Hepatocyte nuclear factor 1alpha controls the expression of terminal complement genes.

The terminal components of the complement system contribute to host defense by forming the multiprotein membrane attack complex (MAC) which is responsible for cell lysis and several noncytotoxic effects. Most of the complement proteins are synthesized in the liver, but the mechanisms controlling their tissue-specific expression have not been elucidated. In this study we show that mice lacking the hepatic transcription factor hepatocyte nuclear factor 1alpha (HNF1alpha) fail to transcribe C5 and C8A complement genes. In addition, mRNAs encoding for several other terminal complement components or subunits are expressed at lower levels, including C8beta, C8gamma, and C9. We next used a reconstitution assay involving human sera with selective complement deficiencies to assess mouse complement activity. Sera from HNF1alpha-deficient mice showed negligible hemolytic activity of both C5 and C8alpha-gamma subunits. The activity of C8beta was severely affected despite only a 50% reduction in C8beta mRNA levels in the liver. This is reminiscent of C8alpha-gamma-deficient patients who accumulate extremely low levels of the C8beta subunit. Our results demonstrate that HNF1alpha plays a key role in the expression of C5 and C8A genes, two terminal complement component genes that are essential for the assembly of MAC as a result of complement activation.

Loss of HNF-1alpha function in mice leads to abnormal expression of genes involved in pancreatic islet development and metabolism.

Mutations in hepatocyte nuclear factor 1alpha (HNF-1alpha) lead to maturity-onset diabetes of the young type 3 as a result of impaired insulin secretory response in pancreatic beta-cells. The expression of 50 genes essential for normal beta-cell function was studied to better define the molecular mechanism underlying the insulin secretion defect in Hnf-1alpha(-/-) mice. We found decreased steady-state mRNA levels of genes encoding glucose transporter 2 (Glut2), neutral and basic amino acid transporter, liver pyruvate kinase (L-Pk), and insulin in Hnf-1alpha(-/-) mice. In addition, we determined that the expression of several islet-enriched transcription factors, including Pdx-1, Hnf-4alpha, and Neuro-D1/Beta-2, was reduced in Hnf-1alpha(-/-) mice. These changes in pancreatic islet mRNA levels were already apparent in newborn animals, suggesting that loss of Hnf-1alpha function rather than chronic hyperglycemia is the primary cause of the altered gene expression. This expression profile was pancreatic islet-specific and distinct from hepatocytes, where we found normal expression of Glut2, L-Pk, and Hnf-4alpha in the liver of Hnf-1alpha(-/-) mice. The expression of small heterodimer partner (Shp-1), an orphan receptor that can heterodimerize with Hnf-4alpha and inhibit its transcriptional activity, was also reduced in Hnf-1alpha(-/-) islets. We characterized a 0.58-kb Shp-1 promoter and determined that the decreased expression of Shp-1 may be indirectly mediated by a downregulation of Hnf-4alpha. We further showed that Shp-1 can repress its own transcriptional activation by inhibiting Hnf-4alpha function, thereby establishing a feedback autoregulatory loop. Our results indicate that loss of Hnf-1alpha function leads to altered expression of genes involved in glucose-stimulated insulin secretion, insulin synthesis, and beta-cell differentiation.

Characterization of the human OATP-C (SLC21A6) gene promoter and regulation of liver-specific OATP genes by hepatocyte nuclear factor 1 alpha.

OATP-C (SLC21A6) is the predominant Na(+)-independent uptake system for bile salts and bilirubin of human liver and is expressed exclusively at the basolateral (sinusoidal) hepatocyte membrane. To investigate the basis of liver-specific expression of OATP-C, we studied promoter function in the two hepatocyte-derived cell lines HepG2 and Huh7 and in nonhepatic HeLa cells. OATP-C promoter constructs containing from 66 to 950 nucleotides of 5'-regulatory sequence were active in HepG2 and Huh7 but not HeLa cells, indicating that determinants of hepatocyte-specific expression reside within the minimal promoter. Deoxyribonuclease I footprint analysis revealed a single region that was protected by HepG2 and Huh7 but not HeLa cell nuclear extracts. The liver-enriched transcription factor hepatocyte nuclear factor 1 alpha (HNF1 alpha) was shown by mobility shift assays to bind within this footprint. Coexpression of HNF1 alpha stimulated OATP-C promoter activity 30-fold in HepG2 and 49-fold in HeLa cells. Mutation of the HNF1 site abolished promoter function, indicating that HNF1 alpha is critical for hepatocyte-specific OATP-C gene expression. The human OATP8 (SLC21A8) and mouse Oatp4 (Slc21a6) promoters were also responsive to HNF1 alpha coexpression in HepG2 cells. These data support a role for HNF1 alpha as a global regulator of liver-specific bile salt and organic anion transporter genes.

