HNF1 Binding Research Articles

The regulation of jejunal induction of the maltase‐glucoamylase gene by a high‐starch/low‐fat diet in mice

Maltase and glucoamylase are derived from the same mRNA and are responsible for digestion of starch in the small intestine. In this study, we examined whether jejunal expression of the maltase-glucoamylase (Mgam) gene is enhanced by a high-starch (HS) diet through changes in the binding of transcription factors and/or histone code on the gene. Seven-week-old male mice of C57BL/6J strain were fed either a low-starch (5% energy as cornstarch) or a HS (70% energy as cornstarch) diet for 7 days, and then the proximal third of jejunoileum was used for analysis of Mgam mRNA levels (real-time RT-PCR) and of the binding of transcription factors and acetylated histones on the Mgam gene (chromatin immunoprecipitation assays). We found that jejunal Mgam mRNA was increased by a HS diet in mice. Chromatin immunoprecipitation assays revealed that the HS-diet increased the acetylations of histones, bindings of a coactivator, Creb binding protein (CREBBP), and the transcriptional factors caudal type homeobox 2 (CDX2) and HNF1 homeobox (HNF1) in the promoter/enhancer and transcriptional regions of Mgam gene. This suggests that the increase in the jejunal activity of maltase and glucoamylase caused by a HS-diet in mice is regulated at the mRNA level through histone acetylation and binding of CREBBP, CDX2 and HNF1 in the promoter/enhancer and transcriptional regions of Mgam gene.

Stimulation of angiotensin‐converting enzyme 2 promoter activity by hepatocyte nuclear factor 1β (HNF1β) in insulinoma cells

We previously showed that Angiotensin-converting enzyme 2 (ACE2) gene therapy increases insulin secretion (Bindom et al, 2010). Moreover, others reported that exogenous expression of hepatocyte nuclear factor 1β (HNF1β) activates the ACE2 promoter in HEK293 cells that have no endogenous HNF1β. However, physical interaction with the ACE2 promoter was not shown and the relevance to diabetes is unclear. Our objective was to determine whether HNF1β directly binds to the ACE2 gene promoter and whether HNF1β overexpression stimulates promoter activity in insulinoma cells. Chromatin immunoprecipitation assays showed binding of HNF1β to the ACE2 gene promoter region in HEK293T cells transfected with an HNF1β expression plasmid. Human and mouse ACE2 promoter regions containing 2 putative HNF1 binding sites were cloned into the luciferase vector pGL3-Basic. The ACE2 promoter sequences were active in driving the expression of luciferase in insulinoma cell lines βTC-3 and 832/13. Furthermore, overexpression of HNF1β markedly induced expression of luciferase (> 20-fold in βTC-3 cells and > 100-fold in 832/13 cells) and ACE2 mRNA (> 10-fold in 832/13 cells). Our data show that HNF1β binds and stimulates ACE2 promoter activity in insulinoma cells. These findings bring new evidence for a role of ACE2 in type 2 diabetes by providing a possible new mechanism leading to insulin secretion. Support: NIH/NIDDK (DK084466).

The nephrogenic potential of the transcription factors osr1, osr2, hnf1b, lhx1 and pax8 assessed in Xenopus animal caps.

BackgroundThe three distinct types of kidneys, pronephros, mesonephros and metanephros, develop consecutively in vertebrates. The earliest form of embryonic kidney, the pronephros, is derived from intermediate mesoderm and the first expressed genes localized in the pronephros anlage are the transcription factors osr1, osr2, hnf1b, lhx1 and pax8, here referred to as the early nephrogenic transcription factors. However, the pathway inducing nephrogenesis and the network of theses factors are poorly understood. Treatment of the undifferentiated animal pole explant (animal cap) of Xenopus with activin A and retinoic acid induces pronephros formation providing a powerful tool to analyze key molecular events in nephrogenesis.ResultsWe have investigated the expression kinetics of the early nephrogenic transcription factors in activin A and retinoic acid treated animal caps and their potential to induce pronephric differentiation. In treated animal caps, expression of osr1, osr2, hnf1b and lhx1 are induced early, whereas pax8 expression occurs later implying an indirect activation. Activin A alone is able to induce osr2 and lhx1 after three hours treatment in animal caps while retinoic acid fails to induce any of these nephrogenic transcription factors. The early expression of the five transcription factors and their interference with pronephros development when overexpressed in embryos suggest that these factors potentially induce nephrogenesis upon expression in animal caps. But no pronephros development is achieved by either overexpression of OSR1, by HNF1B injection with activin A treatment, or the combined application of LHX1 and PAX8, although they influenced the expression of several early nephrogenic transcription factors in some cases. In an additional approach we could show that HNF1B induces several genes important in nephrogenesis and regulates lhx1 expression by an HNF1 binding site in the lhx1 promoter.ConclusionsThe early nephrogenic transcription factors play an important role in nephrogenesis, but have no pronephros induction potential upon overexpression in animal caps. They activate transcriptional cascades that partially reflect the gene activation initiated by activin A and retinoic acid. Significantly, HNF1B activates the lhx1 promoter directly, thus extending the known activin A regulation of the lhx1 gene via an activin A responsive element.

