scholarly journals Transcription Factor CEBPB Inhibits the Expression of the Human HTR1A by Binding to 5’ Regulatory Region in Vitro

Genes ◽  
2019 ◽  
Vol 10 (10) ◽  
pp. 802
Author(s):  
Liu ◽  
Wu ◽  
Meng ◽  
Ding ◽  
Xu ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Fluorescence Intensity ◽  
Mrna Level ◽  
Regulatory Region ◽  
Receptor Expression ◽  
Relative Fluorescence Intensity ◽  
Mobility Shift ◽  
Hek 293 ◽  
Endogenous Expression

This study identified a transcription factor that might bind to the 5’ regulatory region of the HTR1A and explored the potential effect on 5-HT1A receptor expression. Based on JASPAR predictions, the binding of the transcription factor was demonstrated using the electrophoretic mobility shift assay (EMSA). Vectors over-expressing the transcription factor were co-transfected into HEK-293 and SK-N-SH cells with the recombinant pGL3 vector, and relative fluorescence intensity was measured to determine regulatory activity. Additionally, the qRT-PCR and Western blot were also used to identify whether the transcription factor modulated the endogenous expression of 5-HT1A receptor. The results suggest that the transcription factor CCAA/T enhancer binding protein beta (CEBPB) likely binds to the -1219 to -1209 bp (ATG+1) region of the HTR1A. Two sequences located in the -722 to -372 bp and -119 to +99 bp were also identified. Although the effect of CEBPB on endogenous 5-HT1A receptor expression was not significant, it exhibited the strong inhibition on the relative fluorescence intensity and the mRNA level of HTR1A. CEBPB inhibited the human HTR1A expression by binding to the sequence -1219 - -1209 bp. This is useful and informative for ascertaining the regulation of 5-HT1A receptor and mental diseases.

scholarly journals The TLX2 homeobox gene is a transcriptional target of PHOX2B in neural-crest-derived cells

2006 ◽  
Vol 395 (2) ◽  
pp. 355-361 ◽  
Author(s):  
Silvia Borghini ◽  
Tiziana Bachetti ◽  
Monica Fava ◽  
Marco Di Duca ◽  
Francesca Cargnin ◽  
...  
Keyword(s):  
Neural Crest ◽  
Mrna Level ◽  
Regulatory Region ◽  
Neuroblastoma Cells ◽  
Physiological Role ◽  
Mobility Shift ◽  
Functional Link ◽  
Specific Element

The TLX2 (HOX11L1, Ncx, Enx) and PHOX2B genes encode transcription factors crucial in the development of neural-crest-derived cells, leading to ANS (autonomic nervous system) specific neuronal lineages. Moreover, they share a similar expression pattern and are both involved in downstream steps of BMP (bone morphogenetic protein) signalling. In an attempt to reconstruct the gene network sustaining the correct development of the ANS, we have undertaken an in vitro experimental strategy to identify direct upstream regulators of the TLX2 gene. After characterizing a sequence displaying enhancer property in its 5′ flanking region, we confirmed the functional link between the human PHOX2B and TLX2 genes. Transient transfections and electrophoretic-mobility-shift assays suggested that PHOX2B is able to bind the cell-specific element in the 5′ regulatory region of the TLX2 gene, determining its transactivation in neuroblastoma cells. Such interaction was also confirmed in vivo by means of chromatin immunoprecipitation assay and, in addition, up-regulation of endogenous TLX2 mRNA level was demonstrated following PHOX2B over-expression, by quantitative real-time PCR. Finally, PHOX2B proteins carrying mutations responsible for CCHS (congenital central hypoventilation syndrome) development showed a severe impairment in activating TLX2 expression, both in vitro and in vivo. Taken together, these results support the PHOX2B–TLX2 promoter interaction, suggesting a physiological role in the transcription-factor cascade underlying the differentiation of neuronal lineages of the ANS during human embryogenesis.

scholarly journals Functional Analysis of Haplotypes and Promoter Activity at the 5’ Region of the ADH7 Gene

2021 ◽  
Author(s):  
Kuo Zeng ◽  
Ya Li ◽  
Meng Gao ◽  
Yong-ping Liu ◽  
Feng-ling Xu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Cell Lines ◽  
Fluorescence Intensity ◽  
Regulation Of Gene Expression ◽  
Regulatory Region ◽  
Luciferase Reporter ◽  
Relative Fluorescence Intensity ◽  
Hek 293 ◽  
Regulated Gene Expression

