scholarly journals Silencing of the Gene for the α-Subunit of Human Chorionic Gonadotropin by the Embryonic Transcription Factor Oct-3/4

1997 ◽  
Vol 11 (11) ◽  
pp. 1651-1658 ◽  
Author(s):  
Limin Liu ◽  
Douglas Leaman ◽  
Michel Villalta ◽  
R. Michael Roberts
Keyword(s):  
Transcription Factor ◽  
Binding Site ◽  
Gene Promoter ◽  
Site Directed Mutagenesis ◽  
Mobility Shift ◽  
Α Subunit ◽  
Choriocarcinoma Cells ◽  
Human Choriocarcinoma Cells ◽  
Electrophoretic Mobility Shift Assays ◽  
Jar Cells

Abstract CG is required for maintenance of the corpus luteum during pregnancy in higher primates. As CG is a heterodimeric molecule, some form of coordinated control must be maintained over the transcription of its two subunit genes. We recently found that expression of human CG β-subunit (hCGβ) in JAr human choriocarcinoma cells was almost completely silenced by the embryonic transcription factor Oct-3/4, which bound to a unique ACAATAATCA octameric sequence in the hCGβ gene promoter. Here we report that Oct-3/4 is also a potent inhibitor of hCG α-subunit (hCGα) expression in JAr cells. Oct-3/4 reduced human GH reporter expression from the −170 hCGα promoter in either the presence or absence of cAMP by about 70% in transient cotransfection assays, but had no effect on expression from either the −148 hCGα or the −99 hCGα promoter. Unexpectedly, no Oct-3/4-binding site was identified within the −170 to −148 region of the hCGα promoter, although one was found around position −115 by both methylation interference footprinting and electrophoretic mobility shift assays. Site-directed mutagenesis of this binding site destroyed the affinity of the promoter for Oct-3/4, but did not affect repression of the promoter. Therefore, inhibition of hCGα gene transcription by Oct-3/4 appears not to involve direct binding of this factor to the site responsible for silencing. When stably transfected into JAr cells, Oct-3/4 reduced the amounts of both endogenous hCGα mRNA and protein by 70–80%. Oct-3/4 is therefore capable of silencing both hCGα and hCGβ gene expression. We suggest that as the trophoblast begins to form, reduction of Oct-3/4 expression permits the coordinated onset of transcription from the hCGα and hCGβ genes.

Negative regulatory domains in a trophoblast interferon promoter

1996 ◽  
Vol 16 (2) ◽  
pp. 99-106 ◽  
Author(s):  
F M J Guesdon ◽  
H J Stewart ◽  
A P F Flint
Keyword(s):  
Gene Promoter ◽  
Promoter Sequence ◽  
Negative Control ◽  
Type I ◽  
Regulatory Domains ◽  
Murine Fibroblast ◽  
Expression Characteristic ◽  
Choriocarcinoma Cells ◽  
Human Choriocarcinoma Cells ◽  
Jar Cells

ABSTRACT A bovine trophoblast interferon (IFN-τ) gene promoter sequence (− 450 to +26 bp relative to the transcription start site) led to expression of reporter gene (CAT) constructs transfected into L929 (murine fibroblast) or JAR (human choriocarcinoma) cells. Expression depended on the presence of an exogenous (SV40) enhancer. Poly(I)(C) activated endogenous IFN production in L929 and JAR cells but had no consistent effect on CAT expression. Similar results were obtained in L929 cells with inactivated Newcastle disease virus. There was no 'priming' effect of exogenous Type I IFN. Deletion mutants revealed sites exerting negative control on expression between −338 and −247 bp, and between −150 and −71 bp; these regions contained sequences resembling previously identified negative regulatory domains. In the absence of viral inducibility it is proposed that negative regulation contributes towards the stringent control of expression characteristic of IFN-τ genes.

scholarly journals Transcription factor C/EBPβ promotes the transcription of the porcine GPR120 gene

