HNF1 Binding Research Articles

Loss of ARID3A perturbs intestinal epithelial proliferation-differentiation ratio and regeneration.

Intestinal stem cells at the crypt divide and give rise to progenitor cells that proliferate and differentiate into various mature cell types in the transit-amplifying (TA) zone. Here, we showed that the transcription factor ARID3A regulates intestinal epithelial cell proliferation and differentiation at the TA progenitors. ARID3A forms an expression gradient from the villus tip to the upper crypt mediated by TGF-β and WNT. Intestinal-specific deletion of Arid3a reduces crypt proliferation, predominantly in TA cells. Bulk and single-cell transcriptomic analysis shows increased enterocyte and reduced secretory differentiation in the Arid3a cKO intestine, accompanied by enriched upper-villus gene signatures of both cell lineages. We find that the enhanced epithelial differentiation in the Arid3a-deficient intestine is caused by increased binding and transcription of HNF1 and HNF4. Finally, we show that loss of Arid3a impairs irradiation-induced regeneration with sustained cell death and reprogramming. Our findings imply that Arid3a functions to fine-tune the proliferation-differentiation dynamics at the TA progenitors, which are essential for injury-induced regeneration.

Hepatic Expression of the Mouse Angptl8 Gene Is Regulated by Insulin Through Increases in Hepatocyte Nuclear Factor-1

Angiopoietin-like proteins (ANGPTLs) are a family of proteins structurally similar to angiopoietins. ANGPTL8 is an important regulator of circulating triglyceride (TG) levels in mammals. Increasing evidence revealed an association between ANGPTL8 expression and serum lipid profiles, especially in subjects with metabolic syndrome. Several mice studies demonstrated that Angptl8 is suppressed by fasting and induced by long term refeeding, however the detailed mechanism is still unclear. In humans, ANGPTL8 is mainly expressed in the liver. Therefore, this study aims to investigate the mechanisms that control the refeeding induced increase in the hepatic Angptl8 gene expression. Methods and Results: Twenty-week-old male C57/BL6 mice were used in this study. Mice were fasted for 12h during the dark cycle and re-fed for 30, 60, 120, 240 and 360 minutes during the light cycle. Mice were euthanized after each refeeding time course and tissues were collected. We found even short refeeding times (~60 min) enhanced the expression levels of hepatic Angptl8 in mice. We cloned the mouse Angptl8 gene promoter region. Promoter deletion analyses showed that the basal promoter activity was significantly attenuated by a deletion of -309/-60 region in hepatocytes. A computational motif search revealed the presence of a potential binding motif for hepatocyte nuclear factor 1α/1β (HNF-1α/β) at -84/-68 bp of the promoter. Mutations of the HNF-1 binging site significantly decreased the promoter activity in mouse hepatoma cells (Hepa1-6) and mouse primary hepatocytes, and the promoter carrying the mutated HNF-1 site was not transactivated by co-transfected HNF-1 in a non-hepatic cell line. These findings indicated that HNF-1 was essential and critical factor for the basal expression of Angptl8 in murine liver. In fact, knockdown of Hnf-1 using siRNA method in mouse Hepa1-6 and mouse primary hepatocytes reduced Angptl8 protein levels. We also performed Electrophoretic mobility-shift assays and confirmed the direct binding of Hnf-1 to its Angptl8 promoter binding motif. To elucidate whether refeeding could enhance HNF-1, we checked the expression levels of Hnf-1 in mouse liver. Hnf-1 expression levels of both mRNA and protein were increased after short-term refeeding, paralleling the enhanced expression of the Angptl8. Moreover, insulin-stimulated primary hepatocytes showed increased expression of Angptl8 protein, but knockdown of Hnf-1 completely abolished this enhancement by insulin. Chromatin immunoprecipitation (ChIP) analyses confirmed the recruitment of endogenous Hnf-1 to the Angptl8 promoter region and it was strongly induced by insulin. Conclusion: HNF-1 plays essential role in hepatocyte-specific and refeeding-induced rapid increases in Angptl8 expression via insulin.

