YB-1 represses AP1-dependent gene transactivation and interacts with an AP-1 DNA sequence

Samuel, Shaija; Twizere, Jean-Claude; Bernstein, Lori R.

doi:10.1042/bj20041497

Cited by 28 publications

(45 citation statements)

References 33 publications

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“…Cells were TPA treated and assayed for luciferase and β-galactosidase (β-gal) activities as described [19]. The ratio of luciferase to β-gal activity for each transfection was normalized to wild type luciferase reporter activity and expressed as relative luciferase activity.…”

Section: Transient Transfections and Reporter Gene Assaysmentioning

confidence: 99%

“…Both constructs were a gift of Dr. Constance Brinckerhoff (Dartmouth College; [7]). pSVβgal, pcDNAFlag-YB-1 (YB-1 overexpression construct), and pcDNA3.1(+) vector control plasmid are as described [19].…”

Section: Plasmid Constructsmentioning

confidence: 99%

“…Reagents and supplies not described herein were purchased from vendors cited [19,26]. Adherent human HeLa cervical carcinoma cells were cultured as described [19].…”

Section: Reagents and Cell Linesmentioning

confidence: 99%

“…Western immunoblotting was performed according to published procedures using 0.3 μg/ml anti YB-1 (αYB-1) antibody (custom prepared by Bethyl Laboratories) or 0.5 μg/ml αJunD antibody from Santa Cruz Biotechnologies (Santa-Cruz, CA, USA) [19,26].…”

Section: Antibodies and Immunoblottingmentioning

confidence: 99%

“…In our effort to identify new proteins that bind and regulate gene expression at the AP-1 DNA binding site, we previously identified Y box binding protein-1 (YB-1) as a novel AP-1 DNA binding protein [19]. YB-1 is a member of the Y-box family of DNA binding proteins that are defined by the presence of a cold-shock domain in the N-terminal region [20].…”

Section: Introductionmentioning

confidence: 99%

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YB-1 binds to the MMP-13 promoter sequence and represses MMP-13 transactivation via the AP-1 site

Samuel

Beifuss

Bernstein

2007

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expres

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Matrix metalloproteinases (MMPs) are key enzymes that implement degradation of the extracellular matrix during cellular invasion in development, tissue remodeling, and pathogenic disease states. MMP-13 has pivotal roles in the pathogenesis of invasive cancers and arthritis. Here we report the identification of Y-box binding protein-1 (YB-1) as a new repressor of MMP-13 transactivation. YB-1 binds in vitro in DNA affinity chromatography to the activator protein-1 (AP-1) DNA sequence within the MMP-13 promoter. Chromatin immunoprecipitation assays reveal that YB-1 binds in living cells to the MMP-13 gene promoter, to a region of the MMP-13 promoter containing the AP-1 site. YB-1 represses tumor promoter-induced MMP-13 promoter transactivation at the AP-1 site. This is the first report demonstrating YB-1 binding in vitro and in living cells to a mammalian AP-1 target gene, and the first report of YB-1 regulation of the MMP-13 promoter. KeywordsActivator protein-1 (AP-1); chromatin immunoprecipitation (ChIP); matrix metalloproteinase-13 (MMP-13); NAPSTER; transactivation; Y-box binding protein-1 (YB-1)

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Section: Transient Transfections and Reporter Gene Assaysmentioning

confidence: 99%

Section: Plasmid Constructsmentioning

confidence: 99%

“…Reagents and supplies not described herein were purchased from vendors cited [19,26]. Adherent human HeLa cervical carcinoma cells were cultured as described [19].…”

Section: Reagents and Cell Linesmentioning

confidence: 99%

Section: Antibodies and Immunoblottingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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YB-1 binds to the MMP-13 promoter sequence and represses MMP-13 transactivation via the AP-1 site

Samuel

Beifuss

Bernstein

2007

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expres

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Nucleolin binds specifically to an AP‐1 DNA sequence and represses AP1‐dependent transactivation of the matrix metalloproteinase‐13 gene

