Novel monogenic diabetes mutations in the P2 promoter of the HNF4A gene are associated with impaired function in vitro.

AIMS: Mutations in HNF4A cause a form of monogenic beta-cell diabetes. We aimed to identify mutations in the pancreas-specific P2 promoter of HNF4A in families with suspected HNF4A diabetes and to show that they impaired the function of the promoter in vitro. METHODS: We screened families with a clinical suspicion of HNF4A monogenic beta-cell diabetes for mutations in the HNF4A P2 promoter. We investigated the function of the previously reported HNF4A P2 promoter mutation -192C>G linked to late-onset diabetes in several families, along with two new segregating mutations, in vitro using a modified luciferase reporter assay system with enhanced sensitivity. RESULTS: We identified two novel HNF4A P2 promoter mutations that co-segregate with diabetes in two families, -136A>G and -169C>T. Both families displayed phenotypes typical of HNF4A monogenic beta-cell diabetes, including at least two affected generations, good response to sulphonylurea treatment and increased birthweight and/or neonatal hypoglycaemia. We show that both of these novel mutations and -192C>G impair the function of the promoter in transient transfection assays. CONCLUSIONS: Two novel mutations identified here and the previously identified late-onset diabetes mutation, -192C>G, impair the function of the HNF4A P2 promoter in vitro.

Rare variants identified in the HNF- 4 alpha beta-cell-specific promoter and alternative exon 1 lack biological significance in maturity onset diabetes of the young and young onset Type II diabetes.

AIMS/HYPOTHESIS: The recently identified alternative promoter (P2) of HNF-4 alpha is the major HNF-4 alpha transcription start site in pancreatic beta cells. The significance of the P2 promoter was shown by the identification of a mutation in the IPF-1 binding site of the alternative promoter which cosegregated with diabetes in a large MODY family. The role of the P2 promoter and the associated alternative exon 1 in both MODY and polygenic Type II (non-insulin-dependent) diabetes mellitus is not known. Linkage to this region in studies of Type II diabetes makes the P2 region a strong candidate for a role in Type II diabetes susceptibility. METHODS: To assess the role of the P2 region we screened MODY, young-onset Type II diabetic subjects, and probands from Type II diabetes families linked to chromosome 20 for variants of the P2 promoter and associated exon of HNF-4 alpha. RESULTS: Two variants were found that were not present in the control subjects. The -79 C/T substitution was present in a MODY family but did not perfectly cosegregate with diabetes. A -276 G/T substitution was identified in two UK young-onset diabetes probands but did not co-segregate with diabetes. Reporter gene studies did not indicate changes in transcriptional activity caused by either the -79 C/T or -276 G/T single nucleotide substitutions. CONCLUSION/INTERPRETATION: We found no evidence to suggest that variation in the P2 proximal promoter region and associated alternative exon 1 of HNF-4 alpha contribute to young onset Type II diabetes susceptibility in Northern Europeans.

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.

A distant upstream promoter of the HNF-4alpha gene connects the transcription factors involved in maturity-onset diabetes of the young.

Maturity-onset diabetes of the young (MODY) is a monogenic, autosomal dominant subtype of early-onset diabetes mellitus due to defective insulin secretion by the pancreatic beta-cell in humans. Five different genes have been identified including those encoding the tissue-specific transcription factors expressed in pancreatic beta-cells, i.e. HNF-4alpha (MODY1), HNF-1alpha (MODY3), IPF-1 (also known as PDX-1, MODY4) and HNF-1beta (MODY5). Analyzing the transcription of the HNF-4alpha gene, we now identify an alternative promoter, P2, which is 46 kb 5' to the previously identified P1 promoter of the human gene. Based on RT-PCR this distant upstream P2 promoter represents the major transcription site in pancreatic beta-cells, but is also used in hepatic cells. Transfection assays with various deletions and mutants of the P2 promoter reveal functional binding sites for HNF-1alpha, HNF-1beta and IPF-1, the other transcription factors known to encode MODY genes. We demonstrate the significance of this alternative promoter in a large MODY family where a mutated IPF-1 binding site in the P2 promoter of the HNF-4alpha gene co-segregates with diabetes (LOD score 3.25). These data suggest a regulatory network of the four MODY transcription factors interconnected at the distant upstream P2 promoter of the HNF-4alpha gene.

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

Mutations in the human genes encoding the tissue-specific transcription factors hepatocyte nuclear factor (HNF)1alpha, HNF1beta and HNF4alpha are responsible for maturity onset diabetes of the young (MODY), a monogenic dominant inherited form of diabetes mellitus characterized by defective insulin secretion of the pancreatic beta-cells. In addition, the mutated HNF1beta gene causes defective development of the kidney and genital malformation. This review summarizes the main features of these transcription factors and discusses potential events leading to the specific disease phenotypes.

