scholarly journals Threshold Levels of Hepatocyte Nuclear Factor 6 (HNF-6) Acting in Synergy with HNF-4 and PGC-1α Are Required for Time-Specific Gene Expression during Liver Development

2006 ◽  
Vol 26 (16) ◽  
pp. 6037-6046 ◽  
Author(s):  
Jean-Bernard Beaudry ◽  
Christophe E. Pierreux ◽  
Graham P. Hayhurst ◽  
Nicolas Plumb-Rudewiez ◽  
Mary C. Weiss ◽  
...  
Keyword(s):  
Gene Expression ◽  
Specific Gene ◽  
Liver Development ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Loss Of Function ◽  
Specific Expression ◽  
Threshold Levels ◽  
Time Specific

ABSTRACT During liver development, hepatocytes undergo a maturation process that leads to the fully differentiated state. This relies at least in part on the coordinated action of liver-enriched transcription factors (LETFs), but little is known about the dynamics of this coordination. In this context we investigate here the role of the LETF hepatocyte nuclear factor 6 (HNF-6; also called Onecut-1) during hepatocyte differentiation. We show that HNF-6 knockout mouse fetuses have delayed expression of glucose-6-phosphatase (g6pc), which catalyzes the final step of gluconeogenesis and is a late marker of hepatocyte maturation. Using a combination of in vivo and in vitro gain- and loss-of-function approaches, we demonstrate that HNF-6 stimulates endogenous g6pc gene expression directly via a synergistic and interdependent action with HNF-4 and that it involves coordinate recruitment of the coactivator PGC-1α. The expression of HNF-6, HNF-4, and PGC-1α rises steadily during liver development and precedes that of g6pc. We provide evidence that threshold levels of HNF-6 are required to allow synergism between HNF-6, HNF-4, and PGC-1α to induce time-specific expression of g6pc. Our observations on the regulation of g6pc by HNF-6 provide a model whereby synergism, interdependency, and threshold concentrations of LETFs and coactivators determine time-specific expression of genes during liver development.

scholarly journals Inhibition of hepatocyte nuclear factor 4 transcriptional activity by the nuclear factor κB pathway

2006 ◽  
Vol 398 (3) ◽  
pp. 439-450 ◽  
Author(s):  
Varvara Nikolaidou-Neokosmidou ◽  
Vassilis I. Zannis ◽  
Dimitris Kardassis
Keyword(s):  
Gene Expression ◽  
Inflammatory Cytokine ◽  
Transcriptional Activity ◽  
Binding Protein ◽  
Nuclear Factor Κb ◽  
Specific Gene ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Response Element ◽  
Hepatocyte Nuclear Factor 4

HNF-4 (hepatocyte nuclear factor 4) is a key regulator of liver-specific gene expression in mammals. We have shown previously that the activity of the human APOC3 (apolipoprotein C-III) promoter is positively regulated by the anti-inflammatory cytokine TGFβ (transforming growth factor β) and its effectors Smad3 (similar to mothers against decapentaplegic 3) and Smad4 proteins via physical and functional interactions between Smads and HNF-4. We now show that the pro-inflammatory cytokine TNFα (tumour necrosis factor α) antagonizes TGFβ for the regulation of APOC3 gene expression in hepatocytes. TNFα was a strong inhibitor of the activity of apolipoprotein promoters that harbour HNF-4 binding sites and this inhibition required HNF-4. Using specific inhibitors of TNFα-induced signalling pathways, it was shown that inhibition of the APOC3 promoter by TNFα involved NF-κB (nuclear factor κB). Latent membrane protein 1 of the Epstein–Barr virus, which is an established potent activator of NF-κB as well as wild-type forms of various NF-κB signalling mediators, also inhibited strongly the APOC3 promoter and the transactivation function of HNF-4. TNFα had no effect on the stability or the nuclear localization of HNF-4 in HepG2 cells, but inhibited the binding of HNF-4 to the proximal APOC3 HRE (hormone response element). Using the yeast-transactivator-GAL4 system, we showed that both AF-1 and AF-2 (activation functions 1 and 2) of HNF-4 are inhibited by TNFα and that this inhibition was abolished by overexpression of different HNF-4 co-activators, including PGC-1 (peroxisome-proliferator-activated-receptor-γ co-activator 1), CBP [CREB (cAMP-response-element-binding protein) binding protein] and SRC3 (steroid receptor co-activator 3). In summary, our findings indicate that TNFα, or other factors that trigger an NF-κB response in hepatic cells, inhibit the transcriptional activity of the APOC3 and other HNF-4-dependent promoters and that this inhibition could be accounted for by a decrease in DNA binding and the down-regulation of the transactivation potential of the AF-1 and AF-2 domains of HNF-4.

