Differential Gene Expression from Two Transcriptional Units in the cag Pathogenicity Island ofHelicobacter pylori

ABSTRACT Infection with Helicobacter pylori strains containing the cag Pathogenicity Island (cag PAI) is strongly correlated with the development of severe gastric disease, including gastric and duodenal ulceration, mucosa-associated lymphoid tissue lymphoma, and gastric carcinoma. Although in vitro studies have demonstrated that the expression of genes within the cag PAI leads to the activation of a strong host inflammatory response, the functions of mostcag gene products and how they work in concert to promote an immunological response are unknown. We developed a transcriptional reporter that utilizes urease activity and in which nine putative regulatory sequences from the cag PAI were fused to theH. pylori ureB gene. These fusions were introduced in single copies onto the H. pylori chromosome without disruption of the cag PAI. Our analysis indicated that while each regulatory region confers a reproducible amount of promoter activity under laboratory conditions, they differ widely in levels of expression. Transcription initiating upstream of cag15 and upstream of cag21 is induced when the respective fusion strains are cocultured with an epithelial cell monolayer. Results of mouse colonization experiments with an H. pylori strain carrying the cag15-ureB fusion suggested that this putative regulatory region appears to be induced in vivo, demonstrating the importance of the urease reporter as a significant development toward identifying in vivo-induced gene expression in H. pylori.

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First insights into the molecular basis association between promoter polymorphisms of the IL1B gene and Helicobacter pylori infection in the Sudanese population: computational approach

10.21203/rs.3.rs-60531/v3 ◽

2020 ◽

Author(s):

Abeer Babiker Idris ◽

Einas Babiker Idris ◽

Amany Eltayib Ataelmanan ◽

Ali Elbagir Ali Mohamed ◽

Bashir M. Osman Arbab ◽

...

Keyword(s):

Gene Expression ◽

In Silico ◽

Expression Patterns ◽

Regulatory Region ◽

Regulatory Elements ◽

Computer Algorithms ◽

Promoter Polymorphisms ◽

H Pylori

Abstract Background: Helicobacter pylori (H. pylori) infects nearly half of the world’s population with a variation in incidence among different geographic regions. Genetic variants in the promoter regions of the IL1B gene can affect cytokine expression and creates a condition of hypoacidity which favors the survival and colonization of H. pylori. Therefore, the aim of this study was to characterize the polymorphic sites in the 5’- region [-687_+297] of IL1B in H. pylori infection using in silico tools.Results: A total of five nucleotide variations were detected in the 5’-regulatory region [-687_+297] of IL1B which led to the addition or alteration of transcription factor binding sites (TFBSs) or composite regulatory elements (CEs). Genotyping of IL1B-31 C>T revealed a significant association between -31T and susceptibility to H. pylori infection in the studied population (P=0.0363). Comparative analysis showed conservation rates of IL1B upstream [−368_+10] region above 70% in chimpanzee, rhesus monkey, a domesticated dog, cow and rat.Conclusions: In H. pylori-infected patients, three detected SNPs (-338, -155 and -31) located in the IL1B promoter were predicted to alter TFBSs and CE, which might affect the gene expression. These in silico predictions provide insight for further experimental in vitro and in vivo studies of the regulation of IL1B expression and its relationship to H. pylori infection. However, the recognition of regulatory motifs by computer algorithms is fundamental for understanding gene expression patterns.

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Modulatory Effects of Osthole on Lipopolysaccharides-Induced Inflammation in Caco-2 Cell Monolayer and Co-Cultures with THP-1 and THP-1-Derived Macrophages

Nutrients ◽

10.3390/nu13010123 ◽

2020 ◽

Vol 13 (1) ◽

pp. 123

Author(s):

Natalia K. Kordulewska ◽

Justyna Topa ◽

Małgorzata Tańska ◽

Anna Cieślińska ◽

Ewa Fiedorowicz ◽

...

Keyword(s):

Gene Expression ◽

Inflammatory Reaction ◽

Wide Spectrum ◽

Cell Monolayer ◽

In Vivo Studies ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Tnf Α

Lipopolysaccharydes (LPS) are responsible for the intestinal inflammatory reaction, as they may disrupt tight junctions and induce cytokines (CKs) secretion. Osthole has a wide spectrum of pharmacological effects, thus its anti-inflammatory potential in the LPS-treated Caco-2 cell line as well as in Caco-2/THP-1 and Caco-2/macrophages co-cultures was investigated. In brief, Caco-2 cells and co-cultures were incubated with LPS to induce an inflammatory reaction, after which osthole (150–450 ng/mL) was applied to reduce this effect. After 24 h, the level of secreted CKs and changes in gene expression were examined. LPS significantly increased the levels of IL-1β, -6, -8, and TNF-α, while osthole reduced this effect in a concentration-dependent manner, with the most significant decrease when a 450 ng/mL dose was applied (p < 0.0001). A similar trend was observed in changes in gene expression, with the significant osthole efficiency at a concentration of 450 ng/μL for IL1R1 and COX-2 (p < 0.01) and 300 ng/μL for NF-κB (p < 0.001). Osthole increased Caco-2 monolayer permeability, thus if it would ever be considered as a potential drug for minimizing intestinal inflammatory symptoms, its safety should be confirmed in extended in vitro and in vivo studies.

