scholarly journals Foxa3 (HNF-3γ) binds to and activates the rat proglucagon gene promoter but is not essential for proglucagon gene expression

2002 ◽  
Vol 366 (2) ◽  
pp. 633-641 ◽  
Author(s):  
Yuanfang LIU ◽  
Wei SHEN ◽  
Patricia L. BRUBAKER ◽  
Klaus H. KAESTNER ◽  
Daniel J. DRUCKER
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Forkhead Box ◽  
Mild Hypoglycaemia ◽  
Element Binding Protein ◽  
Prolonged Fast

Members of the Forkhead box a (Foxa) transcription factor family are expressed in the liver, pancreatic islets and intestine and both Foxa1 and Foxa2 regulate proglucagon gene transcription. As Foxa proteins exhibit overlapping DNA-binding specificities, we examined the role of Foxa3 [hepatocyte nuclear factor (HNF)-3γ] in control of proglucagon gene expression. Foxa3 was detected by reverse transcriptase PCR in glucagon-producing cell lines and binds to the rat proglucagon gene G2 promoter element in GLUTag enteroendocrine cells. Although Foxa3 increased rat proglucagon promoter activity in BHK fibroblasts, augmentation of Foxa3 expression did not increase proglucagon promoter activity in GLUTag cells. Furthermore, adenoviral Foxa3 expression did not affect endogenous proglucagon gene expression in islet or intestinal endocrine cell lines. Although Foxa3-/- mice exhibit mild hypoglycaemia during a prolonged fast, the levels of proglucagon-derived peptides and proglucagon mRNA transcripts were comparable in tissues from wild-type and Foxa3-/- mice. These findings identify Foxa3 as a member of the proglucagon gene G2 element binding-protein family that, unlike Foxa1, is not essential for control of islet or intestinal proglucagon gene expression in vivo.

scholarly journals Functional analysis of haplotypes and promoter activity at the 5' region of the DRD2 gene and associations with schizophrenia.

2020 ◽  
Author(s):  
Xicen Zhang ◽  
Mei Ding ◽  
Yi Liu ◽  
Yongping Liu ◽  
Jiaxin Xing ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Promoter Region ◽  
Regulatory Region ◽  
Gene Promoter ◽  
Luciferase Reporter ◽  
Drd2 Gene ◽  
Functional Regions

Abstract Background: In previous studies, we researched the association of the DRD2 gene promoter region SNP loci rs7116768, rs1047479195, rs1799732, rs1799978 and schizophrenia using Sanger sequencing. rs7116768 and rs1799978 were found to be slightly associated with schizophrenia. This study investigated the effects of haplotypes consisted of the four SNPs on protein expression level in vitro and identified the functional sequence in the 5’ regulatory region of DRD2 gene which has a potential link with schizophrenia.Methods: Recombinant plasmids with haplotypes, SNPs and 13 recombinant vectors containing deletion fragments from the DRD2 gene 5' regulatory region were transfected into HEK293 and SK-N-SH cell lines. Relative luciferase activity of the haplotypes, SNPs and different sequences was compared using a dual luciferase reporter assay system.Results: Haplotype H4(G-C-InsC-G) could significantly increase the gene expression in SK-N-SH cell lines. Allele C of rs7116768, allele A of rs1047479195 and allele del C of rs1799732 could up-regulate the gene expression. There were 5~7 functional regions in the promoter region of DRD2 gene that could affect the level of gene expression.Conclusion: We cannot rule out the possibility that different haplotypes may influence DRD2 gene expression in vivo. We observed that allele C of rs7116768, allele A of rs1047479195 and allele del C of rs1799732 could up-regulate gene expression. The truncation results confirmed the existence of functional regions in the promoter region of DRD2 gene that could affect the level of gene expression.

scholarly journals Foxa1 and Foxa2 Regulate α-Cell Differentiation, Glucagon Biosynthesis, and Secretion

Endocrinology ◽  
2014 ◽  
Vol 155 (10) ◽  
pp. 3781-3792 ◽  
Author(s):  
Mounia Heddad Masson ◽  
Caroline Poisson ◽  
Audrey Guérardel ◽  
Aline Mamin ◽  
Jacques Philippe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Differentiation ◽  
Target Genes ◽  
Adult Mouse ◽  
Glucagon Secretion ◽  
Mrna Levels ◽  
Forkhead Box ◽  
Interfering Rna