Hepatocyte nuclear factor‐4α interacts with other hepatocyte nuclear factors in regulating transthyretin gene expression

Transthyretin is a negative acute phase protein whose serum level decreases during the acute phase response. Transthyretin gene expression in the liver is regulated at the transcriptional level, and is controlled by hepatocyte nuclear factor (HNF)-4α and other HNFs. The site-directed mutagenesis of HNF-4, HNF-1, HNF-3 and HNF-6 binding sites in the transthyretin proximal promoter dramatically decreases transthyretin promoter activity. Interestingly, the mutation of the HNF-4 binding site not only abolishes the response to HNF-4α, but also reduces significantly the response to other HNFs. However, mutation of the HNF-4 binding site merely affects the specific binding of HNF-4α, but not other HNFs, suggesting that an intact HNF-4 binding site not only provides a platform for specific interaction with HNF-4α, but also facilitates the interaction of HNF-4α with other HNFs. In a cytokine-induced acute phase response cell culture model, we observed a significant reduction in the binding of HNF-4α, HNF-1α, HNF-3β and HNF-6α to the transthyretin promoter, which correlates with a decrease in transthyretin expression after injury. These findings provide new insights into the mechanism of the negative transcriptional regulation of the transthyretin gene after injury caused by a decrease in the binding of HNFs and a modulation in their coordinated interactions.

HNF1α and SREBP2 are important regulators of NPC1L1 in human liver

HNF1α and SREBP2 are important regulators of NPC1L1 in human liver

HNF1 and SREBP2 are important regulators of NPC1L1 in human liver

Niemann-Pick C1-like 1 (NPC1L1), a key regulator of intestinal cholesterol absorption, is highly expressed in human liver. Here, we aimed to gain more insight into mechanisms participating in its hepatic regulation in humans. Correlation analysis in livers from Chinese patients with and without gallstone disease revealed strong positive correlations between NPC1L1 and sterol regulatory element binding protein 2 (SREBP2) (r = 0.74, P < 0.05) and between NPC1L1 and hepatic nuclear factor alpha (HNF4alpha) (r = 0.53, P < 0.05) mRNA expression. HNF4alpha is an upstream regulator of HNF1alpha; thus, we also tested whether HNF1alpha participates in the regulation of NPC1L1. We showed a dose-dependent regulation by SREBP2 on the NPC1L1 promoter activity and mRNA expression in HuH7 cells. Chromatin immunoprecipitation assay confirmed the binding of SREBP2 to the promoter in vivo. Surprisingly, HNF4alpha slightly decreased the NPC1L1 promoter activity but had no effect on its gene expression. By contrast, HNF1alpha increased the promoter activity and the gene expression, and an important HNF1 binding site was identified within the human NPC1L1 promoter. ChIP assays confirmed that HNF1alpha can bind to the NPC1L1 promoter in vivo.

