scholarly journals Regulation of the Human Secretin Gene Is Controlled by the Combined Effects of CpG Methylation, Sp1/Sp3 Ratio, and the E-Box Element

2004 ◽  
Vol 18 (7) ◽  
pp. 1740-1755 ◽  
Author(s):  
Leo Tsz-On Lee ◽  
Kian-Cheng Tan-Un ◽  
Ronald Ting-Kai Pang ◽  
David Tai-Wai Lam ◽  
Billy Kwok-Chong Chow
Keyword(s):  
Gene Expression ◽  
Hepg2 Cells ◽  
Cpg Island ◽  
Methylation Status ◽  
Gene Promoter ◽  
Mobility Shift ◽  
Specific Pcr ◽  
E Box

Abstract To unravel the mechanisms that regulate the human secretin gene expression, in this study, we have used secretin-expressing (HuTu-80 cells, human duodenal adenocarcinoma) and non-secretin-expressing [PANC-1 (human pancreatic ductile carcinoma) and HepG2 (human hepatocellular carcinoma) cells] cell models for in vitro and in vivo analyses. By transient transfection assays, within the promoter region (−11 to −341 from ATG, relative to the ATG initiation codon), we have initially identified several functional motifs including an E-box and 2 GC-boxes. Results from gel mobility shift and chromatin immunoprecipitation assays confirmed further that NeuroD, E2A, Sp1, and Sp3 bind to these E- and GC-boxes in HuTu-80 cells in vitro and in vivo, whereas only high levels of Sp3 is observed to bind the promoter in HepG2 cells. In addition, overexpression of Sp3 resulted in a dose-dependent repression of the Sp1-mediated transactivation. Collectively, these data suggest that the Sp1/Sp3 ratio is instrumental to controlling secretin gene expression in secretin-producing and non-secretin-producing cells. The functions of GC-box and Sp proteins prompted us to investigate the possible involvement of DNA methylation in regulating this gene. Consistent with this idea, we found a putative CpG island (−336 to 262 from ATG) that overlaps with the human secretin gene promoter. By methylation-specific PCR, all the CpG dinucleo-tides (26 of them) within the CpG island in HuTu-80 cells are unmethylated, whereas all these sites are methylated in PANC-1 and HepG2 cells. The expressions of secretin in PANC-1 and HepG2 cells were subsequently found to be significantly activated by a demethylation agent, 5′-Aza-2′ deoxycytidine. Taken together, our data indicate that the human secretin gene is controlled by the in vivo Sp1/Sp3 ratio and the methylation status of the promoter.

scholarly journals Identification of an Adeno-Associated Virus Rep Protein Binding Site in the Adenovirus E2a Promoter

2005 ◽  
Vol 79 (1) ◽  
pp. 28-38 ◽  
Author(s):  
John M. Casper ◽  
Jennifer M. Timpe ◽  
John David Dignam ◽  
James P. Trempe
Keyword(s):  
Gene Expression ◽  
Random Sequence ◽  
Gene Promoter ◽  
Helper Virus ◽  
Host Cells ◽  
Mobility Shift ◽  
Adeno Associated Virus ◽  
Rep Protein

ABSTRACT Adeno-associated virus (AAV) and other parvoviruses inhibit proliferation of nonpermissive cells. The mechanism of this inhibition is not thoroughly understood. To learn how AAV interacts with host cells, we investigated AAV's interaction with adenovirus (Ad), AAV's most efficient helper virus. Coinfection with Ad and AAV results in an AAV-mediated inhibition of Ad5 gene expression and replication. The AAV replication proteins (Rep) activate and repress gene expression from AAV and heterologous transcription promoters. To investigate the role of Rep proteins in the suppression of Ad propagation, we performed chromatin immunoprecipitation analyses that demonstrated in vivo AAV Rep protein interaction with the Ad E2a gene promoter. In vitro binding of purified AAV Rep68 protein to the Ad E2a promoter was characterized by electrophoretic mobility shift assays (Kd = 200 ± 25 nM). A 38 bp, Rep68-protected region (5′-TAAGAGTCAGCGCGCAGTATTTACTGAAGAGAGCCT-3′) was identified by DNase I footprint analysis. The 38-bp protected region contains the weak E2a TATA box, sequence elements that resemble the Rep binding sites identified by random sequence oligonucleotide selection, and the transcription start site. These results suggest that Rep binding to the E2a promoter contributes to the inhibition of E2a gene expression from the Ad E2a promoter and may affect Ad replication.

scholarly journals hDREF Regulates Cell Proliferation and Expression of Ribosomal Protein Genes