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Requirement of glucose metabolism for regulation of glucose transporter type 2 (GLUT2) gene expression in liver

Biochemical Journal ◽

10.1042/bj3140903 ◽

1996 ◽

Vol 314 (3) ◽

pp. 903-909 ◽

Cited By ~ 77

Author(s):

Franck RENCUREL ◽

Gérard WAEBER ◽

Bénédicte ANTOINE ◽

Francis ROCCHICCIOLI ◽

Paulette MAULARD ◽

...

Keyword(s):

Gene Expression ◽

Glucose Metabolism ◽

Reporter Gene ◽

Glucose Transporter ◽

Regulatory Region ◽

Proximal Fragment ◽

Mrna Accumulation ◽

Glut2 Gene

Previous studies have shown that glucose increases the glucose transporter (GLUT2) mRNA expression in the liver in vivo and in vitro. Here we report an analysis of the effects of glucose metabolism on GLUT2 gene expression. GLUT2 mRNA accumulation by glucose was not due to stabilization of its transcript but rather was a direct effect on gene transcription. A proximal fragment of the 5´ regulatory region of the mouse GLUT2 gene linked to a reporter gene was transiently transfected into liver GLUT2-expressing cells. Glucose stimulated reporter gene expression in these cells, suggesting that glucose-responsive elements were included within the proximal region of the promoter. A dose-dependent effect of glucose on GLUT2 expression was observed over 10 mM glucose irrespective of the hexokinase isozyme (glucokinase Km 16 mM; hexokinase I Km 0.01 mM) present in the cell type used. This suggests that the correlation between extracellular glucose and GLUT2 mRNA concentrations is simply a reflection of an activation of glucose metabolism. The mediators and the mechanism responsible for this response remain to be determined. In conclusion, glucose metabolism is required for the proper induction of the GLUT2 gene in the liver and this effect is transcriptionally regulated.

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Discovery and Validation of Novel Methylation Markers in Helicobacter pylori-Associated Gastric Cancer

Disease Markers ◽

10.1155/2021/4391133 ◽

2021 ◽

Vol 2021 ◽

pp. 1-11

Author(s):

Huan Wang ◽

Nian-Shuang Li ◽

Cong He ◽

Chuan Xie ◽

Yin Zhu ◽

...

Keyword(s):

Gene Expression ◽

Gastric Cancer ◽

Dna Methylation ◽

Helicobacter Pylori ◽

Gene Expression Data ◽

Functional Enrichment ◽

Expression Data ◽

H Pylori

Previous studies have shown that abnormal methylation is an early key event in the pathogenesis of most human cancers, contributing to the development of tumors. However, little attention has been given to the potential of DNA methylation patterns as markers for Helicobacter pylori- (H. pylori-) associated gastric cancer (GC). In this study, an integrated analysis of DNA methylation and gene expression was conducted to identify some potential key epigenetic markers in H. pylori-associated GC. DNA methylation data of 28 H. pylori-positive and 168 H. pylori-negative GC samples were compared and analyzed. We also analyzed the gene expression data of 18 H. pylori-positive and 145 H. pylori-negative GC cases. Finally, the results were verified by in vitro and in vivo experiments. A total of 5609 differentially methylated regions associated with 2454 differentially methylated genes were identified. A total of 228 differentially expressed genes were identified from the gene expression data of H. pylori-positive and H. pylori-negative GC cases. The screened genes were analyzed for functional enrichment. Subsequently, we obtained 28 genes regulated by methylation through a Venn diagram, and we identified five genes (GSTO2, HUS1, INTS1, TMEM184A, and TMEM190) downregulated by hypermethylation. HUS1, GSTO2, and TMEM190 were expressed at lower levels in GC than in adjacent samples ( P < 0.05 ). Moreover, H. pylori infection decreased HUS1, GSTO2, and TMEM190 expression in vitro and in vivo. Our study identified HUS1, GSTO2, and TMEM190 as novel methylation markers for H. pylori-associated GC.

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

10.21203/rs.3.rs-15812/v1 ◽

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.