Abstract The Forkhead box A transcription factors are major regulators of glucose homeostasis. They show both distinct and redundant roles during pancreas development and in adult mouse β-cells. In vivo ablation studies have revealed critical implications of Foxa1 on glucagon biosynthesis and requirement of Foxa2 in α-cell terminal differentiation. In order to examine the respective role of these factors in mature α-cells, we used small interfering RNA (siRNA) directed against Foxa1 and Foxa2 in rat primary pancreatic α-cells and rodent α-cell lines leading to marked decreases in Foxa1 and Foxa2 mRNA levels and proteins. Both Foxa1 and Foxa2 control glucagon gene expression specifically through the G2 element. Although we found that Foxa2 controls the expression of the glucagon, MafB, Pou3f4, Pcsk2, Nkx2.2, Kir6.2, and Sur1 genes, Foxa1 only regulates glucagon gene expression. Interestingly, the Isl1 and Gipr genes were not controlled by either Foxa1 or Foxa2 alone but by their combination. Foxa1 and Foxa2 directly activate and bind the promoter region the Nkx2.2, Kir6.2 and Sur1, Gipr, Isl1, and Pou3f4 genes. We also demonstrated that glucagon secretion is affected by the combined effects of Foxa1 and Foxa2 but not by either one alone. Our results indicate that Foxa1 and Foxa2 control glucagon biosynthesis and secretion as well as α-cell differentiation with both common and unique target genes.

scholarly journals Differential regulation of the TRH gene promoter by triiodothyronine and dexamethasone in pancreatic islets

2001 ◽  
Vol 170 (1) ◽  
pp. 91-98 ◽  
Author(s):  
P Fragner ◽  
SL Lee ◽  
S Aratan de Leon
Keyword(s):  
Gene Expression ◽  
Pancreatic Islets ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Luciferase Reporter ◽  
Flanking Sequence ◽  
Luciferase Reporter Gene ◽  
Neoplastic Cells

TRH was initially found in the hypothalamus and regulates TSH secretion. TRH is also produced by insulin-containing beta-cells. Endogenous TRH positively regulates glucagon secretion and attenuates pancreatic exocrine secretion. We have previously shown that triiodothyronine (T(3)) down-regulates pre-pro-TRH gene expression in vivo and in vitro. The present study was designed to determine the initial impact of T(3) on rat TRH gene promoter and to compare this effect with that of dexamethasone (Dex). Primary islet cells and neoplastic cells (HIT T-15 and RIN m5F) were transiently transfected with fragments of the 5'-flanking sequence of TRH fused to the luciferase reporter gene. The persistence of high TRH concentrations in fetal islets in culture, probably due to transactivating factors, allowed us to explore how T(3) and Dex regulate the TRH promoter activity in transfected cells and whether the hormone effect is dependent on the cell type considered. TRH gene promoter activity is inhibited by T(3) in primary but not neoplastic cells and stimulated by Dex in both primary and neoplastic cells of islets. These findings validate previous in vivo and in vitro studies and indicate the transcriptional impact of these hormones on TRH gene expression in the pancreatic islets.

scholarly journals Real-time imaging of gene promoter activity using an adenoviral reporter construct demonstrates transcriptional dynamics in normal anterior pituitary cells

2003 ◽  
Vol 178 (1) ◽  
pp. 61-69 ◽  
Author(s):  
JA Stirland ◽  
ZC Seymour ◽  
S Windeatt ◽  
AJ Norris ◽  
P Stanley ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Cell Lines ◽  
Promoter Activity ◽  
Expression Patterns ◽  
Gene Promoter ◽  
Luciferase Reporter ◽  
Pituitary Cells ◽  
Clonal Cell ◽  
Clonal Cell Lines