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.

FLP and Cre recombinase function in Xenopus embryos.

The use of the site-specific DNA recombinases FLP and Cre is well-established in a broad range of organisms. Here we investigate the applicability of both recombinases to the Xenopus system where they have not been analyzed yet. We show that injection of FLP mRNA triggers the excision of an FLP recombination target (FRT)-flanked green fluorescent protein (GFP) sequence in a coinjected reporter construct inducing the expression of a downstream beta-galactosidase gene (lacZ). The FLP-mediated gene activation can be controlled in Xenopus embryos by injecting a mRNA encoding a fusion of FLP to the mutant ligand binding domain of the human estrogen receptor whose activity is dependent on 4-hydroxytamoxifen. We also demonstrate that a Cre reporter injected into fertilized eggs is fully recombined by Cre recombinase before zygotic gene transcription initiates. Our results indicate that in Xenopus embryos Cre is more effective than FLP in recombining a given quantity of reporter molecules. Finally, we present FLP-inducible double reporter systems encoding two fluorescence proteins (EYFP, ECFP, DsRed or GFP). These novel gene expression systems enable the continuous analysis of two reporter activities within living embryos and are expected to allow cell-lineage studies based on recombinase-mediated DNA rearrangement in transgenic Xenopus lines.

Inhibitor of the tissue-specific transcription factor HNF4, a potential regulator in early Xenopus development.

Hepatocyte nuclear factor 4alpha (HNF4alpha) 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 ligand-binding 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 HNF1alpha is only marginally expressed, we propose that the HNF4 inhibitor functions in the embryo to restrict the activity of the maternal HNF4 proteins.

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.

Primary structure, chromosomal mapping, expression and transcriptional activity of murine hepatocyte nuclear factor 4gamma.

We demonstrate the presence of a new member of the orphan nuclear receptor hepatocyte nuclear factor 4 (HNF4) subfamily in mouse which is genetically distinct from the previously characterized mouse HNF4alpha gene. The new member of the HNF4 subfamily shows highest amino acid identity, similar tissue distribution and syntenous chromosomal localization to the recently described human HNF4gamma (NR2A2), we therefore classify it as mouse HNF4gamma (mHNF4gamma). A combination of RT-PCR and immunohistochemical analysis showed expression of mHNF4gamma mRNA and protein in the endocrine pancreas, testes, kidney and gut. By co-transfection experiments, we show that mHNF4gamma is able to activate transcription, acting through binding sites that have been previously characterized as HNF4alpha binding sites. The presence of HNFgamma in human and mouse implies that a complex transcriptional network exists in higher vertebrates involving a number of HNF4 members with overlapping yet distinct function and tissue distribution.

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.

Abnormal nephron development associated with a frameshift mutation in the transcription factor hepatocyte nuclear factor-1 beta.

BACKGROUND: The transcription factor hepatocyte nuclear factor (HNF)-1 beta functions as a homodimer or as a heterodimer with the structurally related protein HNF-1 alpha. Both are expressed sequentially in rat kidney development, with HNF-1 beta being detected from the earliest inductory phases. HNF-1 beta gene mutations are associated with a unique disorder characterized by maturity-onset diabetes of the young (MODY) and early-onset and progressive nondiabetic renal dysfunction, which may lead to chronic renal failure. METHODS: The HNF-1 beta gene was screened for mutations in six subjects with early-onset diabetes and a history of renal dysfunction in the subjects or their families. RESULTS: A novel frameshift mutation in exon 4 of the HNF-1 beta gene and a deletion of CCTCT at codons 328 to 329 were detected in one subject. She was diagnosed as diabetic at the age of 21 in her second pregnancy. Glucose tolerance rapidly deteriorated over 18 months as a result of beta-cell dysfunction. The HNF-1 beta mutation arose de novo on a paternal chromosome and cosegregated with renal abnormalities in her family. The proband had bilateral small cysts in normal-sized kidneys and a reduced creatinine clearance of 66 mL/min (NR 80-120). Her first pregnancy was terminated at 17 weeks following an ultrasound diagnosis of bilateral, nonfunctioning cystic kidneys. Her first-born child had a small multicystic, dysplastic right kidney and a dysplastic left kidney with a reduced creatinine clearance (40 mL/min per 1.73 m2). Histologic examination of the large (5.8 vs. 1.4 g), polycystic fetal kidneys showed no normal nephrogenesis. CONCLUSIONS: These studies indicate that HNF-1 beta plays a central role in normal kidney development and pancreatic beta-cell function, and suggest that one mechanism by which HNF-1 beta gene mutations may cause renal dysfunction are by their effects on nephron development.