Abstract Background: The function of the 5’ regulatory region and the role of the different SNP loci have not been well characterized. This study investigated the effect of several ADH7 haplotypes on the regulation of gene expression in vitro and the functional sequences in the 5’ regulatory region of ADH7. Three SNPs (rs17537595, rs2851028, and rs2654847) and four different haplotypes (T-C-T, T-T-T, C-T-T, T-C-A) were identified by cloning and sequencing. Methods: Effects of 4 different haplotypes and 8 truncated fragments of 5’ regulatory region on ADH7 gene expression were detected using a dual-luciferase reporter assay system. All recombinant plasmids were transfected into HEK-293, U87, and SH-SY5Y cells, respectively, and their relative fluorescence intensity was measured.Results: In HEK-293, U87, and SH-SY5Y cell lines, the relative fluorescence intensity of haplotype T-C-T was significantly higher than that of haplotype T-T-T, C-T-T, and T-C-A. Additionally, we found that regions from -83 to -310bp (ATG, +1), -560 to -768bp , and -987bp to -1203bp up-regulated gene expression. In contrast, the region from -768 to -987bp down-regulated gene expression. The gene expression of regions from -1203 to -1369bp and -1369 to -1626bp was down-regulated in U87 and SH-SY5Y cell lines, but the trend was opposite in HEK-293 cell line. The region from -310 to -560bp up-regulated gene expression in SH-SY5Y cell line, but down-regulated gene expression in HEK-293 and U87 cell lines.Conclusions: This study has shown that the polymorphisms of ADH7 5’ regulatory region play an important role in the regulation of gene expression.

Transcription from a murine T-cell receptor V beta promoter depends on a conserved decamer motif similar to the cyclic AMP response element

1989 ◽  
Vol 9 (11) ◽  
pp. 4835-4845
Author(s):  
S J Anderson ◽  
S Miyake ◽  
D Y Loh
Keyword(s):  
Cyclic Amp ◽  
Regulatory Region ◽  
Cell Receptor ◽  
Transcription Activator ◽  
Mobility Shift ◽  
Response Element ◽  
Cyclic Amp Response Element ◽  
Gel Mobility Shift

We identified a regulatory region of the murine V beta promoter by both in vivo and in vitro analyses. The results of transient transfection assays indicated that the dominant transcription-activating element within the V beta 8.3 promoter is the palindromic motif identified previously as the conserved V beta decamer. Elimination of this element, by linear deletion or specific mutation, reduced transcriptional activity from this promoter by 10-fold. DNase I footprinting, gel mobility shift, and methylation interference assays confirmed that the palindrome acts as the binding site of a specific nuclear factor. In particular, the V beta promoter motif functioned in vitro as a high-affinity site for a previously characterized transcription activator, ATF. A consensus cyclic AMP response element (CRE) but not a consensus AP-1 site, can substitute for the decamer in vivo. These data suggest that cyclic AMP response element-binding protein (ATF/CREB) or related proteins activate V beta transcription.

Abstract 1384: Osteogenic Transcription Factor Runx2 Regulates Expression of RANKL during VSMC Calcification

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Chang Hyun Byon ◽  
Jay McDonald ◽  
Yabing Chen
Keyword(s):  
Oxidative Stress ◽  
Transcription Factor ◽  
Molecular Mechanism ◽  
Luciferase Reporter ◽  
Rankl Expression ◽  
Reporter System ◽  
Mobility Shift ◽  
Atherosclerotic Lesions ◽  
Flanking Region

The expression of receptor activator of nuclear factor κ B (RANKL) is up-regulated in calcified atherosclerotic lesions, whereas it is frequently undetectable in normal vessels. The underlying molecular mechanism of increased expression of RANKL in calcified vessels is not known. We have previously demonstrated that oxidative stress induces calcification of vascular smooth muscle cells (VSMC) in vitro . Therefore, we determined whether oxidative stress regulates RANKL expression in VSMC and the underlying molecular mechanism. Consistent with previous observations in vivo , we found that the expression of RANKL in VSMC isolated from mouse. However, hydrogen peroxide (H 2 O 2 ), which induces VSMC calcification, induced a 33-fold increase in the transcripts of RANKL as determined by real-time PCR. Increased expression of RANKL protein was further confirmed by ELISA. Using flow cytometry, we demonstrated that membrane-bound RANKL was increased by oxidative stress. To characterize the molecular mechanism underlying H 2 O 2 -induced RANKL expression, we employed the luciferase reporter system with a series of deletion mutants of the RANKL 5′-flanking region. The H 2 O 2 responsive region is located between −200 to −400 in the 5′-flanking region of RANKL gene. Analyses of the sequence of this region identified multiple binding sites for the key osteogenic transcription factor, Runx2, which we previously reported to be an essential regulator of VSMC calcification. Electrophoretic mobility shift analyses demonstrated increased binding of Runx2 on the RANKL promoter sequence in nuclear extracts from VSMC exposed to H 2 O 2 . To further determine the role of Runx2 in regulating RANKL expression, we generated stable Runx2 knockdown VSMC with the use of lentivirus-carrying shRNA for Runx2 gene. H 2 O 2 -induced RANKL expression was abrogated in VSMC with Runx2 knockdown. In addition, adenovirus-mediated overexpression of Runx2 in VSMC induced the expression of RANKL. In summary, we have demonstrated that H 2 O 2 induces the expression of RANKL in VSMC, which is regulated by the osteogenic transcription factor Runx2. These observations provide novel molecular insights into the regulation of RANKL and its role on the pathogenesis of calcified atherosclerotic lesions.

