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

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.

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Identification of the human beta-actin enhancer and its binding factor

Molecular and Cellular Biology ◽

10.1128/mcb.8.1.267-272.1988 ◽

1988 ◽

Vol 8 (1) ◽

pp. 267-272

Author(s):

T Kawamoto ◽

K Makino ◽

H Niwa ◽

H Sugiyama ◽

S Kimura ◽

...

Keyword(s):

Simian Virus 40 ◽

High Specificity ◽

Simian Virus ◽

Mobility Shift ◽

Beta Actin ◽

Binding Factor ◽

Pair Sequence ◽

Mobility Shift Assay ◽

Dyad Symmetry

An enhancer of the human beta-actin gene and a factor that specifically interacts with it were detected. A mobility shift assay showed that the factor bound to the 25-base-pair sequence (between +759 and +783 downstream from the cap site) with high specificity. This finding correlated with those of DNase I protection and exonuclease III digestion assays. This binding region of the beta-actin enhancer contained a hyphenated dyad symmetry and an enhancer core-like sequence. In vitro competition experiments indicated that the factor did not bind to the simian virus 40 enhancer core region.

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A detailed and radioisotope-free protocol for electrophoretic mobility shift assay (EMSA)

10.21203/rs.3.pex-939/v1 ◽

2021 ◽

Author(s):

NGUYEN HOAI NGUYEN

Keyword(s):

Transcription Factor ◽

Electrophoretic Mobility Shift Assay ◽

Electrophoretic Mobility ◽

Chromatin Immunoprecipitation ◽

Direct Interaction ◽

Luciferase Reporter ◽

Mobility Shift ◽

Dna Fragment ◽

Mobility Shift Assay

Abstract To comprehensively characterize the functions of a transcription factor (TF), it is required to analyze the interaction of this TF with its targeted loci. Several methods such as β-glucuronidase (GUS) or luciferase reporter, yeast one-hybrid (Y1H), chromatin-immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA) assays have been developed. Of these, EMSA is an in vitro method which can prove the direct interaction between TF and targeted DNA fragment. This protocol is to provide a detailed procedure for a safe EMSA assay (without using any radioisotope).

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Identification of the human beta-actin enhancer and its binding factor.

Molecular and Cellular Biology ◽

10.1128/mcb.8.1.267 ◽

1988 ◽

Vol 8 (1) ◽

pp. 267-272 ◽

Cited By ~ 29

Author(s):

T Kawamoto ◽

K Makino ◽

H Niwa ◽

H Sugiyama ◽

S Kimura ◽

...

Keyword(s):

Simian Virus 40 ◽

High Specificity ◽

Simian Virus ◽

Mobility Shift ◽

Beta Actin ◽

Binding Factor ◽

Pair Sequence ◽

Mobility Shift Assay ◽

Dyad Symmetry

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Identification of a transcription factor, an 80-kDa protein that interacts with the HLH recognition motif of the rat p53 promoter

Biochemistry and Cell Biology ◽

10.1139/o01-002 ◽

2001 ◽

Vol 79 (2) ◽

pp. 153-158 ◽

Cited By ~ 1

Author(s):

Haisun Song ◽

Minhyung Lee ◽

Sunhee Yu ◽

Jong-sang Park

Keyword(s):