The regulation of jejunal induction of the maltase–glucoamylase gene by a high‐starch/low‐fat diet in mice

Maltase and glucoamylase are derived from the same mRNA and are responsible for digestion of starch in the small intestine. Their jejunal activities in rodents are induced by a high-starch/low-fat (HS)-diet. However, it is unknown whether jejunal expression of the maltase-glucoamylase (Mgam) gene is enhanced by the HS-diet. In this study, we found that jejunal Mgam mRNA was increased by a HS-diet in mice. We showed that the HS-diet increased acetylation of histones, bindings of a coactivator, Creb binding protein (CREBBP), and the transcriptional factors caudal type homeobox 2 (CDX2) and HNF1 homeobox (HNF1) in the promoter/enhancer and transcriptional regions of Mgam gene. This suggests that the increase in the jejunal activity of maltase and glucoamylase caused by a HS-diet in mice is regulated at the mRNA level through histone acetylation and binding of CREBBP, CDX2 and HNF1 in the promoter/enhancer and transcriptional regions of Mgam gene.

A pharmacogenetics study of the human glucuronosyltransferase UGT1A4

UGT1A4 is primarily expressed in the liver and exhibits catalytic activities for various drugs. Amongst the few UGT1A4 polymorphisms evaluated, studies support the alteration of UGT1A4-mediated glucuronidation by a few variations including the Pro²⁴Thr and Leu⁴⁸Val variants (referred to as UGT1A4*2 and *3). We therefore investigated genetic mechanisms that might contribute to interindividual variation in UGT1A4 expression and activity. The UGT1A4 gene was sequenced from -4963 bp relative to the ATG to 2000 bp after the first exon in 184 unrelated Caucasians and African-Americans. We identified a large number of genetic variations, including 13 intronic, 39 promoter, as well as 14 exonic polymorphisms, with 10 that lead to amino-acid changes. Of the nucleotide variations found in the -5 kb promoter region, five are located in the proximal region (first 500 bp), and positioned in putative HNF-1 and OCT-1 binding sites. Four of these variants, placed at -163, -219, -419 and -463, are in complete linkage disequilibrium with the Leu⁴⁸Val coding region variant and with several variants in the upstream region of the promoter. Transient transfections of reference and variant promoter constructs (from position -500 to +1) in different cell lines with or without co-expression of HNF-1 and/or OCT-1 showed limited effect of these variations. Additional functional studies on promoter variants are still required to predict their potential influence on UGT1A4 expression in vivo. Besides, several coding variants significantly modified the enzyme kinetics for tamoxifen and Z-4-hydroxytamoxifen (Val⁴⁸, Asp⁵⁰, Gln⁵⁶, Phe¹⁷⁶, Asn²⁵⁰, Leu²⁷⁶) and are expected to have a potential in vivo effect.

FoxO1 and HNF-4 Are Involved in Regulation of Hepatic Glucokinase Gene Expression by Resveratrol

Resveratrol, a polyphenol derived from grapes, exerts important effects on glucose and lipid metabolism, yet detailed mechanisms mediating these effects remain unknown. The liver plays a central role in energy homeostasis, and glucokinase (GK) is a key enzyme involved in glucose utilization. Resveratrol activates SIRT1 (sirtuin 1), which promotes deacetylation of the forkhead transcription factor FoxO1. Previously, we reported that FoxO1 can suppress and that HNF-4 can stimulate GK expression in the liver. Here, we examined the role of FoxO1 and HNF-4 in mediating resveratrol effects on liver GK expression. Resveratrol suppressed hepatic GK expression in vivo and in isolated hepatocytes, and knocking down FoxO1 with shRNAs disrupted this effect. Reporter gene, gel shift, supershift assay, and chromatin immunoprecipitation studies show that FoxO1 binds to the GK promoter and that the interplay between FoxO1 and HNF-4 within the GK promoter is essential for mediating the effects of resveratrol. Resveratrol promotes deacetylation of FoxO1 and enhances its recruitment to the FoxO-binding element. Conversely, resveratrol suppresses recruitment of HNF-4 to its binding site, and knockdown of FoxO1 blocks this effect of resveratrol. Coprecipitation and chromatin immunoprecipitation studies show that resveratrol enhances interaction between FoxO1 and HNF-4, reduces binding of HNF-4 to its own site, and promotes its recruitment to the FoxO site in a FoxO1-dependent manner. These results provide the first evidence that resveratrol represses GK expression via FoxO1 and that the interaction between FoxO1 and HNF-4 contributes to these effects of resveratrol.

