Pax5 mediates the transcriptional activation of the CD81 gene

AbstractCD81 is an integral membrane protein of the tetraspanin family and forms complexes with a variety of other cell surface membrane proteins. CD81 is involved in cell migration and B cell activation. However, the mechanism of the transcriptional regulation of the CD81 gene remains unclear. Here, we revealed that CD81 transcriptional activation was required for binding of the transcription factor Pax5 at the Pax5-binding sequence (-54)GCGGGAC(-48) located upstream of the transcriptional start site (TSS) of the CD81 gene. The reporter assay showed that the DNA sequence between − 130 and − 39 bp upstream of the TSS of the CD81 gene had promoter activity for CD81 transcription. The DNA sequence between − 130 and − 39 bp upstream of TSS of CD81 harbors two potential Pax5-binding sequences (-87)GCGTGAG(-81) and (-54)GCGGGAC(-48). Reporter, electrophoresis mobility shift, and chromatin immunoprecipitation (ChIP) assays disclosed that Pax5 bound to the (-54)GCGGGAC(-48) in the promoter region of the CD81 gene in order to activate CD81 transcription. Pax5 overexpression increased the expression level of CD81 protein, while the Pax5-knockdown by shRNA decreased CD81 expression. Moreover, we found that the expression level of CD81 was positively correlated with Pax5 expression in human tumor cell lines. Because CD81 was reported to be involved in cell migration, we evaluated the effects of Pax5 overexpression by wound healing and transwell assays. The data showed that overexpression of either Pax5 or CD81 promoted the epithelial cell migration. Thus, our findings provide insights into the transcriptional mechanism of the CD81 gene through transcription factor Pax5.

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Mechanism of RSV-induced IL-8 gene expression in A549 cells before viral replication

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1996.271.6.l963 ◽

1996 ◽

Vol 271 (6) ◽

pp. L963-L971 ◽

Cited By ~ 11

Author(s):

M. A. Fiedler ◽

K. Wernke-Dollries ◽

J. M. Stark

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Viral Replication ◽

Transcriptional Activation ◽

Mutational Analysis ◽

A549 Cells ◽

Nf Kappa B ◽

Mobility Shift ◽

Syncytial Virus ◽

Time Point

Previous studies demonstrated that respiratory syncytial virus (RSV) infection of A549 cells induced interleukin (IL)-8 gene expression and protein release from the cells as early as 2 h after treatment [M. A. Fiedler, K. Wernke-Dollries, and J. M. Stark. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L865-L872, 1995; J. G. Mastronarde, M. M. Monick, and G. W. Hunninghake. Am. J. Respir. Cell Mol. Biol. 13: 237-244, 1995]. Furthermore, the effects of RSV at the 2-h time point were not dependent on viral replication. The studies reported here were designed to test the hypothesis that active and inactive RSV induce IL-8 gene expression in A549 cells at the 2-h time point by a mechanism dependent on the activation of the nuclear transcription factor NF-kappa B Northern blot analysis indicated that IL-8 gene expression occurred independent of protein synthesis 2 h after A549 cells were treated with RSV. Analysis of nuclear extracts from RSV-treated A549 cells by electrophoretic mobility shift assays demonstrated that NF-kappa B was activated as early as 15 min after RSV was added to the cells and remained activated for at least 90 min. In contrast, baseline levels of NF-IL-6 and activator protein-1 (AP-1) did not change over this period of time. Deoxyribonuclease footprint analysis of a portion of the 5'-flanking region of the IL-8 gene demonstrated two potential regions for transcription factor binding, which corresponded to the potential AP-1 binding site, and potential NF-IL-6 and NF-kappa B binding sites. Mutational analysis of the 200-bp 5'-untranslated region of the IL-8 gene demonstrated that activation of NF-kappa B and NF-IL-6 were required for RSV-induced transcriptional activation of the IL-8 gene.

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

Biochemical Journal ◽

10.1042/bj3110769 ◽

1995 ◽

Vol 311 (3) ◽

pp. 769-773 ◽

Cited By ~ 24

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.

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IL-1β induces eotaxin gene transcription in A549 airway epithelial cells through NF-κB

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2000.279.6.l1058 ◽

2000 ◽

Vol 279 (6) ◽

pp. L1058-L1065 ◽

Cited By ~ 30

Author(s):

Sean Jedrzkiewicz ◽

Hidetoshi Nakamura ◽

Eric S. Silverman ◽

Andrew D. Luster ◽

Naresh Mansharamani ◽

...