Factor VII Deficiency Due to Compound Heterozygosity for the p.Leu13Pro Mutation and a Novel Mutation in the HNF4 Binding Region (-58G>C) in the F7 Promoter.

We investigated the molecular basis of factor VII (FVII) deficiency in a Japanese patient and identified compound heterozygous mutations. Factor VII activity and antigen levels in the patient were less than 5.0% and 6.5% of controls, respectively. All exons, exon-intron boundaries, and the 5' promoter region of F7 from genomic DNA were amplified using polymerase chain reaction (PCR). Sequencing analysis of PCR fragments revealed that the patient was heterozygous for a known T to C substitution at nucleotide position 38, which resulted in the p.Leu13Pro missense mutation (Factor VII Morioka) in the signal peptide region, and a novel mutation in the 5' promoter region (-58G>C). An electrophoretic mobility shift assay showed that the mutation in the promoter region reduced the binding of hepatocyte nuclear factor (HNF). It is presumed that the reduced binding of HNF-4 to the F7 promoter region reduces F7 transcription and thus reduces the synthesis and expression of FVII.

Transcriptional Regulation of the Angptl8 Gene by Hepatocyte Nuclear Factor-1 in the Murine Liver

Brief refeeding times (~60 min) enhanced hepatic Angptl8 expression in fasted mice. We cloned the mouse Angptl8 promoter region to characterise this rapid refeeding-induced increase in hepatic Angptl8 expression. Deletion of the −309/−60 promoter region significantly attenuated basal promoter activity in hepatocytes. A computational motif search revealed a potential binding motif for hepatocyte nuclear factor 1α/1β (HNF-1α/β) at −84/−68 bp of the promoter. Mutation of the HNF-1 binding site significantly decreased the promoter activity in hepatocytes, and the promoter carrying the mutated HNF-1 site was not transactivated by co-transfection of HNF-1 in a non-hepatic cell line. Silencing Hnf-1 in hepatoma cells and mouse primary hepatocytes reduced Angptl8 protein levels. Electrophoretic mobility-shift assays confirmed direct binding of Hnf-1 to its Angptl8 promoter binding motif. Hnf-1α expression levels increased after short-term refeeding, paralleling the enhanced in vivo expression of the Angptl8 protein. Chromatin immunoprecipitation (ChIP) confirmed the recruitment of endogenous Hnf-1 to the Angptl8 promoter region. Insulin-treated primary hepatocytes showed increased expression of Angptl8 protein, but knockdown of Hnf-1 completely abolished this enhancement. HNF-1 appears to play essential roles in the rapid refeeding-induced increases in Angptl8 expression. HNF-1α may therefore represent a primary medical target for ANGPTL8-related metabolic abnormalities. The study revealed the transcriptional regulation of the mouse hepatic Angptl8 gene by HNF-1.

Molecular mechanism linking a novel PCSK9 copy number variant to severe hypercholesterolemia

Background and aimsA 42 year-old male with premature atherosclerosis, severe dyslipidemia and resistance to treatment with high dose statin and a recommended dose of a PCSK9 inhibitor, was found to have a duplication of the PCSK9 gene. However, the clinical phenotype, which included a more than 15-fold elevation in circulating PCSK9, was unexpected given that he had one additional gene copy. MethodsHere we have carried out whole genome sequencing and transcriptional reporter assays to investigate the molecular mechanism leading to this unusual FH phenotype. ResultsThe PCSK9 duplication was found to contain the full coding sequence but with an 829 bp shorter 3′-untranslated region (UTR) sequence. All possible rearrangements include a head-to-head fusion between a completely duplicated PCSK9 and a chromosomal region, normally situated ~80 kb away, that includes HNF4 and USF1 binding sites that could promote transcription of the PCSK9 gene. Transcriptional reporter assays demonstrated that a construct harboring the HNF4 binding site significantly increased the promoter activity by 2.5-fold with a smaller effect noted for a USF1 construct. ConclusionsHere we describe, in a patient with resistant hypercholesterolemia, a novel PCSK9 gene rearrangement that enables upregulation of PCSK9 expression by allowing proximity to an active enhancer binding to HNF4A.