Growth Hormone-Activated STAT5 May Indirectly Stimulate IGF-I Gene Transcription through HNF-3γ

IGF-I is abundantly expressed in the liver under the stimulation of GH. We showed previously that expression of hepatocyte nuclear factor (HNF)-3gamma, a liver-enriched transcription factor, was strongly stimulated by GH in bovine liver. In this study, we determined whether GH-increased HNF-3gamma might contribute to GH stimulation of IGF-I gene expression in bovine liver and the underlying mechanism. A sequence analysis of the bovine IGF-I promoter revealed three putative HNF-3 binding sites, which all appear to be conserved in mammals. Chromatin immunoprecipitation assays showed that GH injection increased binding of HNF-3gamma to the IGF-I promoter in bovine liver. Gel-shift assays indicated that one of the three putative HNF-3 binding sites, HNF-3 binding site 1, bound to the HNF-3gamma protein from bovine liver with high affinity. Cotransfection analyses demonstrated that this HNF-3 binding site was essential for the transcriptional response of the IGF-I promoter to HNF-3gamma in CHO cells and to GH in primary mouse hepatocytes. Using similar approaches, we found that GH increased binding of the signal transducer and activator of transcription 5 (STAT5) to the HNF-3gamma promoter in bovine liver, that this binding occurred at a conserved STAT5 binding site, and that this STAT5 binding site was necessary for the HNF-3gamma promoter to respond to GH. Taken together, these results suggest that in addition to direct action, GH-activated STAT5 may also indirectly stimulate IGF-I gene transcription in the liver by directly enhancing the expression of the HNF-3gamma gene.

Combined cis-regulator elements as important mechanism affecting FXII plasma levels

Introduction Factor XII (FXII) deficiency is a recessive Mendelian trait due to mutations in the F12 gene. There is no bleeding associated with FXII deficiency, but FXII deficiency has been reported to be associated with risk of thrombosis in some studies. Material and Methods We examined the functional effect of two naturally-occurring mutations in two Spanish FXII deficient families: a C/G substitution at position –8, and a C/T substitution at position –13. Both mutations were located on a putative HNF4 binding site of F12 gene promoter. We also analyzed the F12 C46T polymorphism (rs1801020), associated with a decrease in the FXII levels, which also segregated in both families. A fragment containing each one of both –8 and -13 mutations, was cloned 5′ of a reporter gene. We compared the in vitro expression of these constructs to the wild type expression. Results Our analyses confirm that the –8C/G and the –13C/T mutations decreased expression levels, demonstrating that both mutations are involved in the observed FXII deficiency. In addition, electrophoretic shift analyses suggest that they alter the union of nuclear proteins to the promoter. Coinheritance of these mutations with the C46T polymorphism, result in a significant genotype-phenotype correlation. Conclusions We have identified two naturally-occurring mutations in the F12 promoter that drastically reduce FXII levels. Knowing rare genetic alterations in the F12 gene, together with the C46T common variant, may yield further understanding about the genetic architecture of FXII levels, which may have a role in the risk of thrombosis.

Feeding Rats Dietary Resistant Starch Shifts the Peak of SGLT1 Gene Expression and Histone H3 Acetylation on the Gene from the Upper Jejunum toward the Ileum.

Sodium glucose cotransporter 1 (SGLT1) participates in the incorporation of glucose from the lumen to enterocytes in the small intestine. We examined whether dietary resistant starch (RS), an autoclaved high amylose starch that is digested more slowly than regular cornstarch in the small intestine, alters SGLT1 mRNA levels along the jejunum-ileum of rats. The SGLT1 mRNA level was lower in the upper jejunum in rats fed an RS diet than in those fed a regular cornstarch diet, whereas it was higher in the lower jejunum/upper ileum. Furthermore, using chromatin immunoprecipitation (ChIP) assay, we demonstrated that histone H3 acetylation on the promoter/enhancer and transcriptional regions was reduced in the upper jejunum and elevated in the lower jejunum/upper ileum by feeding rats an RS diet. On the other hand, HNF-1 binding on the region around transcription start site of the SGLT1 gene was not altered in each jejunoileal segment by feeding rats an RS diet. Our results suggest that a shift of the expressional peak of the SGLT1 gene from the upper jejunum toward the ileum by dietary RS is associated with a change of histone H3 acetylation rather than that of HNF-1 binding on the gene.