2015 ◽  
Vol 56 (2) ◽  
pp. 91-100 ◽  
Author(s):  
Kun Chen ◽  
Ji-Dan Zhou ◽  
Feng Zhang ◽  
Fang Zhang ◽  
Rui-Rui Zhang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Binding Site ◽  
Vital Role ◽  
Site Directed Mutagenesis ◽  
Mobility Shift ◽  
Protein Levels ◽  
Promoter Deletion Analysis ◽  
Mrna And Protein Expression ◽  
Mobility Shift Assay ◽  
Factor C

G protein-coupled receptor 120 (GPR120), an adipogenic receptor critical for the differentiation and maturation of adipocytes, plays an important role in controlling obesity in both humans and rodents and, thus, is an attractive target of obesity treatment studies. However, the mechanisms that regulate the expression of porcine GPR120 remain unclear. In this study, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) techniques were used to analyze and identify the binding of C/EBPβ (transcription factor CCAAT/enhancer binding protein beta) to the GPR120 promoter. C/EBPβ overexpression and RNA interference studies showed that C/EBPβ regulated GPR120 promoter activity and endogenous GPR120 expression. The binding site of C/EBPβ in the GPR120 promoter region from −101 to −87 was identified by promoter deletion analysis and site-directed mutagenesis. Overexpression of C/EBPβ increased endogenous GPR120 expression in pig kidney cells (PK). Furthermore, when endogenous C/EBPβ was knocked down, GPR120 mRNA and protein levels were decreased. The stimulatory effect of C/EBPβ on GPR120 transcription and its ability to bind the transcription factor-binding site were confirmed by luciferase, ChIP, and EMSA. Moreover, the mRNA and protein expression levels of C/EBPβ were induced by high fat diet feeding. Taken together, it can be concluded that C/EBPβ plays a vital role in regulating GPR120 transcription and suggests HFD-feeding induces GPR120 transcription by influencing C/EBPβ expression.

scholarly journals DNA Methylation of Human Choline Kinase Alpha Promoter-Associated CpG Islands in MCF-7 Cells

Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 853
Author(s):  
Siti Aisyah Faten Mohamed Sa’dom ◽  
Sweta Raikundalia ◽  
Shaharum Shamsuddin ◽  
Wei Cun See Too ◽  
Ling Ling Few
Keyword(s):  
Dna Methylation ◽  
Transcription Factor ◽  
Binding Site ◽  
Promoter Activity ◽  
Cytosine Methylation ◽  
Cpg Island ◽  
Cpg Islands ◽  
Site Directed Mutagenesis ◽  
Choline Kinase ◽  
Mcf 7

Choline kinase (CK) is the enzyme catalyzing the first reaction in CDP-choline pathway for the biosynthesis of phosphatidylcholine. Higher expression of the α isozyme of CK has been implicated in carcinogenesis, and inhibition or downregulation of CKα (CHKA) is a promising anticancer approach. This study aimed to investigate the regulation of CKα expression by DNA methylation of the CpG islands found on the promoter of this gene in MCF-7 cells. Four CpG islands have been predicted in the 2000 bp promoter region of ckα (chka) gene. Six CpG island deletion mutants were constructed using PCR site-directed mutagenesis method and cloned into pGL4.10 vectors for promoter activity assays. Deletion of CpG4C region located between –225 and –56 significantly increased the promoter activity by 4-fold, indicating the presence of important repressive transcription factor binding site. The promoter activity of methylated full-length promoter was significantly lower than the methylated CpG4C deletion mutant by 16-fold. The results show that DNA methylation of CpG4C promotes the binding of the transcription factor that suppresses the promoter activity. Electrophoretic mobility shift assay analysis showed that cytosine methylation at MZF1 binding site in CpG4C increased the binding of putative MZF1 in nuclear extract. In conclusion, the results suggest that DNA methylation decreased the promoter activity by promoting the binding of putative MZF1 transcription factor at CpG4C region of the ckα gene promoter.

scholarly journals Characterization of the promoter elements required for hepatic and intestinal transcription of the human apoB gene: definition of the DNA-binding site of a tissue-specific transcriptional factor.