DNA elements for constitutive androstane receptor- and pregnane X receptor-mediated regulation of bovine CYP3A28 gene.

The regulation of cytochrome P450 3A (CYP3A) enzymes is established in humans, but molecular mechanisms of its basal and xenobiotic-mediated regulation in cattle are still unknown. Here, ~10 kbp of the bovine CYP3A28 gene promoter were cloned and sequenced, and putative transcription factor binding sites were predicted. The CYP3A28 proximal promoter (PP; -284/+71 bp) contained DNA elements conserved among species. Co-transfection of bovine nuclear receptors (NRs) pregnane X and constitutive androstane receptor (bPXR and bCAR) with various CYP3A28 promoter constructs into hepatoma cell lines identified two main regions, the PP and the distal fragment F3 (-6899/-4937 bp), that were responsive to bPXR (both) and bCAR (F3 fragment only). Site-directed mutagenesis and deletion of NR motif ER6, hepatocyte nuclear factor 1 (HNF-1) and HNF-4 binding sites in the PP suggested either the involvement of ER6 element in bPXR-mediated activation or the cooperation between bPXR and liver-enriched transcription factors (LETFs) in PP transactivation. A putative DR5 element within the F3 fragment was involved in bCAR-mediated PP+F3 transactivation. Although DNA enrichment by anti-human NR antibodies was quite low, ChIP investigations in control and RU486-treated BFH12 cells, suggested that retinoid X receptor α (RXRα) bound to ER6 and DR5 motifs and its recruitment was enhanced by RU486 treatment. The DR5 element seemed to be recognized mainly by bCAR, while no clear-cut results were obtained for bPXR. Present results point to species-differences in CYP3A regulation and the complexity of bovine CYP3A28 regulatory elements, but further confirmatory studies are needed.

Epigenetic regulation of hibernation-associated HP-20 and HP-27 gene transcription in chipmunk liver

The chipmunk hibernation-related proteins (HPs) HP-20 and HP-27 are components of a 140-kDa complex that dramatically decreases in the blood during hibernation. The HP-20 and HP-27 genes are expressed specifically in the liver and are downregulated in hibernating chipmunks. Hibernation-associated physiological changes are assumed to be under genetic control. Therefore, to elucidate the molecular mechanisms of hibernation, here we examined the mechanisms behind the altered HP-20 and HP-27 gene expression in nonhibernating versus hibernating chipmunks. Chromatin immunoprecipitation (ChIP) analyses revealed that histone H3 on the HP-20 and HP-27 gene promoters was highly acetylated at lysine (K) 9 and K14 and highly trimethylated at K4 in the liver of nonhibernating chipmunks, while these active histone modifications were nearly absent in hibernating chipmunks. Furthermore, histone acetyltransferases and a histone methyltransferase were associated with the HP-20 and HP-27 gene promoters primarily in nonhibernating chipmunks. Consistent with a previous finding that HNF-1 and USF can activate HP-20 and HP-27 gene transcription by binding to the proximal promoter region, ChIP-quantitative PCR (qPCR) analyses revealed that significantly less HNF-1 and USF were bound to these gene promoters in hibernating than in nonhibernating chipmunks. These findings collectively indicated that the hibernation-associated HP-20 and HP-27 gene expression is epigenetically regulated at the transcriptional level by the binding of HNF-1 and USF to their proximal promoters, and that histone modification has a key role in hibernation-associated transcriptional regulation.

CircularLogo: A lightweight web application to visualize intra-motif dependencies

BackgroundThe sequence logo has been widely used to represent DNA or RNA motifs for more than three decades. Despite its intelligibility and intuitiveness, the traditional sequence logo is unable to display the intra-motif dependencies and therefore is insufficient to fully characterize nucleotide motifs. Many methods have been developed to quantify the intra-motif dependencies, but fewer tools are available for visualization.ResultWe developed CircularLogo, a web-based interactive application, which is able to not only visualize the position-specific nucleotide consensus and diversity but also display the intra-motif dependencies. Applying CircularLogo to HNF6 binding sites and tRNA sequences demonstrated its ability to show intra-motif dependencies and intuitively reveal biomolecular structure. CircularLogo is implemented in JavaScript and Python based on the Django web framework. The program’s source code and user’s manual are freely available at http://circularlogo.sourceforge.net. CircularLogo web server can be accessed from http://bioinformaticstools.mayo.edu/circularlogo/index.html.ConclusionCircularLogo is an innovative web application that is specifically designed to visualize and interactively explore intra-motif dependencies.