Embryonic but not postnatal reexpression of hepatocyte nuclear factor 1alpha (HNF1alpha) can reactivate the silent phenylalanine hydroxylase gene in HNF1alpha-deficient hepatocytes.

The failure to transcribe the phenylalanine hydroxylase (PAH) gene in the liver of hepatocyte nuclear factor 1alpha (HNF1alpha)-deficient mice correlated with DNA hypermethylation and the presence of an inactive chromatin structure (M. Pontoglio, D. M. Faust, A. Doyen, M. Yaniv, and M. C. Weiss, Mol. Cell. Biol. 17:4948-4956, 1997). To evaluate the precise role played by HNF1alpha, DNA methylation, or histone acetylation in PAH gene silencing, we examined conditions that could restore PAH gene expression in HNF1alpha-deficient hepatocytes. We show that reactivation of PAH transcription can be achieved by reexpression of HNF1alpha in embryonic (i.e., embryonic day 12.5 [e12.5] to e13.5) hepatocytes but not in fetal (e17.5), newborn, and adult HNF1alpha-deficient hepatocytes. This defines a temporal competence window during which HNF1alpha can act to (re)program PAH gene transcription. We also show that PAH gene silencing can be partially relieved in HNF1alpha-deficient hepatocytes by treatment with the demethylating agent 5-azacytidine, even in the absence of HNF1alpha. Treatment using 5-azacytidine combined with trichostatin, a histone deacetylase inhibitor, resulted in a synergistic reactivation of the silenced PAH gene in adult hepatocytes, but this activity was not further increased by HNF1alpha reexpression. These results suggest that the HNF1alpha homeoprotein is involved in stage-specific developmental control of the methylation state and chromatin remodeling of the PAH gene.

Transcription factor-dependent regulation of CBP and P/CAF histone acetyltransferase activity.

CREB-binding protein (CBP) and CBP-associated factor (P/CAF) are coactivators possessing an intrinsic histone acetyltransferase (HAT) activity. They are positioned at promoter regions via association with sequence-specific DNA-binding factors and stimulate transcription in a gene-specific manner. The current view suggests that coactivator function depends mainly on the strength and specificity of transcription factor-coactivator interactions. Here we show that two dominant-negative mutants of hepatocyte nuclear factor-1alpha (HNF-1alpha), P447L and P519L, occurring in maturity onset diabetes of the young (MODY3) patients, exhibit paradoxically stronger interactions than the wild-type protein with either CBP or P/CAF. However, CBP and P/CAF recruited by these mutants lack HAT activity. In contrast, wild-type HNF-1alpha and other transcription factors, such as Sp1 or HNF-4, stimulated the HAT activity of CBP. The results suggest a more dynamic role for DNA-binding proteins in the transcription process than was considered previously. They are not only required for the recruitment of coactivators to the promoter but they may also modulate their enzymatic activity.

Nuclear covalently closed circular viral genomic DNA in the liver of hepatocyte nuclear factor 1 alpha-null hepatitis B virus transgenic mice.

The role of hepatocyte nuclear factor 1alpha (HNF1 alpha) in the regulation of hepatitis B virus (HBV) transcription and replication in vivo was investigated using a HNF1 alpha-null HBV transgenic mouse model. HBV transcription was not measurably affected by the absence of the HNF1 alpha transcription factor. However, intracellular viral replication intermediates were increased two- to fourfold in mice lacking functional HNF1 alpha protein. The increase in encapsidated cytoplasmic replication intermediates in HNF1 alpha-null HBV transgenic mice was associated with the appearance of nonencapsidated nuclear covalently closed circular (CCC) viral genomic DNA. Viral CCC DNA was not readily detected in HNF1 alpha-expressing HBV transgenic mice. This indicates the synthesis of nuclear HBV CCC DNA, the proposed viral transcriptional template found in natural infection, is regulated either by subtle alterations in the levels of viral transcripts or by changes in the physiological state of the hepatocyte in this in vivo model of HBV replication.

Hepatocyte nuclear factor 1, a transcription factor at the crossroads of glucose homeostasis.