Transcription Factor ◽

In Vitro Transcription ◽

Mobility Shift ◽

P53 Expression ◽

Dnase I Footprinting ◽

E Box ◽

Molecular Masses ◽

Mobility Shift Assay ◽

Stimulating Factor

The p53 promoter has been shown to contain a number of potential regulatory motifs. It was previously reported that the upstream stimulating factor (USF) played a central role in regulating the p53 expression. The USF binding site, E-box, is located around 40 bp upstream of the major transcription start site. In this study, it was confirmed that the E-box binds to proteins by DNase I footprinting assay. In the electrophoretic mobility shift assay (EMSA), two retarded bands were detected. One band was abolished by the competition of USF consensus oligonucleotide, but the other band was not. This result indicated that a factor, other than USF, was bound to the E-box. The molecular masses of the binding proteins were determined by a Southwestern-blotting assay. As a result, 46- and 80-kDa proteins were detected. The 46-kDa protein was eliminated by the competition of USF consensus oligonucleotide. Also, the Southwestern-blotting assay with 32P-labeled USF consensus oligonucleotide showed only a 46-kDa protein. Therefore, the 46-kDa protein was USF. These results showed that USF and the 80-kDa protein were bound to the E-box. In addition, it was proved by in vitro transcription assay that this 80-kDa protein had a basal transcriptional activity.Key words: E-box, HLH, rat p53 promoter, transcription factor, upstream stimulating factor (USF).

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Nucleotides Critical for the Interaction of the Streptococcus pyogenes Mga Virulence Regulator with Mga-Regulated Promoter Sequences

Journal of Bacteriology ◽

10.1128/jb.00809-12 ◽

2012 ◽

Vol 194 (18) ◽

pp. 4904-4919 ◽

Cited By ~ 10

Author(s):

Lara L. Hause ◽

Kevin S. McIver

Keyword(s):

Binding Site ◽

Streptococcus Pyogenes ◽

Transcriptional Activation ◽

Luciferase Reporter ◽

Mobility Shift ◽

Content Type ◽

Promoter Sequences ◽

Mobility Shift Assay

ABSTRACTThe Mga regulator ofStreptococcus pyogenesdirectly activates the transcription of a core regulon that encodes virulence factors such as M protein (emm), C5a peptidase (scpA), and streptococcal inhibitor of complement (sic) by directly binding to a 45-bp binding site as determined by an electrophoretic mobility shift assay (EMSA) and DNase I protection. However, by comparing the nucleotide sequences of all established Mga binding sites, we found that they exhibit only 13.4% identity with no discernible symmetry. To determine the core nucleotides involved in functional Mga-DNA interactions, the M1T1 Pemm1binding site was altered and screened for nucleotides important for DNA bindingin vitroand for transcriptional activation using a plasmid-based luciferase reporterin vivo. Following this analysis, 34 nucleotides within the Pemm1binding site that had an effect on Mga binding, Mga-dependent transcriptional activation, or both were identified. Of these critical nucleotides, guanines and cytosines within the major groove were disproportionately identified clustered at the 5′ and 3′ ends of the binding site and with runs of nonessential adenines between the critical nucleotides. On the basis of these results, a Pemm1minimal binding site of 35 bp bound Mga at a level comparable to the level of binding of the larger 45-bp site. Comparison of Pemmwith directed mutagenesis performed in the M1T1 Mga-regulated PscpAand Psicpromoters, as well as methylation interference analysis of PscpA, establish that Mga binds to DNA in a promoter-specific manner.

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The transcriptional repressor gene Mad3 is a novel target for regulation by E2F1

Biochemical Journal ◽

10.1042/bj20021583 ◽

2003 ◽

Vol 370 (1) ◽

pp. 307-313 ◽

Cited By ~ 3

Author(s):

Elizabeth J. FOX ◽

Stephanie C. WRIGHT

Keyword(s):

Cell Cycle ◽

Transcriptional Activation ◽

S Phase ◽

Mobility Shift ◽

Binding Factor ◽

Flanking Region ◽

Novel Target ◽

Electrophoretic Mobility Shift Assays

Mad family proteins are transcriptional repressors that antagonize the activity of the c-Myc proto-oncogene product. Mad3 is expressed specifically during the S-phase of the cell cycle in both proliferating and differentiating cells, suggesting that its biological function is probably linked to processes that occur during this period. To determine the mechanisms that regulate the cell-cycle-specific transcription of Mad3, we used reporter gene assays in stably transfected fibroblasts. We show that the activation of Mad3 at the G1—S boundary is mediated by a single E2F (E2 promoter binding factor)-binding site within the 5′-flanking region of the gene. Mutation of this element eliminated transcriptional activation at S-phase, suggesting that the positively acting E2F proteins play a role in Mad3 regulation. Using electrophoretic mobility-shift assays and chromatin immunoprecipitation, we show that E2F1 binds to the Mad3 5′-flanking region both in vitro and in vivo. We thus identify Mad3 as a novel transcriptional target of E2F1.