HNF-4 participates in the hibernation-associated transcriptional regulation of the chipmunk hibernation-related protein gene

The chipmunk hibernation-related protein 25 (HP-25) is involved in the circannual control of hibernation in the brain. The liver-specific expression of the HP-25 gene is repressed in hibernating chipmunks under the control of endogenous circannual rhythms. However, the molecular mechanisms that differentially regulate the HP-25 gene during the nonhibernation and hibernation seasons are unknown. Here, we show that the hibernation-associated HP-25 expression is regulated epigenetically. Chromatin immunoprecipitation analyses revealed that significantly less hepatocyte nuclear receptor HNF-4 bound to the HP-25 gene promoter in the liver of hibernating chipmunks compared to nonhibernating chipmunks. Concurrently in the hibernating chipmunks, coactivators were dissociated from the promoter, and active transcription histone marks on the HP-25 gene promoter were lost. On the other hand, small heterodimer partner (SHP) expression was upregulated in the liver of hibernating chipmunks. Overexpressing SHP in primary hepatocytes prepared from nonhibernating chipmunks caused HNF-4 to dissociate from the HP-25 gene promoter, and reduced the HP-25 mRNA level. These results suggest that hibernation-related HP-25 expression is epigenetically regulated by the binding of HNF-4 to the HP-25 promoter, and that this binding might be modulated by SHP in hibernating chipmunks.

Which Genetics Variants in DNase-Seq Footprints Are More Likely to Alter Binding?

Large experimental efforts are characterizing the regulatory genome, yet we are still missing a systematic definition of functional and silent genetic variants in non-coding regions. Here, we integrated DNaseI footprinting data with sequence-based transcription factor (TF) motif models to predict the impact of a genetic variant on TF binding across 153 tissues and 1,372 TF motifs. Each annotation we derived is specific for a cell-type condition or assay and is locally motif-driven. We found 5.8 million genetic variants in footprints, 66% of which are predicted by our model to affect TF binding. Comprehensive examination using allele-specific hypersensitivity (ASH) reveals that only the latter group consistently shows evidence for ASH (3,217 SNPs at 20% FDR), suggesting that most (97%) genetic variants in footprinted regulatory regions are indeed silent. Combining this information with GWAS data reveals that our annotation helps in computationally fine-mapping 86 SNPs in GWAS hit regions with at least a 2-fold increase in the posterior odds of picking the causal SNP. The rich meta information provided by the tissue-specificity and the identity of the putative TF binding site being affected also helps in identifying the underlying mechanism supporting the association. As an example, the enrichment for LDL level-associated SNPs is 9.1-fold higher among SNPs predicted to affect HNF4 binding sites than in a background model already including tissue-specific annotation.

Hepatocyte nuclear factor 1 coordinates multiple processes in a model of intestinal epithelial cell function

Mutations in hepatocyte nuclear factor 1 transcription factors (HNF1α/β) are associated with diabetes. These factors are well studied in the liver, pancreas and kidney, where they direct tissue-specific gene regulation. However, they also have an important role in the biology of many other tissues, including the intestine. We investigated the transcriptional network governed by HNF1 in an intestinal epithelial cell line (Caco2). We used chromatin immunoprecipitation followed by direct sequencing (ChIP-seq) to identify HNF1 binding sites genome-wide. Direct targets of HNF1 were validated using conventional ChIP assays and confirmed by siRNA-mediated depletion of HNF1, followed by RT-qPCR. Gene ontology process enrichment analysis of the HNF1 targets identified multiple processes with a role in intestinal epithelial cell function, including properties of the cell membrane, cellular response to hormones, and regulation of biosynthetic processes. Approximately 50% of HNF1 binding sites were also occupied by other members of the intestinal transcriptional network, including hepatocyte nuclear factor 4A (HNF4A), caudal type homeobox 2 (CDX2), and forkhead box A2 (FOXA2). Depletion of HNF1 in Caco2 cells increases FOXA2 abundance and decreases levels of CDX2, illustrating the coordinated activities of the network. These data suggest that HNF1 plays an important role in regulating intestinal epithelial cell function, both directly and through interactions with other intestinal transcription factors.