Keyword(s):

Epithelial Cells ◽

Transcriptional Activation ◽

Transcriptional Start Site ◽

Nuclear Factor Κb ◽

Airway Epithelial Cells ◽

Site Directed Mutagenesis ◽

Airway Epithelial ◽

Mobility Shift ◽

Mobility Shift Assay ◽

Necessary And Sufficient

Eotaxin is an asthma-related C-C chemokine that is produced in response to interleukin-1β (IL-1β). We detected an increase in newly transcribed eotaxin mRNA in IL-1β-stimulated airway epithelial cells. Transient transfection assays using promoter-reporter constructs identified a region as essential for IL-1β-induced increases in eotaxin transcription. Using site-directed mutagenesis, we found that a nuclear factor-κB (NF-κB) site located 46 bp upstream from the transcriptional start site was both necessary and sufficient for IL-1β induction of reporter construct activity. Electrophoretic mobility shift assay demonstrated that IL-1β-stimulated airway epithelial cells produced p50 and p65 protein that bound this site in a sequence-specific manner. The functional importance of the NF-κB site was demonstrated by coexpression experiments in which increasing doses of p65 expression vector were directly associated with reporter activity exclusively in constructs with an intact NF-κB site ( r 2 = 0.97, P = 0.002). Moreover, IL-1β-induced increases in eotaxin mRNA expression are inhibited by inhibitors of NF-κB. Our findings implicate NF-κB and its binding sequence in IL-1β-induced transcriptional activation of the eotaxin gene.

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Transcriptional regulation of human abraxas brother protein 1 expression by yin yang 1

Biochemistry and Cell Biology ◽

10.1139/bcb-2019-0279 ◽

2020 ◽

Author(s):

Pan Song ◽

Jian Hong ◽

Yuan Wang ◽

Xuelian Yao ◽

Yiqun Zhan ◽

...

Keyword(s):

Transcription Factor ◽

Transcriptional Regulation ◽

Transcriptional Start Site ◽

Regulatory Elements ◽

Start Codon ◽

Mobility Shift ◽

Cis Acting ◽

Yin Yang 1 ◽

Activating Transcription Factor 4 ◽

Yin Yang

Abraxas brother protein 1 (ABRO1) is a subunit of the deubiquitinating enzyme BRCC36-containing isopeptidase complex and plays important roles in cellular responses to stress by interacting with its binding partners, such as ubiquitin-specific peptidase 7, p53, activating transcription factor 4, THAP-domain containing 5, and serine hydroxymethyltransferase. However, the transcriptional regulation of ABRO1 remains unexplored. In this study, we identified and characterized the core regulatory elements of the human ABRO1 gene and mapped them to the ABRO1 promoter region. Additionally, 5′ rapid amplification of cDNA ends revealed that the transcriptional start site (TSS) was located −13 bp upstream from the start codon. Reporter gene, chromatin immunoprecipitation, and electrophoretic mobility shift assays demonstrated that ABRO1 transcription was regulated through cis-acting elements located in the region −89 to −59 bp upstream of the ABRO1 TSS and that these elements were targeted by yin yang 1 transcription factor (YY1). Moreover, YY1 overexpression increased human ABRO1 mRNA and protein expression, and small-interfering RNA-mediated downregulation of YY1 attenuated ABRO1 expression. These results suggested that YY1 positively regulated human ABRO1 expression by binding to cis-acting elements located in the ABRO1 TSS.

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Intracellular HMGB1 Transactivates the Human IL1B Gene Promoter through Association with an ETS Transcription Factor PU.1.

Blood ◽

10.1182/blood.v108.11.1271.1271 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1271-1271

Author(s):

Junichi Tsukada ◽

Fumihiko Mouri ◽

Takamitsu Mizobe ◽

Takehiro Higashi ◽

Hiroto Izumi ◽

...

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Transcriptional Activation ◽

