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.

Regulatory factors controlling transcription of Saccharomyces cerevisiae IXR1 by oxygen levels: a model of transcriptional adaptation from aerobiosis to hypoxia implicating ROX1 and IXR1 cross-regulation

2009 ◽  
Vol 425 (1) ◽  
pp. 235-243 ◽  
Author(s):  
Raquel  Castro-Prego ◽  
Mónica Lamas-Maceiras ◽  
Pilar Soengas ◽  
Isabel Carneiro ◽  
Isabel González-Siso ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Oxygen Availability ◽  
Mrna Levels ◽  
Mobility Shift ◽  
Hypoxic Conditions ◽  
Cross Links ◽  
Mobility Shift Assay ◽  
Transcriptional Adaptation

Ixr1p from Saccharomyces cerevisiae has been previously studied because it binds to DNA containing intrastrand cross-links formed by the anticancer drug cisplatin. Ixr1p is also a transcriptional regulator of anaerobic/hypoxic genes, such as SRP1/TIR1, which encodes a stress-response cell wall manoprotein, and COX5B, which encodes the Vb subunit of the mitochondrial complex cytochrome c oxidase. However, factors controlling IXR1 expression remained unexplored. In the present study we show that IXR1 mRNA levels are controlled by oxygen availability and increase during hypoxia. In aerobiosis, low levels of IXR1 expression are maintained by Rox1p repression through the general co-repressor complex Tup1–Ssn6. Ixr1p itself is necessary for full IXR1 expression under hypoxic conditions. Deletion analyses have identified the region in the IXR1 promoter responsible for this positive auto-control (nucleotides −557 to −376). EMSA (electrophoretic mobility-shift assay) and ChIP (chromatin immunoprecipitation) assays show that Ixr1p binds to the IXR1 promoter both in vitro and in vivo. Ixr1p is also required for hypoxic repression of ROX1 and binds to its promoter. UPC2 deletion has opposite effects on IXR1 and ROX1 transcription during hypoxia. Ixr1p is also necessary for resistance to oxidative stress generated by H2O2. IXR1 expression is moderately activated by H2O2 and this induction is Yap1p-dependent. A model of IXR1 regulation as a relay for sensing different signals related to change in oxygen availability is proposed. In this model, transcriptional adaptation from aerobiosis to hypoxia depends on ROX1 and IXR1 cross-regulation.

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 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 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 GATA augments GNRH-mediated increases in Adcyap1 gene expression in pituitary gonadotrope cells

2013 ◽  
Vol 51 (3) ◽  
pp. 313-324 ◽  
Author(s):  
Robin L Thomas ◽  
Natalie M Crawford ◽  
Constance M Grafer ◽  
Weiming Zheng ◽  
Lisa M Halvorson
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Proximal Promoter ◽  
Mrna Levels ◽  
Paracrine Factor ◽  
Mobility Shift ◽  
Subunit Expression ◽  
Mobility Shift Assay

Pituitary adenylate cyclase-activating polypeptide 1 (PACAP or ADCYAP1) regulates gonadotropin biosynthesis and secretion, both alone and in conjunction with GNRH. Initially identified as a hypothalamic-releasing factor, ADCYAP1 subsequently has been identified in pituitary gonadotropes, suggesting it may act as an autocrine–paracrine factor in this tissue. GNRH has been shown to increase pituitaryAdcyap1gene expression through the interaction of CREB and jun/fos with CRE/AP1cis-elements in the proximal promoter. In these studies, we were interested in identifying additional transcription factors and cognatecis-elements which regulateAdcyap1gene promoter activity and chose to focus on the GATA family of transcription factors known to be critical for both pituitary cell differentiation and gonadotropin subunit expression. By transient transfection and electrophoretic mobility shift assay analysis, we demonstrate that GATA2 and GATA4 stimulateAdcyap1promoter activity via a GATAcis-element located at position −191 in the ratAdcyap1gene promoter. Furthermore, we show that addition of GATA2 or GATA4 significantly augments GNRH-mediated stimulation ofAdcyap1gene promoter activity in the gonadotrope LβT2 cell line. Conversely, blunting GATA expression with specific siRNA inhibits the ability of GNRH to stimulate ADCYAP1 mRNA levels in these cells. These data demonstrate a complex interaction between GNRH and GATA on ADCYAP1 expression, providing important new insights into the regulation of gonadotrope function.

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.