Melanocyte-specific gene expression: role of repression and identification of a melanocyte-specific factor, MSF

1994 ◽  
Vol 14 (5) ◽  
pp. 3494-3503
Author(s):  
U Yavuzer ◽  
C R Goding
Keyword(s):  
Gene Expression ◽  
Mutational Analysis ◽  
Site Directed Mutagenesis ◽  
Specific Factor ◽  
Specific Gene ◽  
Binding Motif ◽  
Specific Expression ◽  
Tissue Specific ◽  
Specific Gene Expression

For a gene to be transcribed in a tissue-specific fashion, expression must be achieved in the appropriate cell type and also be prevented in other tissues. As an approach to understanding the regulation of tissue-specific gene expression, we have analyzed the requirements for melanocyte-specific expression of the tyrosinase-related protein 1 (TRP-1) promoter. Positive regulation of TRP-1 expression is mediated by both an octamer-binding motif and an 11-bp element, termed the M box, which is conserved between the TRP-1 and other melanocyte-specific promoters. We show here that, consistent with its ability to activate transcription in a non-tissue-specific fashion, the M box binds the basic-helix-loop-helix factor USF in vitro. With the use of a combination of site-directed mutagenesis and chimeric promoter constructs, additional elements involved in regulating TRP-1 expression were identified. These include the TATA region, which appears to contribute to the melanocyte specificity of the TRP-1 promoter. Mutational analysis also identified two repressor elements, one at the start site, the other located at -240, which function both in melanoma and nonmelanoma cells. In addition, a melanocyte-specific factor, MSF, binds to sites which overlap both repressor elements, with substitution mutations demonstrating that binding by MSF is not required for repression. Although a functional role for MSF has not been unequivocally determined, the location of its binding sites leads us to speculate that it may act as a melanocyte-specific antirepressor during transcription of the endogenous TRP-1 gene.

scholarly journals Effects of Natural Progesterone and Synthetic Progestin on Germ Layer Gene Expression in a Human Embryoid Body Model

2020 ◽  
Vol 21 (3) ◽  
pp. 769 ◽  
Author(s):  
Yoon Young Kim ◽  
Hoon Kim ◽  
Chang Suk Suh ◽  
Hung-Ching Liu ◽  
Zev Rosenwaks ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryonic Development ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Germ Layer ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Endometrial Cells ◽  
Natural Progesterone