Samuel

Twizere

Beifuss

et al. 2007

Molecular Carcinogenesis

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Transcriptional regulation via activator protein-1 (AP-1) protein binding to AP-1 binding sites within gene promoter regions of AP-1 target genes plays a key role in controlling cellular invasion, proliferation, and oncogenesis, and is important to pathogenesis of arthritis and cardiovascular disease. To identify new proteins that interact with the AP-1 DNA binding site, we performed the DNA affinity chromatography-based Nucleotide Affinity Preincubation Specificity TEst of Recognition (NAPSTER) assay, and discovered a 97 kDa protein that binds in vitro to a minimal AP-1 DNA sequence element. Mass spectrometric fragmentation sequencing determined that p97 is nucleolin. Immunoblotting of DNA affinity-purified material with anti-nucleolin antibodies confirmed this identification. Nucleolin also binds the AP-1 site in gel shift assays. Nucleolin interacts in NAPSTER with the AP-1 site within the promoter sequence of the metalloproteinase-13 gene (MMP-13), and binds in vivo in chromatin immunoprecipitation assays in the vicinity of the AP-1 site in the MMP-13 promoter. Overexpression of nucleolin in human HeLa cervical carcinoma cells significantly represses AP-1 dependent gene transactivation of a minimal AP-1 reporter construct and of an MMP-13 promoter reporter sequence. This is the first report of nucleolin binding and transregulation at the AP-1 site. ß 2007 Wiley-Liss, Inc.

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Purification and identification of a transcription factor, USF-2, binding to E-box element in the promoter of human telomerase reverse transcriptase (hTERT)

2010

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Controversy remains about the identity of the transcription factor(s) which bind to the two E-box elements (CACGTG, proximal and distal) of the human telomerase (hTERT) gene promoter, the essential elements in the regulation of telomerase. Here, systematic oligonucleotide trapping supplemented with two-dimensional gel electrophoresis (2-DE) and proteomic methods was used to identify E-box binding transcription factors. Although insufficient purity was obtained from the proximal E-box element trapping, further fractionation provided by 2-DE and specific identification from Southwestern blotting analysis allow us to clearly identify an E-box binding transcription factor. The protein spot was cut from 2-DE and in-gel digested with trypsin for LCnanosprayESItandem MS analysis. This identified upstream stimulatory factor-2 (USF-2). Western blotting analysis with specific antibodies clearly shows USF-2 present in the purified fraction and USF-2 antibody supershifts the specific DNA-binding complex on nondenaturing gels. Furthermore, a novel method was developed in which the specific DNA-transcription factor complex was separated on a non-denaturing gel, the band was cut and applied to SDS-PAGE for a second dimension. Western blots of this second gel also confirmed the presence of USF-2. IntroductionCharacterization of the transcription factor proteome is difficult because of the large number of transcription factors and their low abundance in cells. In humans, transcription factors are the second largest group of proteins, only exceeded in number by the metabolic enzymes [1]. The current human transcription factor database (http://dbd.mrc-lmb.cam.ac.uk/DBD/index.cgi?Home) includes 1510 unique transcription factors in humans. Of these, less than 5% have ever been purified and characterized [2]. Here, a systematic oligonucleotide trapping method of purification was coupled to two dimensional gel electrophoresis (2DGE) and LC tandem mass spectrometry to identify the USF2 transcription factor binding to the E-box elements of the human telomerase promoter. This combination of methods proved to be a powerful approach to investigating the transcription factor proteome.Telomeres are approximately 10 kilobase long sequences, consisting of TTAGGG repeats in humans, and situated at the end of chromosomes to protect them from degradation and endto-end fusion. Telomeres are involved in chromosome replication, maintenance of nuclear architecture, chromosome stability, gene expression, aging, and cell division. In somatic cells, each division is associated with the loss of 50-200 base pairs of telomere length. Once the length of telomere is shortened to a critical length, growth arrest or senescence occurs, HIF-1a/ARNT binds proximal and distal E-boxes causing activation in JEG-3 cells under hypoxic conditions [17]. Most of the proteins binding the E-boxes were over expressed either in cancer or transformed cell lines or under abnormal living conditions. In some cases, some of these TF were only tested to bind to one E-box,...

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YB-1 represses AP1-dependent gene transactivation and interacts with an AP-1 DNA sequence

Cited by 28 publications

References 33 publications

YB-1 binds to the MMP-13 promoter sequence and represses MMP-13 transactivation via the AP-1 site

YB-1 binds to the MMP-13 promoter sequence and represses MMP-13 transactivation via the AP-1 site

Nucleolin binds specifically to an AP‐1 DNA sequence and represses AP1‐dependent transactivation of the matrix metalloproteinase‐13 gene

Purification and identification of a transcription factor, USF-2, binding to E-box element in the promoter of human telomerase reverse transcriptase (hTERT)

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