Transcription factor hepatocyte nuclear factor 6 regulates pancreatic endocrine cell differentiation and controls expression of the proendocrine gene ngn3.

Hepatocyte nuclear factor 6 (HNF-6) is the prototype of a new class of cut homeodomain transcription factors. During mouse development, HNF-6 is expressed in the epithelial cells that are precursors of the exocrine and endocrine pancreatic cells. We have investigated the role of HNF-6 in pancreas differentiation by inactivating its gene in the mouse. In hnf6(-/-) embryos, the exocrine pancreas appeared to be normal but endocrine cell differentiation was impaired. The expression of neurogenin 3 (Ngn-3), a transcription factor that is essential for determination of endocrine cell precursors, was almost abolished. Consistent with this, we demonstrated that HNF-6 binds to and stimulates the ngn3 gene promoter. At birth, only a few endocrine cells were found and the islets of Langerhans were missing. Later, the number of endocrine cells increased and islets appeared. However, the architecture of the islets was perturbed, and their beta cells were deficient in glucose transporter 2 expression. Adult hnf6(-/-) mice were diabetic. Taken together, our data demonstrate that HNF-6 controls pancreatic endocrine differentiation at the precursor stage and identify HNF-6 as the first positive regulator of the proendocrine gene ngn3 in the pancreas. They also suggest that HNF-6 is a candidate gene for diabetes mellitus in humans.

Hepatocyte nuclear factor-6: associations between genetic variability and type II diabetes and between genetic variability and estimates of insulin secretion.

AIMS/HYPOTHESIS: The transcription factor hepatocyte nuclear factor (HNF)-6 is an upstream regulator of several genes involved in the pathogenesis of maturity-onset diabetes of the young. We therefore tested the hypothesis that variability in the HNF-6 gene is associated with subsets of Type II (non-insulin-dependent) diabetes mellitus and estimates of insulin secretion in glucose tolerant subjects. METHODS: We cloned the coding region as well as the intron-exon boundaries of the HNF-6 gene. We then examined them on genomic DNA in six MODY probands without mutations in the MODY1, MODY3 and MODY4 genes and in 54 patients with late-onset Type II diabetes by combined single strand conformational polymorphism-heteroduplex analysis followed by direct sequencing of identified variants. An identified missense variant was examined in association studies and genotype-phenotype studies. RESULTS: We identified two silent and one missense (Pro75 Ala) variant. In an association study the allelic frequency of the Pro75Ala polymorphism was 3.2% (95% confidence interval, 1.9-4.5) in 330 patients with Type II diabetes mellitus compared with 4.2% (2.4-6.0) in 238 age-matched glucose tolerant control subjects. Moreover, in studies of 238 middle-aged glucose tolerant subjects, of 226 glucose tolerant offspring of Type II diabetic patients and of 367 young healthy subjects, the carriers of the polymorphism did not differ from non-carriers in glucose induced serum insulin or C-peptide responses. CONCLUSION/INTERPRETATION: Mutations in the coding region of the HNF-6 gene are not associated with Type II diabetes or with changes in insulin responses to glucose among the Caucasians examined.

Hepatocyte nuclear factor 6, a transcription factor that contains a novel type of homeodomain and a single cut domain.

Tissue-specific transcription is regulated in part by cell type-restricted proteins that bind to defined sequences in target genes. The DNA-binding domain of these proteins is often evolutionarily conserved. On this basis, liver-enriched transcription factors were classified into five families. We describe here the mammalian prototype of a sixth family, which we therefore call hepatocyte nuclear factor 6 (HNF-6). It activates the promoter of a gene involved in the control of glucose metabolism. HNF-6 contains two different DNA-binding domains. One of these corresponds to a novel type of homeodomain. The other is homologous to the Drosophila cut domain. A similar bipartite sequence is coded by the genome of Caenorhabditis elegans.

Liver-specific factor binding to the liver promoter of a 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene.