2007 ◽  
Vol 27 (6) ◽  
pp. 2003-2013 ◽  
Author(s):  
Daisuke Yamashita ◽  
Yukako Sano ◽  
Yuka Adachi ◽  
Yuma Okamoto ◽  
Hirotaka Osada ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Ribosomal Proteins ◽  
Human Fibroblasts ◽  
Gene Promoter ◽  
Ribosomal Protein Genes ◽  
Mobility Shift ◽  
Positive Elements

ABSTRACT Although ribosomal proteins (RPs) are essential cellular constituents in all living organisms, mechanisms underlying regulation of their gene expression in mammals remain unclear. We have established that 22 out of 79 human RP genes contain sequences similar to the human DREF (DNA replication-related element-binding factor; hDREF) binding sequence (hDRE) within 200-bp regions upstream of their transcriptional start sites. Electrophoretic gel mobility shift assays and chromatin immunoprecipitation analysis indicated that hDREF binds to hDRE-like sequences in the RP genes both in vitro and in vivo. In addition, transient luciferase assays revealed that hDRE-like sequences act as positive elements for RP gene transcription and cotransfection of an hDREF-expressing plasmid was found to stimulate RP gene promoter activity. Like that of hDREF, expression of RP genes is increased during the late G1 to S phases, and depletion of hDREF using short hairpin RNA-mediated knockdown decreased RP gene expression and cell proliferation in normal human fibroblasts. Knockdown of the RPS6 gene also resulted in impairment of cell proliferation. These data suggest that hDREF is an important transcription factor for cell proliferation which plays roles in cell cycle-dependent regulation of a number of RP genes.

Transcriptional regulation mechanisms of hypoxia-induced neuroglobin gene expression

2012 ◽  
Vol 443 (1) ◽  
pp. 153-164 ◽  
Author(s):  
Ning Liu ◽  
Zhanyang Yu ◽  
Shuanglin Xiang ◽  
Song Zhao ◽  
Anna Tjärnlund-Wolf ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hypoxia Inducible Factor ◽  
Gene Promoter ◽  
Nuclear Factor Κb ◽  
Proximal Promoter ◽  
Mobility Shift ◽  
Hypoxic Conditions ◽  
Mobility Shift Assay

Ngb (neuroglobin) has been identified as a novel endogenous neuroprotectant. However, little is known about the regulatory mechanisms of Ngb expression, especially under conditions of hypoxia. In the present study, we located the core proximal promoter of the mouse Ngb gene to a 554 bp segment, which harbours putative conserved NF-κB (nuclear factor κB)- and Egr1 (early growth-response factor 1) -binding sites. Overexpression and knockdown of transcription factors p65, p50, Egr1 or Sp1 (specificity protein 1) increased and decreased Ngb expression respectively. Experimental assessments with transfections of mutational Ngb gene promoter constructs, as well as EMSA (electrophoretic mobility-shift assay) and ChIP (chromatin immunoprecipitation) assays, demonstrated that NF-κB family members (p65, p50 and cRel), Egr1 and Sp1 bound in vitro and in vivo to the proximal promoter region of the Ngb gene. Moreover, a κB3 site was found as a pivotal cis-element responsible for hypoxia-induced Ngb promoter activity. NF-κB (p65) and Sp1 were also responsible for hypoxia-induced up-regulation of Ngb expression. Although there are no conserved HREs (hypoxia-response elements) in the promoter of the mouse Ngb gene, the results of the present study suggest that HIF-1α (hypoxia-inducible factor-1α) is also involved in hypoxia-induced Ngb up-regulation. In conclusion, we have identified that NF-κB, Egr1 and Sp1 played important roles in the regulation of basal Ngb expression via specific interactions with the mouse Ngb promoter. NF-κB, Sp1 and HIF-1α contributed to the up-regulation of mouse Ngb gene expression under hypoxic conditions.

Alterations in Protein-DNA Interactions in the γ-Globin Gene Promoter in Response to Butyrate Therapy

Blood ◽  
1998 ◽  
Vol 92 (8) ◽  
pp. 2924-2933 ◽  
Author(s):  
Tohru Ikuta ◽  
Yuet Wai Kan ◽  
Paul S. Swerdlow ◽  
Douglas V. Faller ◽  
Susan P. Perrine
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Gene Promoter ◽  
Dna Interactions ◽  
Globin Mrna ◽  
Mobility Shift ◽  
Protein Dna Interactions ◽  
American Society ◽  
Globin Gene Expression