Natural progesterone and synthetic progestin are widely used for the treatment of threatened abortion or in in vitro fertilization (IVF) cycles. This in vitro study aimed to assess whether the treatment with natural progesterone or synthetic progestin influences the germ layer gene expression on the early human embryonic development using human embryonic stem cells (hESCs)-derived embryoid bodies (hEBs) as a surrogate of early stage human embryonic development. Human EBs derived from hESCs were cultured for nine days, and were treated with natural progesterone (P4) or synthetic progestin, medroxyprogesterone acetate (MPA) at 10–7 M for five days. To reverse the effects of treatment, mifepristone (RU486) as progesterone antagonist was added to the hEBs for four days starting one day after the initiation of treatment. Mouse blastocysts (mBLs) were cultured in vitro for 24 h, and P4 or MPA at 10−7 M was treated for an additional 24 h. The treated embryos were further transferred onto in vitro cultured endometrial cells to evaluate chorionic gonadotropin (CG) expression. To analyze the effects of P4 or MPA, the expression of differentiation genes representing the three germ layers was investigated, GATA-binding factor 4 (GATA4), α-fetoprotein (AFP), hepatocyte nuclear factor (HNF)-3β, hepatocyte nuclear factor (HNF)-4α (endoderm), Brachyury, cardiac actin (cACT) (mesoderm), and Nestin (ectoderm), using quantitative reverse transcription PCR (qRT-PCR) and immunostaining. Significantly lower expressions of HNF-3β, HNF-4α, Brachyury, and Nestin were observed in MPA-treated hEBs (all p < 0.05), which was negated by RU486 treatment. This inhibitory effect of MPA was also observed in mouse embryos. Conclusively, the effects of natural progesterone and synthetic progestin may differ in the germ layer gene expression in the hEB model, which suggests that caution is necessary in the use of progestogen.

Regulation of kidney-specific Ksp-cadherin gene promoter by hepatocyte nuclear factor-1β

2002 ◽  
Vol 283 (4) ◽  
pp. F839-F851 ◽  
Author(s):  
Yun Bai ◽  
Marco Pontoglio ◽  
Thomas Hiesberger ◽  
Angus M. Sinclair ◽  
Peter Igarashi
Keyword(s):  
Promoter Activity ◽  
Gene Promoter ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Specific Gene ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Tissue Specific ◽  
Transfected Cells

Kidney-specific cadherin (Ksp-cadherin) is a tissue-specific member of the cadherin family that is expressed exclusively in the kidney and developing genitourinary tract. Recent studies have shown that the proximal 250 bp of the Ksp-cadherin gene promoter are sufficient to direct tissue-specific gene expression in vivo and in vitro. The proximal 120 bp of the promoter are evolutionarily conserved between mouse and human and contain a DNase I hypersensitive site that is kidney cell specific. At position −55, the promoter contains a consensus recognition site for hepatocyte nuclear factor-1 (HNF-1). Mutations of the consensus HNF-1 site and downstream GC-boxes inhibit promoter activity in transfected cells. HNF-1α and HNF-1β bind specifically to the −55 site, and both proteins transactivate the promoter directly. Expression of Ksp-cadherin is not altered in the kidneys of HNF-1α-deficient mice. However, expression of a gain-of-function HNF-1β mutant stimulates Ksp-cadherin promoter activity in transfected cells, whereas expression of a dominant-negative mutant inhibits activity. These studies identify Ksp-cadherin as the first kidney-specific promoter that has been shown to be regulated by HNF-1β. Mutations of HNF-1β, as occur in humans with inherited renal cysts and diabetes, may cause dysregulated Ksp-cadherin promoter activity.

scholarly journals Melanocyte-specific gene expression: role of repression and identification of a melanocyte-specific factor, MSF.

1994 ◽  
Vol 14 (5) ◽  
pp. 3494-3503 ◽  
Author(s):  
U Yavuzer ◽  
C R Goding
Keyword(s):  
Gene Expression ◽  
Mutational Analysis ◽  
Site Directed Mutagenesis ◽  
Specific Factor ◽  
Specific Gene ◽  
Binding Motif ◽  
Specific Expression ◽  
Tissue Specific ◽  
Specific Gene Expression