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 The CDK7-cycH-p36 complex of transcription factor IIH phosphorylates p53, enhancing its sequence-specific DNA binding activity in vitro.

1997 ◽  
Vol 17 (10) ◽  
pp. 5923-5934 ◽  
Author(s):  
H Lu ◽  
R P Fisher ◽  
P Bailey ◽  
A J Levine
Keyword(s):  
Transcription Factor ◽  
Excision Repair ◽  
Binding Activity ◽  
Mobility Shift ◽  
Trimeric Complex ◽  
Dna Binding Activity ◽  
Terminal Amino ◽  
Gel Mobility Shift ◽  
Kinase Phosphorylation

Phosphorylation is believed to be one of the mechanisms by which p53 becomes activated or stabilized in response to cellular stress. Previously, p53 was shown to interact with three components of transcription factor IIH (TFIIH): excision repair cross-complementing types 2 and 3 (ERCC2 and ERCC3) and p62. This communication demonstrates that p53 is phosphorylated by the TFIIH-associated kinase in vitro. The phosphorylation was found to be catalyzed by the highly purified kinase components of TFIIH, the CDK7-cycH-p36 trimeric complex. The phosphorylation sites were mapped to the C-terminal amino acids located between residues 311 and 393. Serines 371, 376, 378, and 392 may be the potential sites for this kinase. Phosphorylation of p53 by this kinase complex enhanced the ability of p53 to bind to the sequence-specific p53-responsive DNA element as shown by gel mobility shift assays. These results suggest that the CDK7-cycH-p36 trimeric complex of TFIIH may play a role in regulating p53 functions in cells.

A novel Sp1-relatedciselement involved in intestinal alkaline phosphatase gene transcription

1999 ◽  
Vol 276 (4) ◽  
pp. G800-G807 ◽  
Author(s):  
Jeong H. Kim ◽  
Shufen Meng ◽  
Amy Shei ◽  
Richard A. Hodin
Keyword(s):  
Alkaline Phosphatase ◽  
Transcriptional Activation ◽  
Molecular Mechanisms ◽  
Regulatory Element ◽  
Gene Activation ◽  
Regulatory Region ◽  
Intestinal Alkaline Phosphatase ◽  
Mobility Shift ◽  
Sv40 Promoter

We have used sodium butyrate-treated HT-29 cells as an in vitro model system to study the molecular mechanisms underlying intestinal alkaline phosphatase (IAP) gene activation. Transient transfection assays using human IAP-CAT reporter genes along with DNase I footprinting were used to localize a critical cis element (IF-III) corresponding to the sequence 5′-GACTGGGCGGGGTCAAGATGGA-3′. Deletion of the IF-III element resulted in a dramatic reduction in reporter gene activity, and IF-III was shown to function in the context of a heterologous (SV40) promoter in a cell type-specific manner, further supporting its functional role in IAP transactivation. Electrophoretic mobility shift assays revealed that IF-III binds Sp1 and Sp3, but these factors comprise only a portion of the total nuclear binding and appear to mediate only a small portion of its transcriptional activity. IF-III does not correspond to any previously characterized regulatory region from other intestine-specific genes. We have thus identified a novel, Sp1-related cis-regulatory element in the human IAP gene that appears to play a role in its transcriptional activation during differentiation in vitro.

scholarly journals Regulation of the dauBAR operon and characterization of d-amino acid dehydrogenase DauA in arginine and lysine catabolism of Pseudomonas aeruginosa PAO1

Microbiology ◽  
2010 ◽  
Vol 156 (1) ◽  
pp. 60-71 ◽  
Author(s):  
Congran Li ◽  
Xiangyu Yao ◽  
Chung-Dar Lu
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Mutation Analysis ◽  
Regulatory Region ◽  
Mobility Shift ◽  
Signal Molecule ◽  
Acid Dehydrogenase ◽  
Amino Acid Dehydrogenase ◽  
Activity Measurements

