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 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.

scholarly journals ACOX1, regulated by C/EBPα and miR-25-3p, promotes bovine preadipocyte adipogenesis

2021 ◽  
Author(s):  
Feng Zhang ◽  
Qi Xiong ◽  
Hu Tao ◽  
Yang Liu ◽  
Nian Zhang ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Binding Sites ◽  
Site Directed Mutagenesis ◽  
Luciferase Assay ◽  
Directed Mutagenesis ◽  
Loss Of Function ◽  
Mobility Shift ◽  
Promoter Deletion Analysis ◽  
Factor C ◽  
Peroxisomal Fatty Acid

Acyl-Coenzyme A oxidase 1 (ACOX1) is the first and rate-limiting enzyme in peroxisomal fatty acid β-oxidation of fatty acids. Previous studies have reported that ACOX1 was correlated with the meat quality of livestock, while the role of ACOX1 in intramuscular adipogenesis of beef cattle and its transcriptional and post-transcriptional regulatory mechanisms remain unclear. In the present study, gain-of-function and loss-of-function assays demonstrated that ACOX1 positively regulated the adipogenesis of bovine intramuscular preadipocytes. The C/EBPα-binding sites in the bovine ACOX1 promoter region at -1142 to -1129 bp, -831 to -826 bp, and -303 to -298 bp were identified by promoter deletion analysis and site-directed mutagenesis. Electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) further showed that these three regions are C/EBPα-binding sites, both in vitro and in vivo, indicating that C/EBPα directly interacts with the bovine ACOX1 promoter and inhibits its transcription. Furthermore, the results from bioinformatics analysis, dual luciferase assay, site-directed mutagenesis, qRT-PCR, and Western blotting demonstrated that miR-25-3p directly targeted the ACOX1 3’untranslated region (3’UTR). Taken together, our findings suggest that ACOX1, regulated by transcription factor C/EBPα and miR-25-3p, promotes adipogenesis of bovine intramuscular preadipocytes via regulating peroxisomal fatty acid β-oxidation.

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.

The Vitamin D Receptor Represses Transcription of the Pituitary Transcription Factor Pit-1 Gene without Involvement of the Retinoid X Receptor

2006 ◽  
Vol 20 (4) ◽  
pp. 735-748 ◽  
Author(s):  
Samuel Seoane ◽  
Roman Perez-Fernandez
Keyword(s):  
Vitamin D ◽  
Transcription Factor ◽  
Vitamin D Receptor ◽  
Retinoid X Receptor ◽  
Breast Adenocarcinoma ◽  
Mobility Shift ◽  
Histone Deacetylase 1 ◽  
Protein Levels ◽  
Repressive Effect ◽  
Mcf 7

Abstract Pituitary transcription factor-1 (Pit-1) plays a key role in cell differentiation during organogenesis of the anterior pituitary, and as a transcriptional activator for the pituitary GH and prolactin genes. However, Pit-1 is also expressed in nonpituitary cell types and tissues. In breast tumors, Pit-1 mRNA and protein levels are increased with respect to normal breast, and in MCF-7 human breast adenocarcinoma cells, Pit-1 increases GH secretion and cell proliferation. We report here that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] administration to MCF-7 cells induces a significant decrease in Pit-1 mRNA and protein levels. By deletion analyses, we mapped a region (located between −147 and −171 bp from the transcription start site of the Pit-1 gene) that is sufficient for the repressive response to 1,25-(OH)2D3. Gel mobility shift and chromatin immunoprecipitation assays confirmed the direct interaction between the vitamin D receptor (VDR) as homodimer (without the retinoid X receptor), and the Pit-1 promoter, supporting the view that Pit-1 is a direct transcriptional target of VDR. Our data also indicate that recruitment of histone deacetylase 1 is involved in this repressive effect. This ligand-dependent Pit-1 gene inhibition by VDR in the absence of the retinoid X receptor seems to indicate a new mechanism of transcriptional repression by 1,25-(OH)2D3.

Identification of a killer cell-specific regulatory element of the mouse perforin gene: an Ets-binding site-homologous motif that interacts with Ets-related proteins

1993 ◽  
Vol 13 (11) ◽  
pp. 6690-6701
Author(s):  
H Koizumi ◽  
M F Horta ◽  
B S Youn ◽  
K C Fu ◽  
B S Kwon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Binding Site ◽  
Protein Interactions ◽  
Regulatory Element ◽  
Killer Cell ◽  
Mobility Shift ◽  
Specific Expression ◽  
Native Molecular Mass ◽  
Mobility Shift Assay