Although analysis of luciferase activity using luminescence imaging has provided new insights into the dynamic regulation of gene expression in living tIssues, studies in vitro have relied on stably transfected clonal cell lines, limiting the choice of cell type and species, or DNA microinjection, which is arduous and highly selective. We report here the first use of a recombinant adenovirus in which the firefly luciferase reporter gene was regulated by the prolactin gene promoter, to study temporal dynamics of promoter activity. This vector was used to infect the pituitary GH3 cell line, and also primary cultures of Syrian hamster pituitary cells. We show that adenovirally transduced cells retained normal regulation of the promoter-reporter transgene by appropriate signals. Furthermore, microscopic imaging studies indicated that both clonal and primary pituitary cells were transduced efficiently, giving readily detectable luminescence signals in real-time over long periods. Finally, analysis of single-cell expression patterns indicated that prolactin promoter activity was highly dynamic with pulses in gene expression, revealing that the transcriptional instability seen in clonal cells is a feature of normal pituitary cells. Adenoviral vectors offer a valuable tool for studies of gene regulation where conventional transgenesis and clonal cell lines are not available.

scholarly journals Progression and Differentiation of Alveolar Rhabdomyosarcoma Is Regulated by PAX7 Transcription Factor—Significance of Tumor Subclones

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1870
Author(s):  
Klaudia Skrzypek ◽  
Grażyna Adamek ◽  
Marta Kot ◽  
Bogna Badyra ◽  
Marcin Majka
Keyword(s):  
Transcription Factor ◽  
Cell Lines ◽  
Migration Activity ◽  
Id Proteins ◽  
Rh30 Cell ◽  
Str Profile ◽  
And Migration

Rhabdomyosarcoma (RMS), is the most frequent soft tissue tumor in children that originates from disturbances in differentiation process. Mechanisms leading to the development of RMS are still poorly understood. Therefore, by analysis of two RMS RH30 cell line subclones, one subclone PAX7 negative, while the second one PAX7 positive, and comparison with other RMS cell lines we aimed at identifying new mechanisms crucial for RMS progression. RH30 subclones were characterized by the same STR profile, but different morphology, rate of proliferation, migration activity and chemotactic abilities in vitro, as well as differences in tumor morphology and growth in vivo. Our analysis indicated a different level of expression of adhesion molecules (e.g., from VLA and ICAM families), myogenic microRNAs, such as miR-206 and transcription factors, such as MYOD, MYOG, SIX1, and ID. Silencing of PAX7 transcription factor with siRNA confirmed the crucial role of PAX7 transcription factor in proliferation, differentiation and migration of RMS cells. To conclude, our results suggest that tumor cell lines with the same STR profile can produce subclones that differ in many features and indicate crucial roles of PAX7 and ID proteins in the development of RMS.

scholarly journals Hyperglycaemia up-regulates placental growth factor (PlGF) expression and secretion in endothelial cells via suppression of PI3 kinase-Akt signalling and activation of FOXO1

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Samir Sissaoui ◽  
Stuart Egginton ◽  
Ling Ting ◽  
Asif Ahmed ◽  
Peter W. Hewett
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Endothelial Dysfunction ◽  
Akt Activation ◽  
Forkhead Box ◽  
Pi3k Activity ◽  
Binding Sequence

AbstractPlacenta growth factor (PlGF) is a pro-inflammatory angiogenic mediator that promotes many pathologies including diabetic complications and atherosclerosis. Widespread endothelial dysfunction precedes the onset of these conditions. As very little is known of the mechanism(s) controlling PlGF expression in pathology we investigated the role of hyperglycaemia in the regulation of PlGF production in endothelial cells. Hyperglycaemia stimulated PlGF secretion in cultured primary endothelial cells, which was suppressed by IGF-1-mediated PI3K/Akt activation. Inhibition of PI3K activity resulted in significant PlGF mRNA up-regulation and protein secretion. Similarly, loss or inhibition of Akt activity significantly increased basal PlGF expression and prevented any further PlGF secretion in hyperglycaemia. Conversely, constitutive Akt activation blocked PlGF secretion irrespective of upstream PI3K activity demonstrating that Akt is a central regulator of PlGF expression. Knock-down of the Forkhead box O-1 (FOXO1) transcription factor, which is negatively regulated by Akt, suppressed both basal and hyperglycaemia-induced PlGF secretion, whilst FOXO1 gain-of-function up-regulated PlGF in vitro and in vivo. FOXO1 association to a FOXO binding sequence identified in the PlGF promoter also increased in hyperglycaemia. This study identifies the PI3K/Akt/FOXO1 signalling axis as a key regulator of PlGF expression and unifying pathway by which PlGF may contribute to common disorders characterised by endothelial dysfunction, providing a target for therapy.