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Accurate and sensitive quantification of protein-DNA binding affinity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1714376115 ◽

2018 ◽

Vol 115 (16) ◽

pp. E3692-E3701 ◽

Cited By ~ 32

Author(s):

Chaitanya Rastogi ◽

H. Tomas Rube ◽

Judith F. Kribelbauer ◽

Justin Crocker ◽

Ryan E. Loker ◽

...

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Binding Sites ◽

Biophysical Model ◽

Regulatory Sequences ◽

Binding Modes ◽

Perfect Agreement ◽

Dna Binding Sites

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes.

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HIF-2α regulates murine hematopoietic development in an erythropoietin-dependent manner

Blood ◽

10.1182/blood-2004-05-1695 ◽

2005 ◽

Vol 105 (8) ◽

pp. 3133-3140 ◽

Cited By ~ 139

Author(s):

Marzia Scortegagna ◽

Kan Ding ◽

Quiyang Zhang ◽

Yavuz Oktay ◽

Michael J. Bennett ◽

...

Keyword(s):

Gene Expression ◽

Hypoxia Inducible Factor ◽

Regulatory Region ◽

Dependent Manner ◽

Wild Type ◽

Hematopoietic Development ◽

Erythropoietin Production ◽

Hypoxia Treatment

AbstractErythropoiesis in the adult mammal depends critically on erythropoietin, an inducible cytokine with pluripotent effects. Erythropoietin gene expression increases under conditions associated with lowered oxygen content such as anemia and hypoxia. HIF-1α, the founding member of the hypoxia-inducible factor (HIF) alpha class, was identified by its ability to bind and activate the hypoxia-responsive enhancer in the erythropoietin regulatory region in vitro. The existence of multiple HIF alpha members raises the question of which HIF alpha member or members regulates erythropoietin expression in vivo. We previously reported that mice lacking wild-type HIF-2α, encoded by the EPAS1 gene, exhibit pancytopenia. In this study, we have characterized the etiology of this hematopoietic phenotype. Molecular studies of EPAS1-null kidneys reveal dramatically decreased erythropoietin gene expression. EPAS1-null as well as heterozygous mice have impaired renal erythropoietin induction in response to hypoxia. Treatment of EPAS1-null mice with exogenous erythropoietin reverses the hematopoietic and other defects. We propose that HIF-2α is an essential regulator of murine erythropoietin production. Impairments in HIF signaling, involving either HIF-1α or HIF-2α, may play a prominent role in conditions involving altered hematopoietic or erythropoietin homeostasis.

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DNA Hypermethylation Downregulates Telomerase Reverse Transcriptase (TERT) during H. pylori-Induced Chronic Inflammation

Journal of Oncology ◽

10.1155/2019/5415761 ◽

2019 ◽

Vol 2019 ◽

pp. 1-13 ◽

Cited By ~ 1

Author(s):

Françoise I. Bussière ◽

Valérie Michel ◽

Julien Fernandes ◽

Lionel Costa ◽

Vania Camilo ◽

...

Keyword(s):

Gene Expression ◽

Reverse Transcriptase ◽

Chronic Inflammation ◽

Telomerase Activity ◽

Telomerase Reverse Transcriptase ◽

Mrna Levels ◽

Dna Hypermethylation ◽

H Pylori

Helicobacter pylori infection causes chronic gastritis and is the major risk factor of gastric cancer. H. pylori induces a chronic inflammation-producing reactive oxygen species (ROS) which is a source of chromosome instabilities and contributes to the development of malignancy. H. pylori also promotes DNA hypermethylation, known to dysregulate essential genes that maintain genetic stability. The maintenance of telomere length by telomerase is essential for chromosome integrity. Telomerase reverse transcriptase (TERT) is the catalytic component of telomerase activity and an important target during host-pathogen interaction. We aimed to investigate the consequences of H. pylori on the regulation of TERT gene expression and telomerase activity. In vitro, hTERT mRNA levels and telomerase activity were analysed in H. pylori-infected human gastric epithelial cells. In addition, C57BL/6 and INS-GAS mice were used to investigate the influence of H. pylori-induced inflammation on TERT levels. Our data demonstrated that, in vitro, H. pylori inhibits TERT gene expression and decreases the telomerase activity. The exposure of cells to lycopene, an antioxidant compound, restores TERT levels in infected cells, indicating that ROS are implicated in this downregulation. In vivo, fewer TERT-positive cells are observed in gastric tissues of infected mice compared to uninfected, more predominantly in the vicinity of large aggregates of lymphocytes, suggesting an inflammation-mediated regulation. Furthermore, H. pylori appears to downregulate TERT gene expression through DNA hypermethylation as shown by the restoration of TERT transcript levels in cells treated with 5′-azacytidine, an inhibitor of DNA methylation. This was confirmed in infected mice, by PCR-methylation assay of the TERT gene promoter. Our data unraveled a novel way for H. pylori to promote genome instabilities through the inhibition of TERT levels and telomerase activity. This mechanism could play an important role in the early steps of gastric carcinogenesis.