Hepatocyte nuclear factor 1 (HNF1) is a transcription factor involved in the regulation of a large set of hepatic genes, including albumin, beta-fibrinogen, and alpha1-antitrypsin. HNF1 is expressed in the liver, digestive tract, pancreas, and kidney. Mice lacking HNF1 exhibit hepatic, pancreatic, and renal dysfunctions. HNF1-deficient mice fail to express the hepatic phenylalanine hydroxylase gene, giving rise to hyperphenylalaninemia. Renal proximal tubular reabsorption of glucose, phosphate, arginine, and other metabolites is affected, producing severe renal glucosuria, phosphaturia, and amino aciduria. Homozygous mutant mice also exhibit a dramatic insulin secretion defect. This dysfunction resembles that exhibited by patients with maturity-onset diabetes mellitus of the young type 3, who carry mutations in the human HNF1 gene in the heterozygous state. These data show that HNF1 is a major regulator of glucose homeostasis, regulating the expression of genes that are expressed in the liver, kidney, and pancreas.

HNF1alpha controls renal glucose reabsorption in mouse and man.

Recently it has been shown that dominant mutations in the human hepatocyte nuclear factor 1alpha (HNF1alpha) gene, encoding for a homeoprotein that is expressed in liver, kidney, pancreas and intestine, result in maturity onset diabetes of the young type 3 (MODY3). HNF1alpha-null mice are diabetic, but at the same time suffer from a renal Fanconi syndrome characterized by urinary glucose loss. Here we show that MODY3 patients are also characterized by a reduced tubular reabsorption of glucose. The renal murine defect is due to reduced expression of the low affinity/high capacity glucose cotransporter (SGLT2). Our results show that HNF1alpha directly controls SGLT2 gene expression. Together these data indicate that HNF1alpha plays a key role in glucose homeostasis in mammals.

Anatomy of a homeoprotein revealed by the analysis of human MODY3 mutations.

Hepatocyte nuclear factor 1alpha (HNF1alpha) is an atypical dimeric homeodomain-containing protein that is expressed in liver, intestine, stomach, kidney, and pancreas. Mutations in the HNF1alpha gene are associated with an autosomal dominant form of non-insulin-dependent diabetes mellitus called maturity-onset diabetes of the young (MODY3). More than 80 different mutations have been identified so far, many of which involve highly conserved amino acid residues among vertebrate HNF1alpha. In the present work, we investigated the molecular mechanisms by which MODY3 mutations could affect HNF1alpha function. For this purpose, we analyzed the properties of 10 mutants resulting in amino acid substitutions or protein truncation. Some mutants have a reduced protein stability, whereas others are either defective in the DNA binding or impaired in their intrinsic trans-activation potential. Three mutants, characterized by a complete loss of trans-activation, behave as dominant negatives when transfected with the wild-type protein. These data define a clear causative relationship between MODY3 mutations and functional defects in HNF1alpha trans-activation. In addition, our analysis sheds new light on the structure of a homeoprotein playing a key role in pancreatic beta cell function.

Defective pancreatic beta-cell glycolytic signaling in hepatocyte nuclear factor-1alpha-deficient mice.

Mutations in the hepatocyte nuclear factor-1alpha (HNF-1alpha) gene cause maturity onset diabetes of the young type 3, a form of type 2 diabetes mellitus. In mice lacking the HNF-1alpha gene, insulin secretion and intracellular calcium ([Ca2+]i) responses were impaired following stimulation with nutrient secretagogues such as glucose and glyceraldehyde but normal with non-nutrient stimuli such as potassium chloride. Patch clamp recordings revealed ATP-sensitive K+ currents (KATP) in beta-cells that were insensitive to suppression by glucose but normally sensitive to ATP. Exposure to mitochondrial substrates suppressed KATP, elevated [Ca2+]i, and corrected the insulin secretion defect. NAD(P)H responses to glucose were substantially reduced, and inhibitors of glycolytic NADH generation reproduced the mutant phenotype in normal islets. Flux of glucose through glycolysis in islets from mutant mice was reduced, as a result of which ATP generation in response to glucose was impaired. We conclude that hepatocyte nuclear factor-1alpha diabetes results from defective beta-cell glycolytic signaling, which is potentially correctable using substrates that bypass the defect.

Defective insulin secretion in hepatocyte nuclear factor 1alpha-deficient mice.