The nuclear orphan receptors COUP-TFII and Ear-2 act as silencers of the human oxytocin gene promoter

1997 ◽  
Vol 19 (2) ◽  
pp. 163-172 ◽  
Author(s):  
K Chu ◽  
HH Zingg
Keyword(s):  
Receptor Binding ◽  
Promoter Activity ◽  
Transcriptional Activity ◽  
Gene Promoter ◽  
Fine Tuning ◽  
Orphan Receptor ◽  
Expression Vectors ◽  
Mobility Shift ◽  
Interaction Sites

We have previously shown that COUP-TFII and Ear-2, two members of the nuclear orphan receptor family, are able to repress oestrogen-stimulated transcriptional activity of the human oxytocin (OT) gene promoter by binding to a site that overlaps with the oestrogen response element (ERE) present in the 5' flanking region of the gene. Although most nuclear receptor-mediated transcriptional repression conforms with the paradigm of passive repression and involves competitive binding to an activator site, active repression, i.e. silencing of basal promoter activity, has been observed in a limited number of cases. Here we show by co-transfection experiments using COUP-TFII and Ear-2 expression vectors and reporter constructs containing OT gene promoter fragments linked to the chloramphenicol acetyltransferase gene that both COUP-TFII and Ear-2 are capable of silencing basal OT gene promoter activity by 54 and 75% respectively. 5' Deletion and footprint analyses revealed two areas of functionally important interaction sites: (1) a direct TGACC(T/C) repeat overlapping the ERE and (2) a more promoter-proximal area centred at - 90 containing three imperfect direct repeats (R1-R3) spaced by four nucleotides each. Mutagenesis of reporter constructs as well as electrophoretic mobility-shift assays demonstrated that each of the three proximal repeats R1-R3 contributed to orphan receptor binding and the silencing effect. Inasmuch as the orphan receptor-binding sites are not involved in mediating basal transcriptional activity of the OT gene promoter, the observed effects are best interpreted as active repression or promoter silencing. Moreover, since COUP-TFII and Ear-2 are both co-expressed in OT-expressing uterine epithelial cells, the novel transcriptional effects described here are likely to be of functional importance in the fine-tuning of uterine OT gene expression in vivo.

scholarly journals Positive Regulation of fur Gene Expression via Direct Interaction of Fur in a Pathogenic Bacterium, Vibrio vulnificus

2007 ◽  
Vol 189 (7) ◽  
pp. 2629-2636 ◽  
Author(s):  
Hyun-Jung Lee ◽  
So Hyun Bang ◽  
Kyu-Ho Lee ◽  
Soon-Jung Park
Keyword(s):  
Gene Expression ◽  
Pathogenic Bacteria ◽  
Vibrio Vulnificus ◽  
Iron Concentration ◽  
Direct Interaction ◽  
Repeated Sequence ◽  
Upstream Region ◽  
Fur Protein

ABSTRACT In pathogenic bacteria, the ability to acquire iron, which is mainly regulated by the ferric uptake regulator (Fur), is essential to maintain growth as well as its virulence. In Vibrio vulnificus, a human pathogen causing gastroenteritis and septicemia, fur gene expression is positively regulated by Fur when the iron concentration is limited (H.-J. Lee et al., J. Bacteriol. 185:5891-5896, 2003). Footprinting analysis revealed that an upstream region of the fur gene was protected by the Fur protein from DNase I under iron-depleted conditions. The protected region, from −142 to −106 relative to the transcription start site of the fur gene, contains distinct AT-rich repeats. Mutagenesis of this repeated sequence resulted in abolishment of binding by Fur. To confirm the role of this cis-acting element in Fur-mediated control of its own gene in vivo, fur expression was monitored in V. vulnificus strains using a transcriptional fusion containing the mutagenized Fur-binding site (fur mt::luxAB). Expression of fur mt::luxAB showed that it was not regulated by Fur and was not influenced by iron concentration. Therefore, this study demonstrates that V. vulnificus Fur acts as a positive regulator under iron-limited conditions by direct interaction with the fur upstream region.