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Maturation Stage-Specific Regulation of Megakaryocytic Genes by Pointed-Domain Ets Proteins.

Blood ◽

10.1182/blood.v106.11.1737.1737 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1737-1737

Author(s):

Liyan Pang ◽

Xun Wang ◽

Yuhuan Wang ◽

Gerd Blobel ◽

Mortimer Poncz

Keyword(s):

Gene Expression ◽

Fetal Liver ◽

Critical Role ◽

Regulatory Region ◽

Maturation Stage ◽

Specific Gene ◽

Mrna Levels ◽

Specific Regulation

Abstract The pointed-domain Ets transcription factor Fli-1 has a critical role during megakaryocyte-specific gene expression. Previously, we demonstrated that Fli-1 occupies the early megakaryocyte-specific gene αIIb in vivo. Moreover, our work suggested a mechanism for Fli-1 function by showing that Fli-1 facilitates GATA-1/FOG-1 dependent expression of the αIIb gene. However, studies by others with a targeted disruption of the Fli-1 gene in mice showed that while Fli-1 is essential for normal megakaryocyte maturation, αIIb mRNA levels were not significantly reduced in the resulting megakaryocytes, suggesting that a related Ets factor(s) might compensate for the loss of Fli-1. Here we show that the widely expressed pointed domain Ets protein GABPα specifically binds in vitro to Ets elements from two early megakaryocyte-specific genes, αIIb and c-mpl. Chromatin immunoprecipitation (ChIP) experiments using primary murine fetal liver-derived megakaryocytes reveal that GABPα associates with αIIb and c-mpl in vivo. Moreover, GABPα is capable of mediating GATA-1/FOG-1 synergy in the context of αIIb promoter constructs. These results suggest that GABPα contributes to megakaryocyte-restricted gene expression and is capable of at least partially compensating for the loss of Fli-1. However, loss of Fli-1 leads to a pronounced decrease in the expression of the late megakaryocyte-specific gene GPIX, indicating that compensation by GABPα is incomplete. Consistent with this observation, ChIP experiments fail to detect significant levels of GABPα at the regulatory region of GPIX while Fli-1 is readily detected there. Together, these results point to a model in which Fli-1 and GABPα serve overlapping, but distinct roles, during the development of megakaryocytes. GABPα may be important during early megakaryopoiesis, but Fli-1 exerting an essential role during late stages of maturation.

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Methylation of Transcription Factor Binding Sites in the Epstein-Barr Virus Latent Cycle Promoter Wp Coincides with Promoter Down-Regulation during Virus-Induced B-Cell Transformation

Journal of Virology ◽

10.1128/jvi.74.22.10468-10479.2000 ◽

2000 ◽

Vol 74 (22) ◽

pp. 10468-10479 ◽

Cited By ~ 51

Author(s):

R. J. Tierney ◽

H. E. Kirby ◽

J. K. Nagra ◽

J. Desmond ◽

A. I. Bell ◽

...

Keyword(s):

B Cells ◽

Binding Sites ◽

Epstein Barr Virus ◽

Regulatory Region ◽

Nuclear Antigen ◽

Regulatory Sequences ◽

Barr Virus ◽

Epstein Barr

ABSTRACT Two Epstein-Barr virus latent cycle promoters for nuclear antigen expression, Wp and Cp, are activated sequentially during virus-induced transformation of B cells to B lymphoblastoid cell lines (LCLs) in vitro. Previously published restriction enzyme studies have indicated hypomethylation of CpG dinucleotides in the Wp and Cp regions of the viral genome in established LCLs, whereas these same regions appeared to be hypermethylated in Burkitt's lymphoma cells, where Wp and Cp are inactive. Here, using the more sensitive technique of bisulfite genomic sequencing, we reexamined the situation in established LCLs with the typical pattern of dominant Cp usage; surprisingly, this showed substantial methylation in the 400-bp regulatory region upstream of the Wp start site. This was not an artifact of long-term in vitro passage, since, in cultures of recently infected B cells, we found progressive methylation of Wp (but not Cp) regulatory sequences occurring between 7 and 21 days postinfection, coincident with the period in which dominant nuclear antigen promoter usage switches from Wp to Cp. Furthermore, in the equivalent in vivo situation, i.e., in the circulating B cells of acute infectious mononucleosis patients undergoing primary EBV infection, we again frequently observed selective methylation of Wp but not Cp sequences. An effector role for methylation in Wp silencing was supported by methylation cassette assays of Wp reporter constructs and by bandshift assays, where the binding of two sets of transcription factors important for Wp activation in B cells, BSAP/Pax5 and CREB/ATF proteins, was shown to be blocked by methylation of their binding sites.

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