Proinflammatory Cytokine ◽

Expression Vector ◽

High Mobility ◽

Gene Promoter ◽

Physical Interaction ◽

Chromosomal Protein ◽

Mobility Shift

Abstract High mobility group box chromosomal protein 1 (HMGB1) was recently identified as a late proinflammatory mediator of endotoxin lethality, which amplifies and sustains the inflammatory processes via macrophage/monocyte activation by extracellular release of HMGB1. However, it was originally described as a nonhistone nuclear protein to bind and distort the structure of DNA and to regulate transcription through association with several transcription factors. Here we investigated the effect of intracellular HMGB1 on transcriptional gene regulation of an immediate early proinflammatory cytokine IL-1β in transient transfection studies using HMGB1 expression vector (pcDNA3HMGB1) and murine macrophage cells RAW264.7. When pcDNA3HMGB1 was introduced into RAW264.7 cells with pGL3HT reporter containing the -131 to +12 minimal IL-1β gene promoter, IL-1β promoter activity was significantly induced by expression of HMGB1. The gene promoter possesses two important transcription factor binding motifs, one for PU.1, a myeloid and B cell-specific transcription factor that belongs to the ETS family, and the other a binding site for NF-IL6. To verify the functional role of intracellular HMGB1 in transactivation of the IL-1β promoter, a PU.1 expression vector and/or pcDNA3HMGB1 were cotransfected into PU.1-deficient murine thymocytes EL4 cells along with pGL3HT reporter. As a result, HMGB1 synergized with PU.1 to transactivate the IL-1β promoter, but not HMGB1 alone. This argument was supported by our GST-pulldown data, which demonstrated direct physical interaction of HMGB1 with PU.1. In addition, deletion of the PU.1 winged helix-turn-helix DNA-binding domain significantly inhibited the association of PU.1 with HMGB1. To determine whether HMGB1 could affect PU.1 DNA-binding affinity, we performed electrophoretic mobility shift assay using a radiolabeled IL-1β -59 to +12 promoter element (DT), recombinant PU.1 (rPU.1) and GST-HMGB1. Two complexes with slower and faster mobilities were generated by the addition of HMGB1 to a mixture of rPU.1 and DT probe. The two complexes were abrogated by preincubation with anti-PU.1 Ab, while anti-HMGB1 Ab reacted only with the complex with a slower mobility, indicating that the complex with a slower mobility formed by addition of HMGB1 contained both HMGB1 and PU.1, while the band with a faster mobility contained only PU.1. From the present study, we propose that intracellular HMGB1 might function as a coactivator in PU.1-mediated transcriptional activation, which facilitate access of PU.1 to specific DNA targets. The fact that PU.1 is a transcription factor essential to macrophages/monocyte-specific proinflammatory cytokine genes further raise the possibility that interaction of PU.1 with HMGB1 may play an important role in the inflammation cascade in macrophages/monocytes.

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Transcriptional Activation of sclA by Mga Requires a Distal Binding Site in Streptococcus pyogenes

Journal of Bacteriology ◽

10.1128/jb.186.23.7847-7857.2004 ◽

2004 ◽

Vol 186 (23) ◽

pp. 7847-7857 ◽

Cited By ~ 29

Author(s):

Audry C. Almengor ◽

Kevin S. McIver

Keyword(s):

Binding Site ◽

Streptococcus Pyogenes ◽

Transcriptional Activation ◽

Fluorescent Protein ◽

Transcriptional Start Site ◽

Reporter System ◽

Chromosomal Locus ◽

Mobility Shift ◽

Transcriptional Reporter ◽

Regulated Promoters

ABSTRACT Streptococcus pyogenes (the group A streptococcus [GAS]) is a medically significant pathogen of humans, causing a range of diseases from pharyngitis to necrotizing fasciitis. Several important GAS virulence genes are under the control of a pleiotropic regulator called Mga, or the multiple gene regulator of GAS, including the gene encoding the streptococcal collagen-like protein, or sclA. Analysis of the genome sequence upstream of sclA revealed two potential Mga-binding sites with homology to the published Mga-binding element, which were called PsclA-I (distal) and PsclA-II (proximal) based on their location relative to a predicted start of transcription. Primer extension was used to confirm that the Mga-dependent transcriptional start site for sclA was located adjacent to the proximal PsclA-II binding site. By using overlapping PsclA promoter probes and purified Mga-His fusion protein, it was shown by electrophoretic mobility shift assays that, unlike other Mga-regulated promoters, Mga binds only to a distal DNA-binding site (PsclA-I). Binding of Mga to PsclA-I could be competed with cold probes corresponding to known Mga-regulated promoters (Pemm, PscpA, and Pmga) but not with a nonspecific probe or the proximal PsclA-II fragment. With the use of a plasmid-based green fluorescent protein transcriptional reporter system, the full-length PsclA was not sufficient to reproduce normal Mga-regulated activation. However, studies using a single-copy gusA transcriptional reporter system integrated at the native sclA chromosomal locus clearly demonstrated that the distal PsclA-I binding site is required for Mga regulation. Therefore, PsclA represents a new class of Mga-regulated promoters that requires a single distal binding site for activation.

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Transcriptional stimulation of the human NHE3 promoter activity by PMA: PKC independence and involvement of the transcription factor EGR-1

Biochemical Journal ◽

10.1042/bj20051391 ◽

2006 ◽

Vol 396 (2) ◽

pp. 327-336 ◽

Cited By ~ 26

Author(s):

Jaleh Malakooti ◽

Ricardo Sandoval ◽

Md. Ruhul Amin ◽

Jeremy Clark ◽

Pradeep K. Dudeja ◽

...