Abstract 9772: Antagonism of Rosuvastatin-induced Elevation of Circulating PCSK9 in Hamsters by Liver-specific Knockdown of HNF1α or HNF1β

Proprotein convertase subtilisin/kexin type 9 (PCSK9) induces hepatic LDL receptor (LDLR) degradation and lowers the removal of LDL-cholesterol from circulation. Thus, PCSK9 has become an important therapeutic target for the treatment of hypercholesterolemia. The transcription of PCSK9 is prominently controlled by the HNF1 binding motif embedded in its proximal promoter region that binds to HNF1α or HNF1β as homodimers or heterodimers. Our previous in vivo studies showed that only HNF1α but not HNF1β regulates PCSK9 transcription in mouse liver. Statin has been shown to elevate circulating PCSK9 levels through stimulating PCSK9 gene transcription, which reduces the clinical efficacy of statin in LDL-cholesterol reduction. In this study, we utilized adenoviral shRNA expression vectors (Ad-shHNF1α and Ad-shHNF1β) to generate liver specific knockdown of HNF1α or HNF1β in hamsters to examine the impact of reduced expression of HNF1 transcription factors on statin-induced elevation of PCSK9. Administration of rosuvastatin (RSV) at a daily dose of 15 mg/kg to hamsters infected with a control adenovirus significantly increased liver PCSK9 mRNA levels by 94% and serum PCSK9 levels by 37%. However, injection of Ad-shHNF1α into hamsters largely abolished the RSV-induced elevation of PCSK9 serum levels and hepatic mRNA levels, which was accompanied by a significant increase in liver LDLR protein abundance by 64% as compared to hamsters injected with control virus. Surprisingly, injection of Ad-shHNF1β produced similar inhibitory effects on the RSV-induced PCSK9 expression as that of Ad-shHNF1α. This was different from the negative results of HNF1β knockdown conducted in mice. In addition to changes in PCSK9 levels, we observed a modest but significant reduction in serum non-HDL cholesterol level after knockdown of HNF1α (27%) or HNF1β (32%) in hamsters treated with RSV. Altogether, our study demonstrates that unlike mice, both HNF1α and HNF1β are positive regulators of hepatic PCSK9 transcription in hamster species and that transient, liver specific knockdown of either HNF1α or HNF1β could antagonize the RSV-induced elevation of serum PCSK9 and non-HDL cholesterol levels.

Abstract P046: Interplay of Intronic and Promoter Region Polymorphisms Up-regulate Human Angiotensinogen and Cause Hypertension in Transgenic Mice