For a gene to be transcribed in a tissue-specific fashion, expression must be achieved in the appropriate cell type and also be prevented in other tissues. As an approach to understanding the regulation of tissue-specific gene expression, we have analyzed the requirements for melanocyte-specific expression of the tyrosinase-related protein 1 (TRP-1) promoter. Positive regulation of TRP-1 expression is mediated by both an octamer-binding motif and an 11-bp element, termed the M box, which is conserved between the TRP-1 and other melanocyte-specific promoters. We show here that, consistent with its ability to activate transcription in a non-tissue-specific fashion, the M box binds the basic-helix-loop-helix factor USF in vitro. With the use of a combination of site-directed mutagenesis and chimeric promoter constructs, additional elements involved in regulating TRP-1 expression were identified. These include the TATA region, which appears to contribute to the melanocyte specificity of the TRP-1 promoter. Mutational analysis also identified two repressor elements, one at the start site, the other located at -240, which function both in melanoma and nonmelanoma cells. In addition, a melanocyte-specific factor, MSF, binds to sites which overlap both repressor elements, with substitution mutations demonstrating that binding by MSF is not required for repression. Although a functional role for MSF has not been unequivocally determined, the location of its binding sites leads us to speculate that it may act as a melanocyte-specific antirepressor during transcription of the endogenous TRP-1 gene.

scholarly journals Development of an Efficient Transient Gene Expression Assay Based on Tobacco ((Nicotiana tabacum var. Xanthi) Male Gametophytes

1970 ◽  
Vol 19 (1) ◽  
pp. 9-23 ◽  
Author(s):  
M. A. Y. Akhond ◽  
G. C. Machray
Keyword(s):  
Gene Expression ◽  
Nicotiana Tabacum ◽  
Expression System ◽  
Transient Gene Expression ◽  
Specific Gene ◽  
Loss Of Function ◽  
Pollen Selection ◽  
Gene Expression Assay ◽  
Study Gene Expression

Optimization of direct DNA delivery into tobacco ((Nicotiana tabacum var. Xanthi) male gametophytes was devised together with development of an efficient transient expression system to study gene expression under controlled conditions. Use of a GFP gene driven by strong promoter and enhancer sequences allowed an efficient non-lethal transient gene expression assay with an overall transient gene expression frequency of > 4% for uninucleate microspores and between 10 and 20% for binucleate pollen. The technique demonstrated its suitability for analysis of developmental stage-specific gene expression. The assay allowed observation of real-time transgene expression during microspore maturation proving useful for in vitro pollen selection. We have also used this protocol to determine the recombination potential of tobacco male gametic cells by assessing the frequency of extra-chromosomal homologous recombination events after co-delivery of two loss-of-function GFP genes. No increase of extrachromosomal recombination was observed in assays for transient transformation. Key words: Biolistic, GFP, Microspore, Tobacco, Nicotiana tabacum, Transformation D.O.I. 10.3329/ptcb.v19i1.4078 Plant Tissue Cult. & Biotech. 19(1): 9-23, 2009 (June)

scholarly journals Identification of hepatocyte nuclear factor-3 binding sites in the Clara cell secretory protein gene

1993 ◽  
Vol 295 (1) ◽  
pp. 227-232 ◽  
Author(s):  
C D Bingle ◽  
J D Gitlin
Keyword(s):  
Gene Expression ◽  
Binding Sites ◽  
Secretory Protein ◽  
Clara Cell ◽  
Mouse Lung ◽  
Specific Gene ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Mobility Shift ◽  
Clara Cell Secretory Protein

To determine the mechanisms of cell-specific gene expression in the developing pulmonary epithelium the Clara cell secretory protein (CCSP) gene promoter was analysed by DNAase I footprinting. A prominent site of protein-DNA interaction was detected from nucleotides -132 to -76 using nuclear extract from mouse lung and human H441 cells. Mobility shift analysis revealed that an oligonucleotide corresponding to this region interacted with multiple proteins from lung and H441 cell nuclear extracts. Analysis of the nucleotide sequence of this region identified two potential binding sites for hepatocyte nuclear factor 3 (HNF-3), and consistent with this finding binding to this CCSP oligonucleotide was specifically competed for by an oligonucleotide corresponding to the HNF-3-binding site from the mouse transthyretin gene. Mobility shift of the CCSP oligonucleotide was supershifted using antisera specific to HNF-3 alpha and HNF-3 beta, and HNF-3 alpha and HNF-3 beta translated in vitro were found to bind specifically to this same oligonucleotide. Co-transfection of HNF-3 alpha- and HNF-3 beta-expression plasmids increased cell-specific reporter gene activity in H441 cells transfected with a CCSP-CAT gene chimeric construct containing this -132 to -76 region. Taken together, these results suggest a role for HNF-3 in mediating cell-specific CCSP gene expression within the bronchiolar epithelium. These findings support the hypothesis that members of the HNF-3 ‘forkhead’ family of transcription factors determine gene expression and cell fate in multiple cell lineages derived from the primitive gut endoderm.