Abstract 314: BRaf Plays a Major Role in Gene Expression in Cardiomyocytes in Mouse Hearts in vivo

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Michelle A Hardyman ◽  
Stephen J Fuller ◽  
Daniel N Meijles ◽  
Kerry A Rostron ◽  
Sam J Leonard ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cardiac Hypertrophy ◽  
Cardiac Function ◽  
Left Ventricular ◽  
Braf Mutations ◽  
Gene Markers ◽  
Lv Mass ◽  
Cardiac Volumes

Introduction: Raf kinases lie upstream of ERK1/2 with BRaf being the most highly expressed and having the highest basal activity. V600E BRaf mutations constitutively activate ERK1/2 and are common in cancer. The role of BRaf in the adult heart is yet to be established. ERK1/2 regulate cardiomyocyte gene expression, promoting cardiac hypertrophy and cardioprotection, but effects of ERK1/2 may depend on signal strength. Hypothesis: Our hypotheses are that BRaf is critical in regulating ERK1/2 signaling in cardiomyocytes and, whilst moderate ERK1/2 activity is beneficial, excessive ERK1/2 activity is detrimental to the heart. Methods: We generated heterozygote mice for tamoxifen- (Tam-) inducible cardiomyocyte-specific knockin of V600E in the endogenous BRaf gene. Mice (12 wks) received 2 injections of Tam or vehicle on consecutive days (n=4-10 per group). Kinase activities and mRNA expression were assessed by immunoblotting and qPCR. Echocardiography was performed (Vevo2100). M-mode images (short axis view) were analyzed; data for each mouse were normalized to the mean of 2 baseline controls. Results: V600E knockin did not affect overall BRaf or cRaf levels in mouse hearts, but significantly increased ERK1/2 activities within 48 h (1.51±0.05 fold). Concurrently, mRNAs for hypertrophic gene markers including BNP and immediate early genes (IEGs) increased signficantly. At 72 h, expression of BNP, Fosl1, Myc, Ereg and CTGF increased further, other IEGs (Jun, Fos, Egr1, Atf3) declined, and ANF was upregulated. In contrast, expression of α and β myosin heavy chain mRNAs was substantially downregulated (0.46/0.41±0.05 relative to controls). Within 72 h, left ventricular (LV) mass and diastolic LV wall thickness had increased (1.23±0.05 relative to controls), but cardiac function was severely compromised with significant decreases in ejection fraction and cardiac output (0.53/0.68±0.09 relative to controls) associated with increased LV internal diameters and cardiac volumes. Conclusions: Endogenous cardiomyocyte BRaf is sufficient to activate ERK1/2 in mouse hearts and induce cardiac hypertrophy associated with dynamic temporal changes in gene expression. However, excessive activation of ERK1/2 in isolation is detrimental to cardiac function.

Bcl-2 protein localizes to the chromosomes of mitotic nuclei and is correlated with the cell cycle in cultured epithelial cell lines

1994 ◽  
Vol 107 (2) ◽  
pp. 363-371
Author(s):  
Q.L. Lu ◽  
A.M. Hanby ◽  
M.A. Nasser Hajibagheri ◽  
S.E. Gschmeissner ◽  
P.J. Lu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Epithelial Cell ◽  
Cell Lines ◽  
Cytoplasmic Localization ◽  
Human Epithelial Cell ◽  
Daughter Cells ◽  
Epithelial Cell Lines ◽  
Cell Immortalization

bcl-2 gene expression confers a survival advantage by preventing cells from entering apoptosis. In contrast to the previously described cytoplasmic localization of Bcl-2 in epithelial cells in vivo, in this study we have demonstrated, in a series of human epithelial cell lines, that Bcl-2 also localizes to mitotic nuclei. Both immunocytochemical and immunoelectron microscopical examinations localize this protein to nuclei and in particular to chromosomes. Nuclear Bcl-2 expression in these cell lines is correlated with the cell cycle. There is relatively strong expression during mitosis, most intense during prophase and metaphase, declining in telophase and then the protein becomes undetectable soon after separation of the two daughter cells. The expression and distribution of Bcl-2 is influenced by treatment with excessive thymidine. These results indicate that Bcl-2 may protect the cells from apoptosis occurring during mitosis and suggest a possible role for the protein in cell immortalization.

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