1990 ◽  
Vol 10 (6) ◽  
pp. 2653-2659 ◽  
Author(s):  
D Kardassis ◽  
M Hadzopoulou-Cladaras ◽  
D P Ramji ◽  
R Cortese ◽  
V I Zannis ◽  
...  
Keyword(s):  
Binding Site ◽  
Regulatory Elements ◽  
Apob Gene ◽  
Mobility Shift ◽  
Promoter Elements ◽  
Nuclear Extracts ◽  
Dna Binding Site ◽  
Gene Definition ◽  
Definition Of ◽  
Electrophoretic Mobility Shift Assays

The promoter elements important for intestinal and hepatic transcription of the human apoB gene have been localized downstream of nucleotide -150. Footprinting analysis using hepatic nuclear extracts identified four protected regions, -124 to -100, -97 to -93, -86 to -33, and +33 to +52. Gel electrophoretic mobility shift assays showed that multiple factors interact with the apoB sequence -86 to -33, while the region -88 to -61 binds a single nuclear factor. Methylation interference analysis and nucleotide substitution mutagenesis identified the binding site of the factor between residues -78 and -68. Binding competition experiments indicate that this factor recognizes the regulatory elements of other liver-specific genes.

scholarly journals Regulation of the expression of whiB1 in Mycobacterium tuberculosis: role of cAMP receptor protein

Microbiology ◽  
2006 ◽  
Vol 152 (9) ◽  
pp. 2749-2756 ◽  
Author(s):  
Nisheeth Agarwal ◽  
Tirumalai R. Raghunand ◽  
William R. Bishai
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Binding Site ◽  
Receptor Protein ◽  
Camp Receptor Protein ◽  
Mobility Shift ◽  
Camp Analogue ◽  
Camp Receptor ◽  
Fusion Analysis ◽  
Electrophoretic Mobility Shift Assays

The wbl (whiB-like) genes encode putative transcription factors unique to actinomycetes. This study characterized the promoter element of one of the seven wbl genes of Mycobacterium tuberculosis, whiB1 (Rv3219c). The results reveal that whiB1 is transcribed by a class I-type cAMP receptor protein (CRP)-dependent promoter, harbouring a CRP-binding site positioned at −58.5 with respect to its transcription start point. In vivo promoter activity analysis and electrophoretic mobility shift assays suggest that the expression of whiB1 is indeed regulated by cAMP-dependent binding of CRPM (encoded by the M. tuberculosis gene Rv3676) to the whiB1 5′ untranslated region (5′UTR). β-Galactosidase gene fusion analysis revealed induction of the whiB1 promoter in M. tuberculosis on addition of exogenous dibutyric cAMP (a diffusible cAMP analogue) only when an intact CRP-binding site was present. These results indicate that M. tuberculosis whiB1 transcription is regulated in part by cAMP levels via direct binding of cAMP-activated CRPM to a consensus CRP-binding site in the whiB1 5′UTR.

Distinct functional contributions of 2 GABP–NRF-2 recognition sites within the context of the human TOMM70 promoter

2006 ◽  
Vol 84 (5) ◽  
pp. 813-822 ◽  
Author(s):  
José R. Blesa ◽  
José Hernández-Yago
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Site Directed Mutagenesis ◽  
Outer Mitochondrial Membrane ◽  
Mobility Shift ◽  
A Subunit ◽  
Expression Evidence ◽  
Electrophoretic Mobility Shift Assays

TOMM70 is a subunit of the outer mitochondrial membrane translocase that plays a major role as a receptor of hydrophobic preproteins targeted to mitochondria. We have previously reported 2 binding sites for the transcription factor GABP–NRF-2 in the promoter region of the human TOMM70 gene that are important in activating transcription. To assess the functionality and actual role of these sites, chromatin immunoprecipitation, site-directed mutagenesis, and electrophoretic mobility shift assays were carried out. We conclude that GABP–NRF-2 binds in vivo to the TOMM70 promoter, and that the 2 GABP–NRF-2 binding sites of the promoter have different functional contributions in promoting TOMM70 expression. Evidence is provided that they work in an additive manner as single sites.