Control of ACAT2 Liver Expression by HNF4α

ACAT2 is thought to be responsible for cholesteryl ester production in chylomicron and VLDL assembly. Recently, we identified HNF1alpha as an important regulator of the human ACAT2 promoter. Thus, we hypothesized that MODY3 (HNF1alpha gene mutations) and possibly MODY1 (HNF4alpha, upstream regulator of HNF1alpha, gene mutations) subjects may have lower VLDL esterified cholesterol. Serum analysis and lipoprotein separation using size-exclusion chromatography were performed in controls and MODY1 and MODY3 subjects. In vitro analyses included mutagenesis and cotransfections in HuH7 cells. Finally, the relevance in vivo of these findings was tested by ChIP assays in human liver. Whereas patients with MODY3 had normal lipoprotein composition, those with MODY1 had lower levels of VLDL and LDL esterified cholesterol, as well as of VLDL triglyceride. Mutagenesis revealed one important HNF4 binding site in the human ACAT2 promoter. ChIP assays and protein-to-protein interaction studies showed that HNF4alpha, directly or indirectly (via HNF1alpha), can bind to the ACAT2 promoter. We identified HNF4alpha as an important regulator of the hepatocyte-specific expression of the human ACAT2 promoter. Our results suggest that the lower levels of esterified cholesterol in VLDL- and LDL-particles in patients with MODY1 may-at least in part-be attributable to lower ACAT2 activity in these patients.

Specific Expression and Regulation of Hepassocin in the Liver and Down-regulation of the Correlation of HNF1α with Decreased Levels of Hepassocin in Human Hepatocellular Carcinoma

Hepassocin (HPS), is a liver-specific gene with mitogenic activity on isolated hepatocytes. It is up-regulated following partial hepatectomy and down-regulated frequently in heptocellular carcinoma (HCC). However, very little is known about the HPS transcription regulation mechanism. In this study, we identified HNF1alpha (hepatocyte nuclear factor-1alpha) as an important liver-specific cis-acting element for HPS using in vivo luciferase assays. Deletion of the HNF1 binding site not only led to a complete loss of HPS promoter activity in vivo but also abolished the induction of the HPS promoter by HNF1alpha. An electrophoretic mobility shift assay demonstrated that HNF1alpha interacted with the HPS gene promoter in vitro. Chromatin immunoprecipitation showed that HNF1alpha interacted with HMGB1 and CREB-binding protein, and all of them were recruited to the HPS promoter in vivo. Moreover, HNF1alpha expression was lower in HCC cell lines and tissues and correlated significantly with the down-regulation of HPS expression. Re-expression of HNF1alpha in human hepatoma HepG2 cells reinduced HPS expression. In contrast, knockdown of endogenous HNF1alpha expression by small interfering RNA resulted in a significant reduction of HPS expression. Furthermore, we found that partial hepatectomy and IL-6 significantly induced promoter activity of HPS, depending on STAT3 and HNF1 binding sites in the HPS promoter. These results demonstrate that the HNF1 binding site and HNF1alpha are critical to liver-specific expression of HPS, and down-regulation or loss of HNF1alpha causes, at least in part, the transcriptional down-regulation of HPS in HCC.

ZHX2 is a repressor of α‐fetoprotein expression in human hepatoma cell lines

The zinc-fingers and homeoboxes 2 (ZHX2) protein was shown previously to be involved in postnatal repression of α-fetoprotein (AFP) in mice. More recently, loss of ZHX2 expression was often found in human hepatcellular carcinoma (HCC), where AFP is frequently reactivated. Using HepG2 and HepG2.2.15, which express high AFP levels, we show that ZHX2 overexpression significantly decreases of AFP secretion in a dose dependent manner. Furthermore, using LO2 and SMMC7721 cells, which express low AFP levels, we use siRNA inhibition to show that AFP is de-repressed when ZHX2 levels are reduced. This represents the first direct evidence that ZHX2 represses AFP. Co-transfections of ZHX2 and AFP-luciferase reporter genes demonstrate ZHX2 repression is governed by the AFP promoter and requires intact HNF1 binding sites. These data support the idea that ZHX2 contributes to AFP repression in the liver after birth and may also be involved in AFP reactivation in liver cancer.