The gene encoding the cytolytic protein perforin is selectively expressed by activated killer lymphocytes. To understand the mechanisms underlying the cell-type-specific expression of this gene, we have characterized the regulatory functions and the DNA-protein interactions of the 5'-flanking region of the mouse perforin gene (Pfp). A region extending from residues +62 through -141, which possesses the essential promoter activity, and regions further upstream, which are able to either enhance or suppress gene expression, were identified. The region between residues -411 and -566 was chosen for further characterization, since it contains an enhancer-like activity. We have identified a 32-mer sequence (residues -491 to -522) which appeared to be capable of enhancing gene expression in a killer cell-specific manner. Within this segment, a 9-mer motif (5'-ACAGGAAGT-3', residues -505 to -497; designated NF-P motif), which is highly homologous to the Ets proto-oncoprotein-binding site, was found to interact with two proteins, NF-P1 and NF-P2. NF-P2 appears to be induced by reagents known to up-regulate the perforin message level and is present exclusively in killer cells. Electrophoretic mobility shift assay and UV cross-linking experiments revealed that NF-P1 and NF-P2 may possess common DNA-binding subunits. However, the larger native molecular mass of NF-P1 suggests that NF-P1 contains an additional non-DNA-binding subunit(s). In view of the homology between the NF-P motif and other Ets proto-oncoprotein-binding sites, it is postulated that NF-P1 and NF-P2 belong to the Ets protein family. Results obtained from the binding competition assay, nevertheless, suggest that NF-P1 and NF-P2 are related to but distinct from Ets proteins, e.g., Ets-1, Ets-2, and NF-AT/Elf-1, known to be expressed in T cells.

PDGF Up-regulates CSF-1 Gene Transcription in Ameloblast-like Cells

2008 ◽  
Vol 87 (1) ◽  
pp. 33-38 ◽  
Author(s):  
Y. Wittrant ◽  
B. Sriniketan Bhandari ◽  
H. Abboud ◽  
N. Benson ◽  
K. Woodruff ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Binding Motif ◽  
Rt Pcr ◽  
Mobility Shift ◽  
Protein Levels ◽  
Macrophage Colony Stimulating Factor ◽  
Cellular Pathways ◽  
Mobility Shift Assay ◽  
Macrophage Colony ◽  
Stimulating Factor

Macrophage colony-stimulating factor (CSF-1) is a key regulatory cytokine for amelogenesis, and ameloblasts synthesize CSF-1. We hypothesized that PDGF stimulates DNA synthesis and regulates CSF-1 in these cells. We determined the effect of PDGF on CSF-1 expression using MEOE-3M ameloblasts as a model. By RT-PCR, MEOE-3M expressed PDGFRs and PDGF A- and B-chain mRNAs. PDGF-BB increased DNA synthesis and up-regulated CSF-1 mRNA and protein in MEOE-3M. Cells transfected with CSF-1 promoter deletion constructs were analyzed. A PDGF-responsive region between −1.7 and −0.795 kb, containing a consensus Pea3 binding motif, was identified. Electrophoretic mobility shift assay (EMSA) showed that PDGF-BB stimulated protein binding to this motif that was inhibited in the presence of anti-Pea3 antibody. Analysis of these data provides the first evidence that PDGF-BB is a mitogen for MEOE-3M and increases CSF-1 protein levels, predominantly by transcription. Elucidation of the cellular pathways that control CSF-1 expression may provide novel strategies for the regulation of enamel matrix formation.

scholarly journals Transcriptional activation of the H-ferritin gene in differentiated Caco-2 cells parallels a change in the activity of the nuclear factor Bbf

1995 ◽  
Vol 311 (3) ◽  
pp. 769-773 ◽  
Author(s):  
M A Bevilacqua ◽  
M C Faniello ◽  
P D′Agostino ◽  
B Quaresima ◽  
M T Tiano ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Mobility Shift ◽  
Differentiated Cells ◽  
Binding Factor ◽  
H Gene ◽  
Ferritin Gene ◽  
Mobility Shift Assay ◽  
Promoter Interaction

In this paper, we examine the mechanisms that regulate the expression of the heavy (H) ferritin subunit in the colon carcinoma Caco-2 cell line allowed to differentiate spontaneously in vitro. The differentiation process of these cells in continuous culture is accompanied by an accumulation of the mRNA coding for the apoferritin H chain. The analysis of Caco-2 subclones stably transfected with an H-chain promoter-chloramphenicol acetyltransferase (CAT) construct revealed that the mRNA increase is paralleled by an enhanced transcription of the H gene, driven by the -100 to +4 region of the H promoter. The H gene transcriptional activation seems to be a specific feature of differentiated Caco-2 cells, since the activity of other promoters did not change upon differentiation. The -100 to +4 region of the H promoter binds a transcription factor called Bbf (B-box binding factor); electrophoretic-mobility-shift-assay analyses showed that the retarded complex due to Bbf-H promoter interaction is significantly increased in the differentiated cells. We propose that the activation of H-ferritin gene expression may be associated with the establishment of a differentiated phenotype in Caco-2 cells, and that the H-ferritin gene transcriptional up-regulation is accompanied by a modification in the activity of the transcription factor Bbf.

scholarly journals PgMYB2, a MeJA-Responsive Transcription Factor, Positively Regulates the Dammarenediol Synthase Gene Expression in Panax Ginseng