A unique d-to-l racemization of arginine by coupled arginine dehydrogenases DauA and DauB encoded by the dauBAR operon has been recently reported as a prerequisite for d-arginine utilization as the sole source of carbon and nitrogen through l-arginine catabolic pathways in P. aeruginosa. In this study, enzymic properties of the catabolic FAD-dependent d-amino acid dehydrogenase DauA and the physiological functions of the dauBAR operon were further characterized with other d-amino acids. These results establish DauA as a d-amino acid dehydrogenase of broad substrate specificity, with d-Arg and d-Lys as the two most effective substrates, based on the kinetic parameters. In addition, expression of dauBAR is specifically induced by exogenous d-Arg and d-Lys, and mutations in the dauBAR operon affect utilization of these two amino acids alone. The function of DauR as a repressor in the control of the dauBAR operon was demonstrated by dauB promoter activity measurements in vivo and mobility shift assays with purified His-tagged protein in vitro. The potential effect of 2-ketoarginine (2-KA) derived from d-Arg deamination by DauA as a signal molecule in dauBAR induction was first revealed by mutation analysis and further supported by its in vitro effect on alleviation of DauR–DNA interactions. Through sequence analysis, putative DauR operators were identified and confirmed by mutation analysis. Induction of the dauBAR operon to the maximal level was found to require the l-arginine-responsive regulator ArgR, as supported by the loss of inductive effect by l-Arg on dauBAR expression in the argR mutant and binding of purified ArgR to the dauB regulatory region in vitro. In summary, this study establishes that optimal induction of the dauBAR operon requires relief of DauR repression by 2-KA and activation of ArgR by l-Arg as a result of d-Arg racemization by the encoded DauA and DauB.

A nuclease-hypersensitive element of the human c-myc promoter interacts with a transcription initiation factor

1989 ◽  
Vol 9 (11) ◽  
pp. 5123-5133
Author(s):  
E H Postel ◽  
S E Mango ◽  
S J Flint
Keyword(s):  
Transcription Factor ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Initiation Factor ◽  
Mobility Shift ◽  
Cis Acting ◽  
Factor Binding ◽  
Myc Oncogene ◽  
Transcription In Vitro

Transcription of the human c-myc oncogene is elaborately regulated, but the relevant molecular mechanisms are not yet understood. To begin to define elements and enzyme systems responsible for c-myc transcription in vitro, we partially purified a transcription factor essential for efficient and accurate in vitro initiation from the principal myc promoter, P2. DNA mobility shift assays located the factor binding domain at -142 to -115 with respect to the P1 promoter. This region contains pur/pyr sequences (predominantly purines in one strand), nuclease-hypersensitive sites (U. Siebenlist, L. Henninghausen, J. Battey, and P. Leder, Cell 37:381-391, 1984; C. Boles and M. Hogan, Biochemistry 26:367-376, 1987), and a triple-helix-forming element (M. Cooney, G. Czernuszewicz, E. Postel, S. Flint, and M. Hogan, Science 241:456-459, 1988). Methylation interference mapping established that the factor, termed PuF, directly contacts the repeated palindromic sequence GGGTGGG of the -142/-115 element. The interaction of PuF with this cis-acting element is necessary for P2 transcription in vitro, for (i) deletion of this 5' region from the myc promoter greatly reduced transcription efficiency and (ii) a synthetic duplex oligonucleotide corresponding to the -142/-115 sequence completely repressed c-myc transcription in the presence of the partially purified factor. These observations lend support to the hypothesis that pur/pyr sequences perform important biological roles in the regulation of c-myc gene expression, most likely by serving as transcription factor binding sites.

scholarly journals Transcription from a murine T-cell receptor V beta promoter depends on a conserved decamer motif similar to the cyclic AMP response element.

1989 ◽  
Vol 9 (11) ◽  
pp. 4835-4845 ◽  
Author(s):  
S J Anderson ◽  
S Miyake ◽  
D Y Loh
Keyword(s):  
Cyclic Amp ◽  
Regulatory Region ◽  
Cell Receptor ◽  
Transcription Activator ◽  
Mobility Shift ◽  
Response Element ◽  
Cyclic Amp Response Element ◽  
Gel Mobility Shift

We identified a regulatory region of the murine V beta promoter by both in vivo and in vitro analyses. The results of transient transfection assays indicated that the dominant transcription-activating element within the V beta 8.3 promoter is the palindromic motif identified previously as the conserved V beta decamer. Elimination of this element, by linear deletion or specific mutation, reduced transcriptional activity from this promoter by 10-fold. DNase I footprinting, gel mobility shift, and methylation interference assays confirmed that the palindrome acts as the binding site of a specific nuclear factor. In particular, the V beta promoter motif functioned in vitro as a high-affinity site for a previously characterized transcription activator, ATF. A consensus cyclic AMP response element (CRE) but not a consensus AP-1 site, can substitute for the decamer in vivo. These data suggest that cyclic AMP response element-binding protein (ATF/CREB) or related proteins activate V beta transcription.

Sign in / Sign up

Export Citation Format

Share Document