scholarly journals Persistent NF-κB activation in renal epithelial cells in a mouse model of HIV-associated nephropathy

2006 ◽  
Vol 290 (3) ◽  
pp. F657-F665 ◽  
Author(s):  
Scott Martinka ◽  
Leslie A. Bruggeman
Keyword(s):  
Transcription Factor ◽  
Epithelial Cells ◽  
Cell Types ◽  
Nuclear Accumulation ◽  
Mobility Shift ◽  
Glomerular Epithelial Cells ◽  
Host Virus Interactions ◽  
Virus Interactions ◽  
Electrophoretic Mobility Shift Assays ◽  
Hiv 1

Human immunodeficiency virus (HIV)-associated nephropathy (HIVAN) is caused, in part, by direct infection of kidney epithelial cells by HIV-1. In the spectrum of pathogenic host-virus interactions, abnormal activation or suppression of host transcription factors is common. NF-κB is a necessary host transcription factor for HIV-1 gene expression, and it has been shown that NF-κB activity is dysregulated in many naturally infected cell types. We show here that renal glomerular epithelial cells (podocytes) expressing the HIV-1 genome, similar to infected immune cells, also have a dysregulated and persistent activation of NF-κB. Although podocytes produce p50, p52, RelA, RelB, and c-Rel, electrophoretic mobility shift assays and immunocytochemistry showed a predominant nuclear accumulation of p50/RelA-containing NF-κB dimers in HIV-1-expressing podocytes compared with normal. In addition, the expression level of a transfected NF-κB reporter plasmid was significantly higher in HIVAN podocytes. The mechanism of NF-κB activation involved increased phosphorylation of IκBα, resulting in an enhanced turnover of the IκBα protein. There was no evidence for regulation by IκBβ or the alternate pathway of NF-κB activation. Altered activation of this key host transcription factor likely plays a role in the well-described cellular phenotypic changes observed in HIVAN, such as proliferation. Studies with inhibitors of proliferation and NF-κB suggest that NF-κB activation may contribute to the proliferative mechanism in HIVAN. In addition, because NF-κB regulates many aspects of inflammation, this dysregulation may also contribute to disease severity and progression through regulation of proinflammatory processes in the kidney microenvironment.

Fusion of the ets Transcription Factor TEL to Jak2 Results in Constitutive Jak-Stat Signaling

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4354-4364 ◽  
Author(s):  
Jen M.-Y. Ho ◽  
Bryan K. Beattie ◽  
Jeremy A. Squire ◽  
David A. Frank ◽  
Dwayne L. Barber
Keyword(s):  
Transcription Factor ◽  
Fusion Protein ◽  
Janus Kinase ◽  
Chimeric Proteins ◽  
Mobility Shift ◽  
Specific Antibodies ◽  
Hematopoietic Cell Line ◽  
Ets Transcription Factor ◽  
Electrophoretic Mobility Shift Assays ◽  
Insight Into

Abstract To study constitutive Janus kinase signaling, chimeric proteins were generated between the pointed domain of the etstranscription factor TEL and the cytosolic tyrosine kinase Jak2. The effects of these proteins on interleukin-3 (IL-3)–dependent proliferation of the hematopoietic cell line, Ba/F3, were studied. Fusion of TEL to the functional kinase (JH1) domain of Jak2 resulted in conversion of Ba/F3 cells to factor-independence. Importantly, fusion of TEL to the Jak2 pseudokinase (JH2) domain or a kinase-inactive Jak2 JH1 domain had no effect on IL-3–dependent proliferation of Ba/F3 cells. Active TEL-Jak2 constructs (consisting of either Jak2 JH1 or Jak2 JH2+JH1 domain fusions) were constitutively tyrosine-phosphorylated but did not affect phosphorylation of endogeneous Jak1, Jak2, or Jak3. TEL-Jak2 activation resulted in the constitutive tyrosine phosphorylation of Stat1, Stat3, and Stat5 as determined by detection of phosphorylation using activation-specific antibodies and by binding of each protein to a preferential GAS sequence in electrophoretic mobility shift assays. Elucidation of signaling events downstream of TEL-Jak2 activation may provide insight into the mechanism of leukemogenesis mediated by this oncogenic fusion protein.