The liver-type and muscle-type isozymes of 6-phosphofructo-2-kinase/fructose- 2,6-bisphosphatase are encoded by the same gene from different promoters. We have found earlier that the liver-type promoter is controlled by ubiquitous and liver-enriched transcription factors and that two cis-acting sequences, called sites III and IV, account for about half of the activity of this promoter (Lemaigre, F. P., Durviaux, S. M., and Rousseau, G. G. (1991) Mol. Cell. Biol. 11, 1099-1106). Site III contains the TGTTTGC sequence, i.e. the hepatocyte nuclear factor 5 motif, hepatocyte nuclear factor 5-TGTTTGC sequence motif, which is known to be functionally important in several liver-specific genes. We now show that site III actually binds the ubiquitous octamer factor 1 and the liver-enriched hepatocyte nuclear factor 3 (HNF-3) in a mutually exclusive way. We also show that site IV binds CCAAT/enhancer-binding protein (C/EBP)-related factors as well as another factor that is liver-specific. This factor is more abundant or has a higher affinity for site IV than the C/EBP-related proteins. Although it can bind to a high affinity HNF-3 site, this factor differs from known members of the HNF-3 family and from other liver-specific transcription factors.

Identification of regulatory sequences and protein-binding sites in the liver-type promoter of a gene encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

The liver-type and muscle-type isozymes of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase are encoded by one gene that uses two alternative promoters. We have identified cis-acting sequences and protein-binding sites on the liver-type promoter. Transfection assays with deleted promoters showed that maximal promoter activity is contained within 360 bp upstream of the cap site. DNase I footprinting experiments with liver and spleen nuclear extracts and with purified proteins revealed several protein-binding sites in this region. These included four binding sites for nuclear factor I, one site that contains an octamer consensus but showed a liver-specific footprint pattern, two liver-specific protein-binding sites, and one poly(dG)-containing binding site. Transfection of cells of hepatic origin suggested that all these sites except one are involved in transcriptional regulation. The region between -360 and -2663 contained an element that functioned as a silencer in a nonhepatic cell line. We conclude that in liver transcription from the liver-type promoter of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene is controlled by ubiquitous and tissue-specific factors and involves activating and derepressing mechanisms.

Sp1 can displace GHF-1 from its distal binding site and stimulate transcription from the growth hormone gene promoter.

DNase I footprinting experiments showed that binding activities of Sp1 and of GHF-1 to its distal site on the human growth hormone gene promoter are mutually exclusive. The kinetics of GHF-1 binding were indicative of positive cooperativity. The Sp1 site did not affect promoter activity in cell-free transcription. Still, Sp1 could compensate partially for the decreased stimulation of transcription seen at low GHF-1 concentrations.

Nuclear factor-I and activator protein-2 bind in a mutually exclusive way to overlapping promoter sequences and trans-activate the human growth hormone gene.

Transcription of the human growth hormone (hGH) gene and its regulation are controlled by trans-acting factors that bind to hGH gene promoter sequences. Several DNase I footprints have been described within 500 bp of this promoter, one of which (-289 to -267) has not yet been ascribed to a defined factor. By DNase I footprinting, gel mobility shift, and methylation interference assays with extracts from HeLa cells and GH-producing pituitary tumor (GC) cells, we show that this factor belongs to the NF-I family. When NF-I was competed out of the cell extracts, the trans-acting factor AP-2 bound to the same site as NF-I. AP-2 was present not only in HeLa cells, but also in GC cells albeit at a much lower concentration. Consistent with the mutually exclusive binding of NF-I and AP-2, their methylation interference patterns included four guanine residues that were crucial for binding of both NF-I and AP-2. Cell-free transcription from the hGH gene promoter showed that these two factors can transactivate this gene.

Analysis of cis- and trans-acting elements in the hormone-sensitive human somatotropin gene promoter.

The expression of the human growth hormone (hGH) gene is regulated by several transcription factors. Basal level transcription factors include TATA box-binding proteins, Sp1, USF and CTF/NF-1. The hGH gene is expressed only in pituitary somatotrophs, and the pituitary-specific GHF-1/Pit-1 protein is a potent transcriptional stimulator. Glucocorticoid and thyroid hormones, insulin, and GHRH which acts via cAMP, also control hGH gene transcription via trans-acting factors some of which are the hormone receptors themselves. Three transcription initiation sites were detected when hGH gene promoter activity was studied in a cell-free system. This system enabled us to delineate the respective role of some transcription factors and to propose a model that accounts for the basal, pituitary-specific, and hormonal control of hGH gene expression.