A Combination of HNF-4 and Foxo1 Is Required for Reciprocal Transcriptional Regulation of Glucokinase and Glucose-6-phosphatase Genes in Response to Fasting and Feeding

Glucokinase (GK) and glucose-6-phosphatase (G6Pase) regulate rate-limiting reactions in the physiologically opposed metabolic cascades, glycolysis and gluconeogenesis, respectively. Expression of these genes is conversely regulated in the liver in response to fasting and feeding. We explored the mechanism of transcriptional regulation of these genes by nutritional condition and found that reciprocal function of HNF-4 and Foxo1 plays an important role in this process. In the GK gene regulation, Foxo1 represses HNF-4-potentiated transcription of the gene, whereas it synergizes with HNF-4 in activating the G6Pase gene transcription. These opposite actions of Foxo1 concomitantly take place in the cells under no insulin stimulus, and such gene-specific action was promoter context-dependent. Interestingly, HNF-4-binding elements (HBEs) in the GK and G6Pase promoters were required both for the insulin-stimulated GK gene activation and insulin-mediated G6Pase gene repression. Indeed, mouse in vivo imaging showed that mutating the HBEs in the GK and G6Pase promoters significantly impaired their reactivity to the nutritional states, even in the presence of intact Foxo1-binding sites (insulin response sequences). Thus, in the physiological response of the GK and G6Pase genes to fasting/feeding conditions, Foxo1 distinctly decodes the promoter context of these genes and differently modulates the function of HBE, which then leads to opposite outcomes of gene transcription.

Interaction of hepatocyte nuclear factors in transcriptional regulation of tissue specific hormonal expression of human multidrug resistance-associated protein 2 (abcc2)

Multidrug resistance-associated protein 2 (MRP2) (ABCC2) is an ATP-binding cassette membrane protein located primarily on apical surface of hepatocytes that mediates transport of conjugated xenobiotics and endogenous compounds into bile. MRP2 is highly expressed in hepatocytes, and at lower levels in small intestines, stomach and kidney. Previous reports have characterized mammalian MRP2 promoters, but none have established the molecular mechanism(s) involved in liver enriched expression. This study aims to investigate the mechanism of hepatic MRP2 regulation. A 2130 bp of MRP2 promoter was cloned from PAC-1 clone P108G1–7, to identify putative liver specific/hormone responsive functional DNA binding sites. Using deletion analysis, site specific mutagenesis and co-transfection studies, liver specific expression was determined. MRP2 promoter-LUC constructs were highly expressed in liver cell lines compared to non-liver cells. The region extending from − 3 to+ 458 bp of MRP2 promoter starting from AUG contained the potential binding sites for CAAATT box enhancer binding protein (C/EBP), hepatocytes nuclear factor 1, 3 and 4 (HNF1, HNF3, and HNF4. Only HNF1 and HNF4 co-transfection with MRP2 luciferase increased expression. Site specific mutational analysis of HNF1 binding site indicated an important role for HNF1α. HNF4α induction of MRP2 was independent of HNF1 binding site. C/EBP, HNF3, and HNF6 inhibited HNF1α while HNF4α induced MRP2 luciferase expression and glucocorticoids stimulated MRP2 expression. This study emphasizes the complex regulation of MRP2 with HNF1α and HNF4α playing a central role. The coordinated regulation of xenobiotic transporters and oxidative conjugation may determine the adaptive responses to cellular detoxification processes.

Na+-Glucose Transporter-2 Messenger Ribonucleic Acid Expression in Kidney of Diabetic Rats Correlates with Glycemic Levels: Involvement of Hepatocyte Nuclear Factor-1α Expression and Activity