Naturally occurring mutations in the human HNF4alpha gene impair the function of the transcription factor to a varying degree.

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.

Distinct promoter elements mediate endodermal and mesodermal expression of the HNF1alpha promoter in transgenic Xenopus.

The gene encoding the tissue specific transcription factor HNF1alpha 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 HNF1alpha 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 HNF1alpha promoter we succeeded in dissecting the HNF1alpha 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.

Hepatocyte nuclear factor-4gamma: cDNA sequence, gene organization, and mutation screening in early-onset autosomal-dominant type 2 diabetes.

The aim of this study was to investigate whether mutations in hepatocyte nuclear factor (HNF)-4gamma, a transcription factor homologous to HNF-4alpha, contribute to the etiology of early-onset type 2 diabetes. Linkage between diabetes and two polymorphic markers at the HNF-4gamma locus (D8S286 and D8S548) was evaluated in 32 multigenerational families with early-onset autosomal-dominant type 2 diabetes unlinked to known maturity-onset diabetes of the young genes. Total logarithm of odds (LOD) scores were strongly negative (-50.3 at D8S286 and -46.2 at D8S548), but linkage could not be excluded in 15 families having LOD scores >-2.0. To screen these pedigrees for HNF-4gamma mutations, the gene structure was defined. Because reverse transcriptase-polymerase chain reaction experiments indicated that the first 1,674 bp of the published cDNA sequence (3,248 bp) were a cloning artifact, the correct cDNA sequence was determined by 5' rapid amplification of cDNA ends (RACE) and primer extension assay. Based on the new cDNA sequence (1,731 bp), 11 exons were found. After screening the 5' flanking region and all coding exons for mutations, we identified several polymorphisms, one of which affected the amino acid sequence (M190I). However, no mutations segregating with diabetes could be found in these families. We conclude that genetic variability in the HNF-4gamma gene is unlikely to play a major role in the etiology of early-onset autosomal-dominant type 2 diabetes.

Loss of function of the tissue specific transcription factor HNF1 alpha in renal cell carcinoma and clinical prognosis.

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.

Loss of HNF1alpha function in human renal cell carcinoma: frequent mutations in the VHL gene but not the HNF1alpha gene.

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) 1alpha. Detailed analyses of the 10 exons encoding HNF1alpha 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 HNF1alpha function was detected by analyzing the integrity of the HNF1alpha 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 HNF1alpha in eight of 10 cell lines. As the HNF1alpha-related transcription factor HNF1beta was expressed in all these tumor cell lines, the loss of HNF1alpha expression was a specific event and was maintained in RCC cell lines. The loss of HNF1alpha 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 HNF1alpha gene do not occur in human RCC and that the loss of function is partially due to a transcriptional inactivation of the HNF1alpha gene.

The embryonic expression of the tissue-specific transcription factor HNF1alpha in Xenopus: rapid activation by HNF4 and delayed induction by mesoderm inducers.

The tissue-specific transcription factor HNF1alpha is expressed in kidney, liver, intestine and stomach of Xenopus. We show that the HNF1alpha gene is transcriptionally activated at the onset of zygotic gene transcription and that this transcription is maintained throughout development. Ectodermal explants of blastulae (animal caps) express HNF1alpha mRNA upon stimulation with the mesoderm inducers activin A and BMP4 as well as on overexpression of Smad2 and Smad1, the corresponding members of the intracellular TGF-beta signal transducers, respectively. Beside these factors that mediate their response through serine/threonine kinase receptors, bFGF, which acts via tyrosine kinase receptors, leads to HNF1alpha expression, too. These embryonic inducers result in a delayed appearance of HNF1alpha mRNA, excluding a direct activation of HNF1alpha. In contrast, the maternally expressed nuclear receptors HNF4alpha and HNF4beta activate the initial HNF1alpha transcription, since overexpression of HNF4 leads to a rapid expression of HNF1alpha mRNA in animal caps. Similarly, in entire neurulae HNF4 overexpression results in increased HNF1alpha transcription. Therefore, we assume that the initial activation is dependent on maternal HNF4alpha and HNF4beta transcription factors whereas HNF1alpha induction by growth factors reflects the property of these factors to induce the differentiation of mesodermal and entodermal cell types expressing HNF1alpha.

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.