scholarly journals The transcription factor PITX1 drives astrocyte differentiation by regulating the SOX9 gene

2020 ◽  
Vol 295 (39) ◽  
pp. 13677-13690
Author(s):  
Jeong Su Byun ◽  
Mihee Oh ◽  
Seonha Lee ◽  
Jung-Eun Gil ◽  
Yeajin Mo ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Formation Control ◽  
Synapse Formation ◽  
Embryonic Stem ◽  
Binding Motif ◽  
Mobility Shift ◽  
Differentiation Protocol ◽  
Mature Brain ◽  
Astrocyte Differentiation ◽  
Electrophoretic Mobility Shift Assays

Astrocytes perform multiple essential functions in the developing and mature brain, including regulation of synapse formation, control of neurotransmitter release and uptake, and maintenance of extracellular ion balance. As a result, astrocytes have been implicated in the progression of neurodegenerative disorders such as Alzheimer's disease, Huntington's disease, and Parkinson's disease. Despite these critical functions, the study of human astrocytes can be difficult because standard differentiation protocols are time-consuming and technically challenging, but a differentiation protocol recently developed in our laboratory enables the efficient derivation of astrocytes from human embryonic stem cells. We used this protocol along with microarrays, luciferase assays, electrophoretic mobility shift assays, and ChIP assays to explore the genes involved in astrocyte differentiation. We demonstrate that paired-like homeodomain transcription factor 1 (PITX1) is critical for astrocyte differentiation. PITX1 overexpression induced early differentiation of astrocytes, and its knockdown blocked astrocyte differentiation. PITX1 overexpression also increased and PITX1 knockdown decreased expression of sex-determining region Y box 9 (SOX9), known initiator of gliogenesis, during early astrocyte differentiation. Moreover, we determined that PITX1 activates the SOX9 promoter through a unique binding motif. Taken together, these findings indicate that PITX1 drives astrocyte differentiation by sustaining activation of the SOX9 promoter.

Matrix metalloproteinase-3 induction in rat brain astrocytes: focus on the role of two AP-1 elements

2008 ◽  
Vol 410 (3) ◽  
pp. 605-611 ◽  
Author(s):  
Kwang Soo Kim ◽  
Hee Young Kim ◽  
Eun-hye Joe ◽  
Ilo Jou
Keyword(s):  
Rat Brain ◽  
Interferon Γ ◽  
Site Directed Mutagenesis ◽  
Start Codon ◽  
Activator Protein 1 ◽  
Mobility Shift ◽  
Matrix Metalloproteinase 3 ◽  
Mitogen Activated Protein ◽  
Electrophoretic Mobility Shift Assays

Many brain cells secrete MMPs (matrix metalloproteinases), and increased or misregulated MMP levels are found in neurodegenerative disorders. Here we report that MMP-3 transcription and protein secretion were increased in rat brain astrocytes stimulated with lipopolysaccharide, gangliosides or interferon-γ. Sequential deletion of the MMP-3 promoter revealed that sequences between −0.5 kb and the start codon were crucial for the transcriptional induction of MMP-3. In addition, experiments using pharmacological inhibitors of individual mitogen-activated protein kinases revealed that MMP-3 induction and promoter activity involved Jun N-terminal kinase, a representative upstream signal of AP-1 (activator protein-1). Sequence analyses of the region of the MMP-3 promoter 500 bp from the start codon indicated the presence of three AP-1 binding sequences. Among them, electrophoretic-mobility-shift assays as well as site-directed mutagenesis of individual AP-1 sequences revealed that distal and middle, but not proximal, sequences largely mediated its induction. Together, these results indicate that AP-1 could control MMP-3 induction in brain astrocytes and that its regulation through specific AP-1 elements could be exploited in the treatment of brain pathologies in which increased expression of MMP-3 plays crucial roles.

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