Abstract The mechanisms by which pharmacologic agents stimulate γ-globin gene expression in β-globin disorders has not been fully established at the molecular level. In studies described here, nucleated erythroblasts were isolated from patients with β-globin disorders before and with butyrate therapy, and globin biosynthesis, mRNA, and protein-DNA interactions were examined. Expression of γ-globin mRNA increased twofold to sixfold above baseline with butyrate therapy in 7 of 8 patients studied. A 15% to 50% increase in γ-globin protein synthetic levels above baseline γ globin ratios and a relative decrease in β-globin biosynthesis were observed in responsive patients. Extensive new in vivo footprints were detected in erythroblasts of responsive patients in four regions of the γ-globin gene promoter, designated butyrate-response elements gamma 1-4 (BRE-G1-4). Electrophoretic mobility shift assays using BRE-G1 sequences as a probe demonstrated that new binding of two erythroid-specific proteins and one ubiquitous protein, CP2, occurred with treatment in the responsive patients and did not occur in the nonresponder. The BRE-G1 sequence conferred butyrate inducibility in reporter gene assays. These in vivo protein-DNA interactions in human erythroblasts in which γ-globin gene expression is being altered strongly suggest that nuclear protein binding, including CP2, to the BRE-G1 region of the γ-globin gene promoter mediates butyrate activity on γ-globin gene expression. © 1998 by The American Society of Hematology.

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 Transcriptional Regulation of HumanRev-erbαGene Expression by the Orphan Nuclear Receptor Retinoic Acid-related Orphan Receptor α

2002 ◽  
Vol 277 (51) ◽  
pp. 49275-49281 ◽  
Author(s):  
Eric Raspè ◽  
Gisèle Mautino ◽  
Caroline Duval ◽  
Coralie Fontaine ◽  
Hélène Duez ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Hepg2 Cells ◽  
Gene Promoter ◽  
Orphan Receptor ◽  
Similar Response ◽  
Orphan Nuclear Receptor ◽  
Orphan Nuclear Receptors ◽  
Mrna Accumulation

The Rev-erb and retinoic acid-related orphan receptors (ROR) are two related families of orphan nuclear receptors that recognize similar response elements but have opposite effects on transcription. Recently, theRev-erbαgene promoter has been characterized and shown to harbor a functional Rev-erbα-binding site known as Rev-DR2, responsible for negative feedback down-regulation of promoter activity by Rev-erbα itself. The present study aimed to investigate whetherRev-erbαgene expression is regulated by RORα. Gel shift analysis demonstrated thatin vitrotranslated hRORα1 protein binds to the Rev-DR2 site, both as monomer and dimer. Chromatin immunoprecipitation assays demonstrated that binding of RORα to this site also occurredin vivoin human hepatoma HepG2 cells. The Rev-DR2 site was further shown to be functional as it conferred hRORα1 responsiveness to a heterologous promoter and to the natural humanRev-erbαgene promoter in these cells. Mutation of this site in the context of the naturalRev-erbαgene promoter abolished its activation by RORα, indicating that this site plays a key role in hRORα1 action. Finally, adenoviral overexpression of hRORα1 in HepG2 cells led to enhanced hRev-erbα mRNA accumulation, further confirming the physiological importance of RORα1 in the regulation of Rev-erbα expression.

scholarly journals Identification of a signal transducer and activator of transcription (STAT) binding site in the mouse metallothionein-I promoter involved in interleukin-6-induced gene expression

1998 ◽  
Vol 337 (1) ◽  
pp. 59-65 ◽  
Author(s):  
Dae Kee LEE ◽  
Javier CARRASCO ◽  
Juan HIDALGO ◽  
Glen K. ANDREWS
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Time Course ◽  
Proximal Promoter ◽  
Type I ◽  
Mobility Shift ◽  
Knock Out ◽  
I Gene

Mechanisms of regulation of mouse metallothionein (MT)-I gene expression in response to bacterial endotoxin-lipopolysaccharide (LPS) were examined. Northern blot analysis of hepatic MT-I mRNA in interleukin (IL)-6 or tumour necrosis factor (TNF)-receptor type I knock-out mice demonstrated that IL-6, not TNF-α, is of central importance in mediating hepatic MT-I gene expression in vivo after LPS injection. In vivo genomic footprinting of the MT-I promoter demonstrated a rapid increase, after LPS injection, in the protection of several guanine residues in the -250 to -300 bp region of the MT-I promoter. The protected bases were within sequences which resemble binding sites for the signal transducers and activators of transcription (STAT) transcription factor family. Electrophoretic mobility-shift assays using oligonucleotides from footprinted MT-I promoter regions showed that injection of LPS resulted in a rapid increase in the specific, high-affinity, in vitro binding of STAT1 and STAT3 to a binding site at -297 bp (TTCTCGTAA). Western blotting of hepatic nuclear proteins showed that the time-course for changes of total nuclear STAT1 and STAT3 after LPS injection paralleled the increased complex formation in vitro using this oligonucleotide, and binding was specifically competed for by a functional STAT-binding site from the rat α2-macroglobulin promoter. Furthermore, the MT-I promoter -297 bp STAT-binding site conferred IL-6 responsiveness in the context of a minimal promoter in transient transfection assays using HepG2 cells. This study suggests that the effects of LPS on hepatic MT-I gene expression are mediated by IL-6 and involve the activation of STAT-binding to the proximal promoter.