Mutations in Na(+)-glucose transporters (SGLT)-2 and hepatocyte nuclear factor (HNF)-1alpha genes have been related to renal glycosuria and maturity-onset diabetes of the young 3, respectively. However, the expression of these genes have not been investigated in type 1 and type 2 diabetes. Here in kidney of diabetic rats, we tested the hypotheses that SGLT2 mRNA expression is altered; HNF-1alpha is involved in this regulation; and glycemic homeostasis is a related mechanism. The in vivo binding of HNF-1alpha into the SGLT2 promoter region in renal cortex was confirmed by chromatin immunoprecipitation assay. SGLT2 and HNF-1alpha mRNA expression (by Northern and RT-PCR analysis) and HNF-1 binding activity of nuclear proteins (by EMSA) were investigated in diabetic rats and treated or not with insulin or phlorizin (an inhibitor of SGLT2). Results showed that diabetes increases SGLT2 and HNF-1alpha mRNA expression (~50%) and binding of nuclear proteins to a HNF-1 consensus motif (~100%). Six days of insulin or phlorizin treatment restores these parameters to nondiabetic-rat levels. Moreover, both treatments similarly reduced glycemia, despite the differences in plasma insulin and urinary glucose concentrations, highlighting the plasma glucose levels as involved in the observed modulations. This study shows that SGLT2 mRNA expression and HNF-1alpha expression and activity correlate positively in kidney of diabetic rats. It also shows that diabetes-induced changes are reversed by lowering glycemia, independently of insulinemia. Our demonstration that HNF-1alpha binds DNA that encodes SGLT2 supports the hypothesis that HNF-1alpha, as a modulator of SGLT2 expression, may be involved in diabetic kidney disease.

Association Study of 69 Genes in the Ret Pathway Identifies Low-penetrance Loci in Sporadic Medullary Thyroid Carcinoma

To date, few association studies have been done to better understand the genetic basis for the development of sporadic medullary thyroid carcinoma (sMTC). To identify additional low-penetrance genes, we have done a two-stage case-control study in two European populations using high-throughput genotyping. We selected 417 single nucleotide polymorphisms (SNP) belonging to 69 genes either related to RET signaling pathway/functions or involved in key processes for cancer development. TagSNPs and functional variants were included where possible. These SNPs were initially studied in the largest known series of sMTC cases (n = 266) and controls (n = 422), all of Spanish origin. In stage II, an independent British series of 155 sMTC patients and 531 controls was included to validate the previous results. Associations were assessed by an exhaustive analysis of individual SNPs but also considering gene- and linkage disequilibrium-based haplotypes. This strategy allowed us to identify seven low-penetrance genes, six of them (STAT1, AURKA, BCL2, CDKN2B, CDK6, and COMT) consistently associated with sMTC risk in the two case-control series and a seventh (HRAS) with individual SNPs and haplotypes associated with sMTC in the Spanish data set. The potential role of CDKN2B was confirmed by a functional assay showing a role of a SNP (rs7044859) in the promoter region in altering the binding of the transcription factor HNF1. These results highlight the utility of association studies using homogeneous series of cases for better understanding complex diseases.

Comparison of LTR enhancer elements in sheep betaretroviruses: insights into the basis for tissue-specific expression

Jaagsiekte sheep retrovirus (JSRV), enzootic nasal tumor virus (ENTV), and endogenous sheep retroviruses (ESRVs) are highly related sheep betaretroviruses that display different expression profiles in vivo. JSRV and ENTV are expressed in lungs and nasal adenocarcinomas, respectively, while ESRVs are primarily expressed in the reproductive tract of ewes. Evidence suggests that the cell tropism of JSRV, ENTV, and ESRVs is due to the transcriptional specificity of the LTRs. We have previously found several enhancer elements in the JSRV LTR that are important for lung-specific expression, including binding sites for the lung-specific transcription factor HNF-3beta, as well as binding sites for the ubiquitously expressed transcription factors C/EBP and NF-I. In this study, we have aligned the U3 regions of JSRV, ENTV, and several ESRVs in order to compare the transcriptional enhancer elements of JSRV that are conserved or absent in ESRV and ENTV. All three JSRV U3 sequences examined contain two conserved HNF-3 binding sites, while the ENTV and ESRV U3 regions are not predicted to bind this transcription factor. In addition, the C/EBP binding site is interrupted in the ESRV LTRs, but conserved in the ENTV LTRs. Some enhancer elements are conserved between JSRV and ENTV, but a reporter vector carrying the ENTV-1 LTR showed less activity than a JSRV LTR-driven reporter vector in a lung epithelial cell line. These studies support the importance of LTR enhancer elements in the respective tissue specificities of these exogenous and endogenous betaretroviruses.