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The two activation domains of the CCAAT-binding factor CBF interact with the dTAFII110 component of the Drosophila TFIID complex

Biochemical Journal ◽

10.1042/bj3310291 ◽

1998 ◽

Vol 331 (1) ◽

pp. 291-297 ◽

Cited By ~ 27

Author(s):

Françoise COUSTRY ◽

Satrajit SINHA ◽

Sankar N. MAITY ◽

Benoit de CROMBRUGGHE

Keyword(s):

Transcription Factor ◽

Transcriptional Activation ◽

Direct Interaction ◽

Transcription Activation ◽

Activation Domains ◽

Rich Domain ◽

Eukaryotic Genes ◽

Binding Factor ◽

Tfiid Complex

The CCAAT-binding factor CBF is a heterotrimeric transcription factor that specifically binds to CCAAT sequences in many eukaryotic genes. Previous studies have shown that CBF contains two transcription activation domains: a glutamine-rich, serine-threonine-rich domain present in the CBF-B subunit and a glutamine-rich domain in the CBF-C subunit. In this study, by using a series of deletion mutations of CBF-B and CBF-C in transcription assay in vitro, we further delineated smaller segments in these domains that were sufficient to support transcriptional activation by CBF. To test whether transcription activation by CBF requires co-activators, we examined the interaction between CBF and dTAF110, a component of the Drosophila TFIID complex. Recent work has demonstrated that glutamine-rich domains of the Sp1 transcription factor interact with dTAF110 and that this interaction has an important role in mediating transcription activation. Here we first demonstrate in a direct interaction assay in vitro that CBF binds dTAF110. By using a yeast two-hybrid system we show that both of the transcription activation domains of CBF interact with dTAF110. A deletion analysis suggests that a segment of CBF-B needed for transcription activation is also involved in interaction with dTAF110. In CBF-C the C-terminal portion of the molecule seems to be needed for these two activities. Our results suggest that TAF110 might represent one of the co-activators that mediate transcriptional activation by CBF.

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Androgen response element of the glycine N-methyltransferase gene is located in the coding region of its first exon

Bioscience Reports ◽

10.1042/bsr20130030 ◽

2013 ◽

Vol 33 (5) ◽

Cited By ~ 5

Author(s):

Cheng-Ming Lee ◽

Chia-Hung Yen ◽

Tsai-Yu Tzeng ◽

Yu-Zen Huang ◽

Kuan-Hsien Chou ◽

...

Keyword(s):

Transcriptional Activation ◽

Susceptibility Gene ◽

Luciferase Reporter ◽

Coding Region ◽

Mobility Shift ◽

Methyltransferase Gene ◽

Exon 1 ◽

Mobility Shift Assay

Androgen plays an important role in the pathogenesis of PCa (prostate cancer). Previously, we identified GNMT (glycine N-methyltransferase) as a tumour susceptibility gene and characterized its promoter region. Besides, its enzymatic product-sarcosine has been recognized as a marker for prognosis of PCa. The goals of this study were to determine whether GNMT is regulated by androgen and to map its AREs (androgen response elements). Real-time PCR analyses showed that R1881, a synthetic AR (androgen receptor) agonist induced GNMT expression in AR-positive LNCaP cells, but not in AR-negative DU145 cells. In silico prediction showed that there are four putative AREs in GNMT-ARE1, ARE2 and ARE3 are located in the intron 1 and ARE4 is in the intron 2. Consensus ARE motif deduced from published AREs was used to identify the fifth ARE-ARE5 in the coding region of exon 1. Luciferase reporter assay found that only ARE5 mediated the transcriptional activation of R1881. ARE3 overlaps with a YY1 [Yin and Yang 1 (motif (CaCCATGTT, +1118/+1126)] that was further confirmed by antibody supershift and ChIP (chromatin immunoprecipitation) assays. EMSA (electrophoretic mobility shift assay) and ChIP assay confirmed that AR interacts with ARE5 in vitro and in vivo. In summary, GNMT is an AR-targeted gene with its functional ARE located at +19/+33 of the first exon. These results are valuable for the study of the influence of androgen on the gene expression of GNMT especially in the pathogenesis of cancer.