Mutations in the gene for the transcription factor hepatocyte nuclear factor (HNF) 1alpha cause maturity-onset diabetes of the young (MODY) 3, a form of diabetes that results from defects in insulin secretion. Since the nature of these defects has not been defined, we compared insulin secretory function in heterozygous [HNF-1alpha (+/-)] or homozygous [HNF-1alpha (-/-)] mice with null mutations in the HNF-1alpha gene with their wild-type littermates [HNF-1alpha (+/+)]. Blood glucose concentrations were similar in HNF-1alpha (+/+) and (+/-) mice (7.8+/-0.2 and 7.9+/-0.3 mM), but were significantly higher in the HNF-1alpha (-/-) mice (13.1+/-0.7 mM, P < 0.001). Insulin secretory responses to glucose and arginine in the perfused pancreas and perifused islets from HNF-1alpha (-/-) mice were < 15% of the values in the other two groups and were associated with similar reductions in intracellular Ca2+ responses. These defects were not due to a decrease in glucokinase or insulin gene transcription. beta cell mass adjusted for body weight was not reduced in the (-/-) animals, although pancreatic insulin content adjusted for pancreas weight was slightly lower (0.06+/-0.01 vs. 0.10+/-0.01 microg/mg, P < 0.01) than in the (+/+) animals. In summary, a null mutation in the HNF-1alpha gene in homozygous mice leads to diabetes due to alterations in the pathways that regulate beta cell responses to secretagogues including glucose and arginine. These results provide further evidence in support of a key role for HNF-1alpha in the maintenance of normal beta cell function.

Hepatocyte nuclear factor 1alpha gene inactivation impairs chromatin remodeling and demethylation of the phenylalanine hydroxylase gene.

Hepatocyte nuclear factor 1 alpha (HNF1alpha) is a homeoprotein that is expressed in the liver, kidney, pancreas, and digestive tract. Its inactivation in mouse resulted in decreased transcription of known target genes such as albumin and alpha1-antitrypsin. In contrast, the phenylalanine hydroxylase (PAH) gene was totally silent and unresponsive to normal inducers like glucocorticoids and cyclic AMP in the liver. DNase I and micrococcal nuclease digestion of liver nuclei showed that HNF1alpha inactivation had drastic effects on the chromatin structure of the PAH regulatory regions. Three DNase I-hypersensitive sites (HSSI, HSSII, and HSSIII), typical of the actively transcribed PAH gene, were undetectable in liver from HNF1alpha-deficient animals. Both HSSII and HSSIII elements harbor HNF1 sites, but only the latter has detectable enhancer activity in transient-transfection assays. In addition, the PAH promoter in livers of HNF1alpha-deficient animals was methylated. These results suggest that HNF1alpha could activate transcription through two mechanisms. One implies participation in the recruitment of the general transcription machinery to the promoter, and the second involves the remodeling of chromatin structure and demethylation that would allow transcription factors to interact with their cognate cis-acting elements.

Analysis of the distribution of binding sites for a tissue-specific transcription factor in the vertebrate genome.

Hepatocyte nuclear factor 1 (HNF1) is a dimeric homeoprotein expressed in hepatocytes and in a few other epithelial cells where it helps regulate the expression of a specific subset of genes. In an attempt to identify novel target genes for HNF1 and to assess the distribution of its target sites within the vertebrate genome, we performed a computer-assisted search within the available databases using a weighted matrix. Several hundred potential target sequences were identified within the GenBank and EMBL data banks. DNA binding assays demonstrated that more than 95%, of the new sites tested (52 sites among 54) bound HNF1. Surprisingly many HNF1 target sites were found in genes that are transcribed in cell types that do not contain the protein. On the other hand these sites are 2.5 to five times more frequent in hepatic genes than expected. It seems that the presence of HNF1 sites in liver-specific genes was favoured, but that no counter-selection occurred within the rest of the genome. HNF1 binding sites in liver genes are more often associated in clusters with sites for other transcription factors and the enrichment is more pronounced in promoter regions. We identified more than 100 liver specific genes that are potentially regulated by HNF1.

The HNF1/HNF4-dependent We2 element of woodchuck hepatitis virus controls viral replication and can activate the N-myc2 promoter.

Transcriptional activation of myc family proto-oncogenes through the insertion of viral sequences is the predominant mechanism by which woodchuck hepatitis virus (WHV) induces liver tumors in chronically infected animals. The main target is N-myc2, a functional retroposon of the N-myc gene, but c-myc and N-myc are also marginally involved. Here we identify a major, liver-specific regulatory element in the WHV genome (We2) which efficiently activates the N-myc2 promoter in cultured hepatoma cells. In the context of the episomal viral genome, We2 governs the production of pregenomic RNA and thus plays a central role in the control of viral replication. We2 activity is primarily controlled by the liver-enriched HNF1 and HNF4 transcription factors, although NF1 and Oct proteins were also shown to bind in a central region. The expression of HNF1 and HNF4 appears to be maintained in woodchuck tumors. Thus, We2 is a prime candidate for controlling myc gene cis activation during WHV-induced hepatocarcinogenesis.