scholarly journals The combined detection of Amphiregulin, Cyclin A1 and DDX20/Gemin3 expression predicts aggressive forms of oral squamous cell carcinoma

2021 ◽  
Author(s):  
Ekaterina Bourova-Flin ◽  
Samira Derakhshan ◽  
Afsaneh Goudarzi ◽  
Tao Wang ◽  
Anne-Laure Vitte ◽  
...  
Keyword(s):  
Gene Expression ◽  
Squamous Cell ◽  
Large Scale ◽  
Biomarker Discovery ◽  
Gene Expression Signature ◽  
Predictive Biomarkers ◽  
Specific Gene ◽  
Specific Expression ◽  
Intrinsic Nature ◽  
Independent Cohort

Abstract Background Large-scale genetic and epigenetic deregulations enable cancer cells to ectopically activate tissue-specific expression programmes. A specifically designed strategy was applied to oral squamous cell carcinomas (OSCC) in order to detect ectopic gene activations and develop a prognostic stratification test. Methods A dedicated original prognosis biomarker discovery approach was implemented using genome-wide transcriptomic data of OSCC, including training and validation cohorts. Abnormal expressions of silent genes were systematically detected, correlated with survival probabilities and evaluated as predictive biomarkers. The resulting stratification test was confirmed in an independent cohort using immunohistochemistry. Results A specific gene expression signature, including a combination of three genes, AREG, CCNA1 and DDX20, was found associated with high-risk OSCC in univariate and multivariate analyses. It was translated into an immunohistochemistry-based test, which successfully stratified patients of our own independent cohort. Discussion The exploration of the whole gene expression profile characterising aggressive OSCC tumours highlights their enhanced proliferative and poorly differentiated intrinsic nature. Experimental targeting of CCNA1 in OSCC cells is associated with a shift of transcriptomic signature towards the less aggressive form of OSCC, suggesting that CCNA1 could be a good target for therapeutic approaches.

Serial analysis of gene expression (SAGE) during porcine embryo development

2004 ◽  
Vol 16 (2) ◽  
pp. 87 ◽  
Author(s):  
Le Ann Blomberg ◽  
Kurt A. Zuelke
Keyword(s):  
Gene Expression ◽  
Functional Genomics ◽  
Embryo Development ◽  
Molecular Mechanisms ◽  
Physiological Role ◽  
Transgenic Animals ◽  
Specific Gene ◽  
Research Strategies

Functional genomics provides a powerful means for delving into the molecular mechanisms involved in pre-implantation development of porcine embryos. High rates of embryonic mortality (30%), following either natural mating or artificial insemination, emphasise the need to improve the efficiency of reproduction in the pig. The poor success rate of live offspring from in vitro-manipulated pig embryos also hampers efforts to generate transgenic animals for biotechnology applications. Previous analysis of differential gene expression has demonstrated stage-specific gene expression for in vivo-derived embryos and altered gene expression for in vitro-derived embryos. However, the methods used to date examine relatively few genes simultaneously and, thus, provide an incomplete glimpse of the physiological role of these genes during embryogenesis. The present review will focus on two aspects of applying functional genomics research strategies for analysing the expression of genes during elongation of pig embryos between gestational day (D) 11 and D12. First, we compare and contrast current methodologies that are being used for gene discovery and expression analysis during pig embryo development. Second, we establish a paradigm for applying serial analysis of gene expression as a functional genomics tool to obtain preliminary information essential for discovering the physiological mechanisms by which distinct embryonic phenotypes are derived.

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