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 TAB1 regulates glycolysis and activation of macrophages in diabetic nephropathy

2020 ◽  
Vol 69 (12) ◽  
pp. 1215-1234
Author(s):  
Hanxu Zeng ◽  
Xiangming Qi ◽  
Xingxin Xu ◽  
Yonggui Wu
Keyword(s):  
Diabetic Nephropathy ◽  
Transforming Growth Factor ◽  
Lentiviral Vector ◽  
Hypoxia Inducible Factor ◽  
Damage Index ◽  
Transforming Growth Factor Β ◽  
Nuclear Factor Κb ◽  
Inflammatory Factors

Abstract Objective and design Macrophages exhibit strong phenotypic plasticity and can mediate renal inflammation by polarizing into an M1 phenotype. They play a pivotal role in diabetic nephropathy (DN). Here, we have investigated the regulatory role of transforming growth factor β-activated kinase 1-binding protein 1 (TAB1) in glycolysis and activation of macrophages during DN. Methods TAB1 was inhibited using siRNA in high glucose (HG)-stimulated bone marrow-derived macrophages (BMMs) and lentiviral vector-mediated TAB1 knockdown was used in streptozotocin (STZ)-induced diabetic mice. Western blotting, flow cytometry, qRT-PCR, ELISA, PAS staining and immunohistochemical staining were used for assessment of TAB1/nuclear factor-κB (NF-κB)/hypoxia-inducible factor-1α (HIF-1α), iNOS, glycolysis, inflammation and the clinical and pathological manifestations of diabetic nephropathy. Results We found that TAB1/NF-κB/HIF-1α, iNOS and glycolysis were up-regulated in BMMs under HG conditions, leading to release of further inflammatory factors, Downregulation of TAB1 could inhibit glycolysis/polarization of macrophages and inflammation in vivo and in vitro. Furthermore, albuminuria, the tubulointerstitial damage index and glomerular mesangial expansion index of STZ-induced diabetic nephropathy mice were decreased by TAB1 knockdown. Conclusions Our results suggest that the TAB1/NF-κB/HIF-1α signaling pathway regulates glycolysis and activation of macrophages in DN.

Activating transcription factor 2 increases transactivation and protein stability of hypoxia-inducible factor 1α in hepatocytes

2009 ◽  
Vol 424 (2) ◽  
pp. 285-296 ◽  
Author(s):  
Jeong Hae Choi ◽  
Hyun Kook Cho ◽  
Yung Hyun Choi ◽  
JaeHun Cheong
Keyword(s):  
Transcription Factor ◽  
Protein Stability ◽  
Gene Transcription ◽  
Hypoxia Inducible Factor ◽  
Protein Stabilization ◽  
Hypoxic Conditions ◽  
Tumour Suppressor Protein ◽  
Activating Transcription Factor

HIF-1 (hypoxia inducible factor 1) performs a crucial role in mediating the response to hypoxia. However, other transcription factors are also capable of regulating hypoxia-induced target-gene transcription. In a previous report, we demonstrated that the transcription factor ATF-2 (activating transcription factor 2) regulates hypoxia-induced gene transcription, along with HIF-1α. In the present study, we show that the protein stability of ATF-2 is induced by hypoxia and the hypoxia-mimic CoCl2 (cobalt chloride), and that ATF-2 induction enhances HIF-1α protein stability via direct protein interaction. The knockdown of ATF-2 using small interfering RNA and translation-inhibition experiments demonstrated that ATF-2 plays a key role in the maintenance of the expression level and transcriptional activity of HIF-1α. Furthermore, we determined that ATF-2 interacts directly with HIF-1α both in vivo and in vitro and competes with the tumour suppressor protein p53 for HIF-1α binding. Collectively, these results show that protein stabilization of ATF-2 under hypoxic conditions is required for the induction of the protein stability and transactivation activity of HIF-1α for efficient hypoxia-associated gene expression.

scholarly journals Mutational and in vitro protein-binding studies on centromere DNA from Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (12) ◽  
pp. 4522-4534 ◽  
Author(s):  
R Ng ◽  
J Carbon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Binding ◽  
In Vitro Assays ◽  
Binding Studies ◽  
Mobility Shift ◽  
Sequence Element ◽  
Sequence Elements ◽  
Mobility Shift Assay

Centromeres on chromosomes in the yeast Saccharomyces cerevisiae contain approximately 140 base pairs (bp) of DNA. The functional centromere (CEN) region contains three important sequence elements (I, PuTCACPuTG; II, 78 to 86 bp of high-AT DNA; and III, a conserved 25-bp sequence with internal bilateral symmetry). Various point mutations or deletions in the element III region have a profound effect on CEN function in vivo, indicating that this DNA region is a key protein-binding site. This has been confirmed by the use of two in vitro assays to detect binding of yeast proteins to DNA fragments containing wild-type or mutationally altered CEN3 sequences. An exonuclease III protection assay was used to demonstrate specific binding of proteins to the element III region of CEN3. In addition, a gel DNA fragment mobility shift assay was used to characterize the binding reaction parameters. Sequence element III mutations that inactivate CEN function in vivo also prevent binding of proteins in the in vitro assays. The mobility shift assay indicates that double-stranded DNAs containing sequence element III efficiently bind proteins in the absence of sequence elements I and II, although the latter sequences are essential for optimal CEN function in vivo.

Sign in / Sign up

Export Citation Format

Share Document