Hepatocyte nuclear factor 1 inactivation results in hepatic dysfunction, phenylketonuria, and renal Fanconi syndrome.

HNF1 is a transcriptional activator of many hepatic genes including albumin, alpha1-antitrypsin, and alpha- and beta-fibrinogen. It is related to the homeobox gene family and is predominantly expressed in liver and kidney. Mice lacking HNF1 fail to thrive and die around weaning after a progressive wasting syndrome with a marked liver enlargement. The transcription rate of genes like albumin and alpha1-antitrypsin is reduced, while the gene coding for phenylalanine hydroxylase is totally silent, giving rise to phenylketonuria. Mutant mice also suffer from severe Fanconi syndrome caused by renal proximal tubular dysfunction. The resulting massive urinary glucose loss leads to energy and water wasting. HNF1-deficient mice may provide a model for human renal Fanconi syndrome.

Characterization by yeast artificial chromosome cloning of the linked apolipoprotein(a) and plasminogen genes and identification of the apolipoprotein(a) 5' flanking region.

The apoprotein(a) [apo(a)] gene encodes a protein component of the circulating lipoprotein(a) [Lp(a)]. The apo(a) gene is highly homologous to the plasminogen gene. It encodes one of the most polymorphic human proteins, due to variability in the number of repetitions of structures called kringles. In addition, Lp(a) levels vary among individuals by more than two orders of magnitude, the high levels being highly correlated with predisposition to early atherosclerotic disease. To better understand the genetics and function of the apo(a) gene, we have cloned in yeast artificial chromosome vectors DNA fragments comprising the linked apo(a) and plasminogen genes and other members of the plasminogen family. By a combination of pulsed-field gel electrophoresis and genome walking experiments, we have identified the 5' portion and flanking regions of the apo(a) gene.

Structure of the gene encoding hepatocyte nuclear factor 1 (HNF1).

Genomic clones have been isolated that cover the entire gene for the transcription factor HNF1 (hepatocyte nuclear factor 1). This protein governs the expression of many genes, synthesized in the liver in a tissue-specific manner. We have determined the intron/exon structure of the HNF1 gene, which is strictly conserved between rat and mouse and estimate that it spans not more than 40kb in the rat genome. Whereas most homeoprotein genes do not contain introns within the homeodomain, HNF1 displays an intron between the regions encoding the second and the third helices. We discuss possible evolutionary mechanisms leading to this homeobox intron/exon pattern.

Functional analysis of the human lecithin cholesterol acyl transferase gene promoter.

In this report cis-acting sequences that direct transcription of the lecithin choleterol acyl transferase (LCAT) gene were identified. To assay the promoter activity, fragments from the 5' flanking region were fused upstream to the cloramphenicol acetyl transferase gene and transfected into Hep3B and HeLa cells. The gene sequences were active in both cell lines. A minimal promoter comprising only 71 bp is still fully active and contains a TATA box, a LFAI motif and two Sp1 binding sites. The activity of the promoter was entirely dependent on the Sp1 sites.

Definition of the transcription initiation site of human plasminogen gene in liver and non hepatic cell lines.

We have mapped the cap site of the human plasminogen mRNA by primer extension and PCR techniques and found that it is located at position -161 relative to the first ATG, 97 bases upstream to the 5' end of the previously isolated cDNA clone. Seven human hepatic and non hepatic cell lines and fresh liver cells were tested for human plasminogen mRNA expression: the liver and the liver derived HepG2 cell line represent the major site of plasminogen RNA synthesis while the other cell lines (Hep3B, HeLa, IMR, 293 CaCo and SW626) show much lower levels.

Lecithin cholesterol acyl transferase deficiency: molecular analysis of a mutated allele.

The enzyme, lecithin cholesterol acyltransferase (LCAT), is responsible for the esterification of plasma cholesterol mediating the transfer of an acyl group from lecithin to the 3-hydroxy group of cholesterol. Deficiency of the enzyme is a well-known syndrome with a widespread geographic occurrence. We have cloned an allele from a patient homozygous for the LCAT deficiency. The only change that we could detect is a C to T transition in the fourth exon of the gene; this causes a substitution of Arg for Trp at position 147 of the mature protein. The functional significance of such a substitution with respect to the enzyme defect was demonstrated by transfecting the mutated LCAT gene in the cell line COS-1.