Angiotensinogen (AGT) is the substrate for the RAS-cascade and polymorphisms leading to its overexpression are linked to hypertension. We have shown that SNPs in linkage disequilibrium with -6A of the hAGT gene cause increased AGT expression and hypertension. Recently, two genome wide association studies (GWAS) have linked SNP at the +1164 in intron-I with increased blood pressure. The +1164A allele has stronger homology with HNF-3 binding site as compared to +1164G. HNF3 belongs to the family of “pioneer” transcription factors, which enable chromatin access for other tissue-specific transcription factors. Thus, we propose that a haplotype, where multiple SNP-groups occur together, is a better model to predict and study disease correlation with genetic variations than individual SNPs alone. Intronic SNPs could increase chromatin access thus modulating promoter-transcription factor (TF) interactions. To study these possible interactions between the intronic SNPs in the promoter and intron I of the hAGT gene, we have divided this gene in two major haplotypes: haplotype-I (containing -6A & +1164A) and II (containing -6G & +1164G). Reporter construct containing haplotype-I has increased promoter activity as compared to haplotype-II on transient transfection in human liver cells, without (hap-1: 73±2 vs. hap-II: 23.64±3.21 A.U; p<0.05) and with dexamethasone treatment (hap-1: 1174±68vs. hap-II: 333.3±37 A.U; p<0.05). Double transgenic mice (TG) containing human renin gene (hREN) and haplotype-I of the hAGT gene (containing 2.0 Kb of the promoter, all the introns, exons and 3’UTR of the hAGT gene at the HPRT locus) have increased basal and GR induced expression of the hAGT gene in liver, kidney and fat as compared to haplotype-II. Complementary CHIP analysis shows increased TF-chromatin binding in the liver of haplotype-I TG mice for HNF3β (4.35 folds), C/EBPβ (2.1 folds), STAT-3 (3.6 folds) and GR (4.2 folds). Crucially, haplotype-I TG mice have increased (p<0.05) SBP (137±4 mm Hg) when compared to haplotype-II mice (126±3 mm Hg). In conclusion, intronic SNPs regulate hAGT gene expression via complex interplay with SNPs in the gene-promoter and this underscores the need for a haplotype-based approach to genetic variability and disease correlation.

Cross Talk Between GH-Regulated Transcription Factors HNF6 and CUX2 in Adult Mouse Liver.

Hepatocyte-enriched nuclear factor (HNF)6 and CUX2 are GH and STAT5-regulated homeobox transcription factors. CUX2 shows female-specific expression and contributes to liver sex differences by repressing many male-biased genes and inducing many female-biased genes, whereas HNF6 is expressed at similar levels in male and female liver. In cell-based transfection studies, CUX2 inhibited HNF6 transcriptional regulation of the sex-specific gene promoters CYP2C11 and CYP2C12, blocking HNF6 repression of CYP2C11 and HNF6 activation of CYP2C12. These inhibitory actions of CUX2 can be explained by competition for HNF6 DNA binding, as demonstrated by in vitro EMSA analysis and validated in vivo by global analysis of the HNF6 cistrome. Approximately 40 000 HNF6-binding sites were identified in mouse liver chromatin, including several thousand sites showing significant sex differences in HNF6 binding. These sex-biased HNF6-binding sites showed strong enrichment for correspondingly sex-biased DNase hypersensitive sites and for proximity to genes showing local sex-biased chromatin marks and a corresponding sex-biased expression. Further, approximately 90% of the genome-wide binding sites for CUX2 were also bound by HNF6. These HNF6/CUX2 common binding sites were enriched for genomic regions more accessible in male than in female mouse liver chromatin and showed strongest enrichment for male-biased genes, suggesting CUX2 displacement of HNF6 as a mechanism to explain the observed CUX2 repression of male-biased genes in female liver. HNF6 binding was sex independent at a majority of its binding sites, and HNF6 peaks were frequently associated with cobinding by multiple other liver transcription factors, consistent with HNF6 playing a global regulatory role in both male and female liver.

The transcription factor HNF1α induces expression of angiotensin-converting enzyme 2 (ACE2) in pancreatic islets from evolutionarily conserved promoter motifs

Pancreatic angiotensin-converting enzyme 2 (ACE2) has previously been shown to be critical for maintaining glycemia and β-cell function. Efforts to maintain or increase ACE2 expression in pancreatic β-cells might therefore have therapeutic potential for treating diabetes. In our study, we investigated the transcriptional role of hepatocyte nuclear factor 1α (HNF1α) and hepatocyte nuclear factor 1β (HNF1β) in induction of ACE2 expression in insulin-secreting cells. A deficient allele of HNF1α or HNF1β causes maturity-onset diabetes of the young (MODY) types 3 and 5, respectively, in humans. We found that ACE2 is primarily transcribed from the proximal part of the ACE2 promoter in the pancreas. In the proximal part of the human ACE2 promoter, we further identified three functional HNF1 binding sites, as they have binding affinity for HNF1α and HNF1β and are required for induction of promoter activity by HNF1β in insulinoma cells. These three sites are well-conserved among mammalian species. Both HNF1α and HNF1β induce expression of ACE2 mRNA and lead to elevated levels of ACE2 protein and ACE2 enzymatic activity in insulinoma cells. Furthermore, HNF1α dose-dependently increases ACE2 expression in primary pancreatic islet cells. We conclude that HNF1α can induce the expression of ACE2 in pancreatic islet cells via evolutionarily conserved HNF1 binding sites in the ACE2 promoter. Potential therapeutics aimed at counteracting functional HNF1α depletion in diabetes and MODY3 will thus have ACE2 induction in pancreatic islets as a likely beneficial effect.