scholarly journals Identification of miR-515-3p and its targets, vimentin and MMP3, as a key regulatory mechanism in esophageal cancer metastasis: functional and clinical significance

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Hui-Fang Hu ◽  
Wen Wen Xu ◽  
Wei-Xia Zhang ◽  
Xin Yan ◽  
Yang-Jia Li ◽  
...  
Keyword(s):  
Cancer Metastasis ◽  
Cpg Island ◽  
Treatment Strategies ◽  
Underlying Mechanism ◽  
Systemic Delivery ◽  
Matrix Metalloproteinase 3 ◽  
Specific Pcr ◽  
Tumor Tissues

AbstractMetastasis is the main factor of treatment failure in cancer patients, but the underlying mechanism remains to be elucidated and effective new treatment strategies are urgently needed. This study aims to explore novel key metastasis-related microRNAs (miRNAs) in esophageal squamous cell carcinoma (ESCC). By comparing miRNA profiles of the highly metastatic ESCC cell sublines, we established through serial in vivo selection with the parental cells, we found that the expression level of miR-515-3p was lower in ESCC tumor tissues than adjacent normal tissues, further decreased in metastatic tumors, and moreover, markedly associated with advanced stage, metastasis and patient survival. The in vitro and in vivo assays suggested that miR-515-3p could increase the expression of the epithelial markers as well as decrease the expression of the mesenchymal markers, and more importantly, suppress invasion and metastasis of ESCC cells. Mechanistically, we revealed that miR-515-3p directly regulated vimentin and matrix metalloproteinase-3 (MMP3) expression by binding to the coding sequence and 3′untranslated region, respectively. In addition, the data from whole-genome methylation sequencing and methylation-specific PCR indicated that the CpG island within miR-515-3p promoter was markedly hypermethylated in ESCC cell lines and ESCC tumor tissues, which may lead to deregulation of miR-515-3p expression in ESCC. Furthermore, our preclinical experiment provides solid evidence that systemic delivery of miR-515-3p oligonucleotide obviously suppressed the metastasis of ESCC cells in nude mice. Taken together, this study demonstrates that miR-515-3p suppresses tumor metastasis and thus represents a promising prognostic biomarker and therapeutic strategy in ESCC.

Alterations in Protein-DNA Interactions in the γ-Globin Gene Promoter in Response to Butyrate Therapy

Blood ◽  
1998 ◽  
Vol 92 (8) ◽  
pp. 2924-2933
Author(s):  
Tohru Ikuta ◽  
Yuet Wai Kan ◽  
Paul S. Swerdlow ◽  
Douglas V. Faller ◽  
Susan P. Perrine
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Gene Promoter ◽  
Dna Interactions ◽  
Globin Mrna ◽  
Mobility Shift ◽  
Protein Dna Interactions ◽  
American Society ◽  
Globin Gene Expression

The mechanisms by which pharmacologic agents stimulate γ-globin gene expression in β-globin disorders has not been fully established at the molecular level. In studies described here, nucleated erythroblasts were isolated from patients with β-globin disorders before and with butyrate therapy, and globin biosynthesis, mRNA, and protein-DNA interactions were examined. Expression of γ-globin mRNA increased twofold to sixfold above baseline with butyrate therapy in 7 of 8 patients studied. A 15% to 50% increase in γ-globin protein synthetic levels above baseline γ globin ratios and a relative decrease in β-globin biosynthesis were observed in responsive patients. Extensive new in vivo footprints were detected in erythroblasts of responsive patients in four regions of the γ-globin gene promoter, designated butyrate-response elements gamma 1-4 (BRE-G1-4). Electrophoretic mobility shift assays using BRE-G1 sequences as a probe demonstrated that new binding of two erythroid-specific proteins and one ubiquitous protein, CP2, occurred with treatment in the responsive patients and did not occur in the nonresponder. The BRE-G1 sequence conferred butyrate inducibility in reporter gene assays. These in vivo protein-DNA interactions in human erythroblasts in which γ-globin gene expression is being altered strongly suggest that nuclear protein binding, including CP2, to the BRE-G1 region of the γ-globin gene promoter mediates butyrate activity on γ-globin gene expression. © 1998 by The American Society of Hematology.

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