specific gene expression in mammals. It interacts with the promoter element HP1, which is functionally conserved between mammals and amphibians, suggesting that a homologous factor, XLFB1, also exists in Xenopus laevis. To study the role of LFB1 in early development, we isolated two groups of cDNAs coding for this factor from a Xenopus liver cDNA library by using a rat LFB1 cDNA probe. A comparison of the primary structures of the Xenopus and mammalian proteins shows that the myosin-like dimerization helix, the POU-A-related domain, the homeo-domain-related region, and the serine/threonine-rich activation domain are conserved between X. laevis and mammals, suggesting that all these features typical for LFB1 are essential for function. Using monoclonal antibodies, we demonstrate that XLFB1 is present not only in the liver but also in the stomach, intestine, colon, and kidney. In an analysis of the expression of XLFB1 in the developing Xenopus embryo, XLFB1 transcripts appear at the gastrula stage. The XLFB1 protein can be identified in regions of the embryo in which the liver diverticulum, stomach, gut, and pronephros are localized. The early appearance of XLFB1 expression during embryogenesis suggests that the tissue-specific transcription factor XLFB1 is involved in the determination and/or differentiation of specific cell types during organogenesis.