Integrative Bioinformatics Links HNF1B with Clear Cell Carcinoma and Tumor-Associated Thrombosis

Clear cell carcinoma (CCC) is a histologically distinct carcinoma subtype that arises in several organ systems and is marked by cytoplasmic clearing, attributed to abundant intracellular glycogen. Previously, transcription factor hepatocyte nuclear factor 1-beta (HNF1B) was identified as a biomarker of ovarian CCC. Here, we set out to explore more broadly the relation between HNF1B and carcinomas with clear cell histology. HNF1B expression, evaluated by immunohistochemistry, was significantly associated with clear cell histology across diverse gynecologic and renal carcinomas (P<0.001), as was hypomethylation of the HNF1B promoter (P<0.001). From microarray analysis, an empirically-derived HNF1B signature was significantly enriched for computationally-predicted targets (with HNF1 binding sites) (P<0.03), as well as genes associated with glycogen metabolism, including glucose-6-phophatase, and strikingly the blood clotting cascade, including fibrinogen, prothrombin and factor XIII. Enrichment of the clotting cascade was also evident in microarray data from ovarian CCC versus other histotypes (P<0.01), and HNF1B-associated prothrombin expression was verified by immunohistochemistry (P = 0.015). Finally, among gynecologic carcinomas with cytoplasmic clearing, HNF1B immunostaining was linked to a 3.0-fold increased risk of clinically-significant venous thrombosis (P = 0.043), and with a 2.3-fold increased risk (P = 0.011) in a combined gynecologic and renal carcinoma cohort. Our results define HNF1B as a broad marker of clear cell phenotype, and support a mechanistic link to glycogen accumulation and thrombosis, possibly reflecting (for gynecologic CCC) derivation from secretory endometrium. Our findings also implicate a novel mechanism of tumor-associated thrombosis (a major cause of cancer mortality), based on the direct production of clotting factors by cancer cells.

Impact of Nucleotide Mutations at the HNF3- and HNF4-Binding Sites in Enhancer 1 on Viral Replication in Patients with Chronic Hepatitis B Virus Infection

Background/AimsThe hepatitis B virus (HBV) genome contains binding sites for hepatocyte nuclear factors (HNF) 3 and 4 in the core domain of enhancer 1 (Enh1), and mutations in this domain have a strong impact on virus replication. We aimed to identify frequent base-mutation sites in the core domain of Enh1 and to examine the impact of these mutations on viral replication.MethodsWe studied virological characteristics and genetic sequences in 387 patients with chronic hepatitis B. We evaluated functional differences associated with specific mutations within the core domain of Enh1.ResultsMutations in the core domain were found with significant frequency in C1126 (122/387 [31.5%], the binding site for HNF3) and in C1134 (106/387 [27.4%], the binding site for HNF4). A single mutation at nt 1126 (C1126) was identified in 17/123 (13.8%), and 105/123 (85.4%) had double mutations (C1126/1134). The level of HBV DNA (log10 copies/mL) was lower in single mutants (C1126, 5.81±1.25) than in wild (6.80±1.65) and double mutants (C1126/1134, 6.81±1.54). Similarly, the relative luciferase activity of C1126 and C1126/C1134 was 0.18 and 1.12 times that of the wild-type virus, respectively.ConclusionsMutations in the HNF3 binding site inhibit viral replication, whereas mutations at the HNF4 binding site restore viral replication.