2019 ◽  
Vol 20 (9) ◽  
pp. 2219 ◽  
Author(s):  
Tuo Liu ◽  
Tiao Luo ◽  
Xiangqian Guo ◽  
Xian Zou ◽  
Donghua Zhou ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Panax Ginseng ◽  
Growth Regulation ◽  
Ginseng Root ◽  
Myb Transcription Factor ◽  
Mobility Shift ◽  
Binding Interaction ◽  
Mobility Shift Assay ◽  
Dammarenediol Synthase

The MYB transcription factor family members have been reported to play different roles in plant growth regulation, defense response, and secondary metabolism. However, MYB gene expression has not been reported in Panax ginseng. In this study, we isolated a gene from ginseng adventitious root, PgMYB2, which encodes an R2R3-MYB protein. Subcellular localization revealed that PgMYB2 protein was exclusively detected in the nucleus of Allium cepa epidermis. The highest expression level of PgMYB2 was found in ginseng root and it was significantly induced by plant hormones methyl jasmonate (MeJA). Furthermore, the binding interaction between PgMYB2 protein and the promoter of dammarenediol synthase (DDS) was found in the yeast strain Y1H Gold. Moreover, the electrophoretic mobility shift assay (EMSA) identified the binding site of the interaction and the results of transiently overexpressing PgMYB2 in plants also illustrated that it may positively regulate the expression of PgDDS. Based on the key role of PgDDS gene in ginsenoside synthesis, it is reasonable to believe that this report will be helpful for the future studies on the MYB family in P. ginseng and ultimately improving the ginsenoside production through genetic and metabolic engineering.

scholarly journals Coordination of erythropoiesis by the transcription factor c-Myb

Blood ◽  
2006 ◽  
Vol 107 (12) ◽  
pp. 4703-4710 ◽  
Author(s):  
Alexandros Vegiopoulos ◽  
Paloma García ◽  
Nikla Emambokus ◽  
Jon Frampton
Keyword(s):  
Transcription Factor ◽  
Critical Role ◽  
Leukemia Cell ◽  
Erythroid Cell ◽  
Erythroid Progenitors ◽  
Aberrant Expression ◽  
Protein Levels ◽  
Kit Receptor ◽  
Erythroid Cell Differentiation ◽  
Factor C

Abstract The involvement of the transcription factor c-Myb in promoting the proliferation and inhibition of erythroid cell differentiation has been established in leukemia cell models. The anemia phenotype observed in c-myb knockout and knockdown mice highlights a critical role for c-Myb in erythropoiesis. However, determining the reason for the failure of erythropoiesis in these mice and the precise function of c-Myb in erythroid progenitors remains elusive. We examined erythroid development under conditions of reduced c-Myb protein levels and report an unexpected role for c-Myb in the promotion of commitment to the erythroid lineage and progression to erythroblast stages. c-myb knockdown erythroid colony-forming unit (CFU-E) stage progenitors displayed an immature phenotype and aberrant expression of several hematopoietic regulators. To extend our findings, we analyzed the response of normal enriched erythroid progenitors to inducible disruption of a floxed c-myb allele. In agreement with the c-myb knockdown phenotype, we show that c-Myb is strictly required for expression of the c-Kit receptor in erythroid cells.

Inhibition of transcription factor assembly and structural stability on mitoxantrone binding with DNA

2010 ◽  
Vol 30 (5) ◽  
pp. 331-340 ◽  
Author(s):  
Shahper N. Khan ◽  
Mohd Danishuddin ◽  
Asad U. Khan
Keyword(s):  
Transcription Factor ◽  
Binding Mode ◽  
Cd Spectroscopy ◽  
Sequence Specificity ◽  
Mobility Shift ◽  
Dna Complexes ◽  
Naked Dna ◽  
Plasmid Nicking Assay ◽  
Modelling Studies ◽  
Mobility Shift Assay

MTX (mitoxantrone) is perhaps the most promising drug used in the treatment of various malignancies. Comprehensive literature on the therapeutics has indicated it to be the least toxic in its class, although its mechanism of action is still not well defined. In the present study, we have evaluated the associated binding interactions of MTX with naked DNA. The mechanism of MTX binding with DNA was elucidated by steady-state fluorescence and a static-type quenching mechanism is suggested for this interaction. Thermodynamic parameters from van 't Hoff plots showed that the interaction of these drugs with DNA is an entropically driven phenomenon. The binding mode was expounded by attenuance measurements and competitive binding of a known intercalator. Sequence specificity of these drug–DNA complexes was analysed by FTIR (Fourier-transform infrared) spectroscopy and molecular modelling studies. CD spectroscopy and the plasmid nicking assay showed that the binding of this drug with DNA results in structural and conformational perturbations. EMSA (electrophoretic mobility-shift assay) results showed that these drug–DNA complexes prevent the binding of octamer TF (transcription factor) to DNA. In summary, the study implicates MTX-induced conformational instability and transcription inhibition on DNA binding.

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