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Subnuclear compartmentalization of sequence-specific transcription factors and regulation of eukaryotic gene expression

Biochemistry and Cell Biology ◽

10.1139/o05-062 ◽

2005 ◽

Vol 83 (4) ◽

pp. 535-547 ◽

Cited By ~ 7

Author(s):

Gareth N Corry ◽

D Alan Underhill

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Transcription Factors ◽

Transcriptional Activation ◽

Current Knowledge ◽

Nuclear Architecture ◽

Subnuclear Localization ◽

Modular Structures

To date, the majority of the research regarding eukaryotic transcription factors has focused on characterizing their function primarily through in vitro methods. These studies have revealed that transcription factors are essentially modular structures, containing separate regions that participate in such activities as DNA binding, protein–protein interaction, and transcriptional activation or repression. To fully comprehend the behavior of a given transcription factor, however, these domains must be analyzed in the context of the entire protein, and in certain cases the context of a multiprotein complex. Furthermore, it must be appreciated that transcription factors function in the nucleus, where they must contend with a variety of factors, including the nuclear architecture, chromatin domains, chromosome territories, and cell-cycle-associated processes. Recent examinations of transcription factors in the nucleus have clarified the behavior of these proteins in vivo and have increased our understanding of how gene expression is regulated in eukaryotes. Here, we review the current knowledge regarding sequence-specific transcription factor compartmentalization within the nucleus and discuss its impact on the regulation of such processes as activation or repression of gene expression and interaction with coregulatory factors.Key words: transcription, subnuclear localization, chromatin, gene expression, nuclear architecture.

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Arsenic Directly Binds to and Activates the Yeast AP-1-Like Transcription Factor Yap8

Molecular and Cellular Biology ◽

10.1128/mcb.00842-15 ◽

2015 ◽

Vol 36 (6) ◽

pp. 913-922 ◽

Cited By ~ 17

Author(s):

Nallani Vijay Kumar ◽

Jianbo Yang ◽

Jitesh K. Pillai ◽

Swati Rawat ◽

Carlos Solano ◽

...

Keyword(s):

Transcription Factor ◽

Transcriptional Activation ◽

Transcriptional Response ◽

Transcriptional Regulator ◽

Yeast Protein ◽

Content Type ◽

Arsenic Tolerance ◽

Arsenic Sensing

The AP-1-like transcription factor Yap8 is critical for arsenic tolerance in the yeastSaccharomyces cerevisiae. However, the mechanism by which Yap8 senses the presence of arsenic and activates transcription of detoxification genes is unknown. Here we demonstrate that Yap8 directly binds to trivalent arsenite [As(III)]in vitroandin vivoand that approximately one As(III) molecule is bound per molecule of Yap8. As(III) is coordinated by three sulfur atoms in purified Yap8, and our genetic and biochemical data identify the cysteine residues that form the binding site as Cys132, Cys137, and Cys274. As(III) binding by Yap8 does not require an additional yeast protein, and Yap8 is regulated neither at the level of localization nor at the level of DNA binding. Instead, our data are consistent with a model in which a DNA-bound form of Yap8 acts directly as an As(III) sensor. Binding of As(III) to Yap8 triggers a conformational change that in turn brings about a transcriptional response. Thus, As(III) binding to Yap8 acts as a molecular switch that converts inactive Yap8 into an active transcriptional regulator. This is the first report to demonstrate how a eukaryotic protein couples arsenic sensing to transcriptional activation.

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