Cocoa flavanol metabolites activate HNF‐3β, Sp1, and NFY‐mediated transcription of apolipoprotein AI in human cells

To identify the mechanisms by which cocoa induces HDL levels and since apolipoprotein AI (ApoAI) is the major protein in HDLs, we analyzed, upon incubation with cocoa metabolites, ApoAI mRNA levels, its transcriptional regulation, and the levels of the transcription factors involved in this process. Epicatechin and cocoa metabolites caused an increase in ApoAI expression in HepG2 cells. Electrophoretic mobility shift assays revealed the involvement of Sites A and B of the ApoAI promoter in the induction of ApoAI mRNA upon incubation with cocoa metabolites. Using supershift assays, we demonstrated the binding of HNF-3β, HNF-4, ER-α, and RXR-α to Site A and the binding of HNF-3β, NFY, and Sp1 to Site B. Luciferase assays performed with a construct containing Site B confirmed its role in the upregulation of ApoAI by cocoa metabolites. Incubation with 3-methyl-epicatechin led to an increase in HNF-3β mRNA, HNF-3β, ER-α, Sp1, and NFY protein levels and the activation of ApoAI transcription mediated by NFY, Sp1, and ER-α. The activation of ApoAI transcription through Site B by cocoa flavanol metabolites is mainly mediated by an increase in HNF-3β, with a significant contribution of Sp1 and NFY, as a mechanism for the protective role of these compounds in cardiovascular diseases.

Glis3 regulates neurogenin 3 expression in pancreatic β-cells and interacts with its activator, Hnf6.

The Krüppel-like zinc finger transcription factor, Glis3, has been associated with neonatal diabetes in humans and mice, and implicated in the regulation of pancreatic β-cell generation. However, its precise function in the development of pancreatic β-cells has not yet been elucidated. In this study, we provide evidence that Glis3 regulates Neurogenin 3 (Ngn3) through its distal promoter region. Previous studies showed that the distal region and proximal region of Ngn3 promoter contains various transcription binding sites, including binding sites for pancreatic and duodenal homeobox 1 (Pdx1), Hnf1β and Hnf6. Interestingly, putative Glis3 binding sites (Glis3BS) were found in the distal region of Ngn3 promoter close to the Hnf6 binding sites. This suggested that along with Hnf6, Glis3 may also be involved in the regulation of Ngn3 expression. This hypothesis is supported by data showing that Glis3 can bind to the Ngn3 promoter directly and activate Ngn3 transcriptional activity. Additionally, Glis3 can interact directly with Hnf6 in vitro and in vivo. The amino-terminus in Glis3 and the homeodomain of Hnf6 are critical for this interaction. These data suggest that crosstalk between Glis3 and Hnf6 may play an important role in the regulation of Ngn3 during pancreatic endocrine progenitor cell specification and development.

Lethal factor VII deficiency due to novel mutations in the F7 promoter: Functional analysis reveals disruption of HNF4 binding site

Hereditary factor VII (FVII) deficiency is a rare autosomal recessive disorder. Deleterious mutations that prevent the synthesis of any amount of functional FVII have been associated with life-threatening haemorrhage in neonates. Here we report two infants, of Maghrebian origin, who suffered a fatal spontaneous cerebral haemorrhage. Investigation of the molecular basis for their severe FVII deficiency revealed novel mutations in a homozygous state within the F7 gene promoter: a single nucleotide substitution (c.-65G>C) and a 2bp deletion (c.-60_-59delTT). To determine whether these promoter variants were responsible for the FVII deficiency, computer-assisted sequence analyses were performed. The data predicted a disrupted binding of both HNF4 and COUP-TF transcription factors with each variant. Concordantly, experimental results revealed an altered HNF4-induced transactivation in the promoter mutated variants. The execution of functional tests is critical to ensuring a complete understanding of the effect of any promoter mutant on FVII deficiency. Only then can an accurate molecular diagnosis be made and further genetic counselling and prenatal diagnosis be offered.