Keyword(s):

Transcription Factor ◽

Mrna Expression ◽

Transcriptional Activation ◽

Promoter Region ◽

Promoter Activity ◽

De Novo ◽

Growth Conditions ◽

Mobility Shift ◽

Nuclear Extracts ◽

Stimulation Of

NHE3 (Na+/H+ exchanger 3) is essential for Na+ absorption in the ileum and is expressed in a cell-specific manner in the apical membrane of the intestinal epithelial cells. In the present study, we report the stimulatory effect of PMA on the hNHE3 (human NHE3) transcription. Pretreatment with actinomycin D or cycloheximide blocked the up-regulation of the NHE3 mRNA by PMA, indicating that the increased level of NHE3 mRNA expression is regulated by transcriptional activation and is dependent on de novo protein synthesis. 5′-Deletion of the promoter region and transfection analysis in C2BBe1 cells revealed that the PMA effect is mediated through a GC-rich DNA region between nt −88 and −69. Gel mobility-shift assays demonstrated that in nuclear extracts from C2BBe1 cells grown under the basal growth conditions, Sp1 (stimulating protein-1) and Sp3 interact with this GC-rich DNA region, while, in PMA-treated nuclear extracts, PMA-induced EGR-1 (early growth response gene product 1) transcription factor binds to the same site. Binding of EGR-1 diminished the Sp1 and Sp3 interactions with this promoter region significantly. Co-transfection of Sp1 or Sp3 into SL2 cells activated the NHE3-reporter constructs, suggesting that Sp1 and Sp3 act as positive regulators of the NHE3 expression. In addition, overexpression of EGR-1 was sufficient to transactivate the NHE3-reporter gene activity, and knockdown of EGR-1 with gene-specific small interfering RNA resulted in inhibition of the PMA-induced up-regulation of the endogenous NHE3 mRNA expression. Furthermore, the PKC (protein kinase C) inhibitor chelerythrine chloride did not affect PMA-induced NHE3 promoter activity, suggesting that PMA stimulation of the hNHE3 gene expression may be PKC-independent.

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Phosphorylation of the oncogenic transcription factor ERG in prostate cells dissociates polycomb repressive complex 2, allowing target gene activation

Journal of Biological Chemistry ◽

10.1074/jbc.m117.796458 ◽

2017 ◽

Vol 292 (42) ◽

pp. 17225-17235 ◽

Cited By ~ 7

Author(s):

Vivekananda Kedage ◽

Brady G. Strittmatter ◽

Paige B. Dausinas ◽

Peter C. Hollenhorst

Keyword(s):

Transcription Factor ◽

Cell Migration ◽

Transcriptional Activation ◽

Molecular Mechanisms ◽

Cellular Migration ◽

Serine Residue ◽

Polycomb Repressive Complex ◽

Polycomb Repressive Complex 2 ◽

Prostate Cells ◽

Prostate Cancers

In ∼50% of prostate cancers, chromosomal rearrangements cause the fusion of the promoter and 5′-UTR of the androgen-regulated TMPRSS2 (transmembrane protease, serine 2) gene to the open reading frame of ERG, encoding an ETS family transcription factor. This fusion results in expression of full-length or N-terminally truncated ERG protein in prostate epithelia. ERG is not expressed in normal prostate epithelia, but when expressed, it promotes tumorigenesis via altered gene expression, stimulating epithelial-mesenchymal transition, cellular migration/invasion, and transformation. However, limited knowledge about the molecular mechanisms of ERG function in prostate cells has hampered efforts to therapeutically target ERG. ERK-mediated phosphorylation of ERG is required for ERG functions in prostate cells, but the reason for this requirement is unknown. Here, we report a mechanism whereby ERK-mediated phosphorylation of ERG at one serine residue causes a conformational change that allows ERK phosphorylation at a second serine residue, Ser-96. We found that the Ser-96 phosphorylation resulted in dissociation of EZH2 and SUZ12, components of polycomb repressive complex 2 (PRC2), transcriptional activation of ERG target genes, and increased cell migration. Conversely, loss of ERG phosphorylation at Ser-96 resulted in recruitment of EZH2 across the ERG-cistrome and a genome-wide loss of ERG-mediated transcriptional activation and cell migration. In conclusion, our findings have identified critical molecular mechanisms involving ERK-mediated ERG activation that could be exploited for therapeutic intervention in ERG-positive prostate cancers.

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Identification of a Novel Melon Transcription Factor CmNAC60 as a Potential Regulator of Leaf Senescence

Genes ◽

10.3390/genes10080584 ◽

2019 ◽

Vol 10 (8) ◽

pp. 584 ◽

Cited By ~ 1

Author(s):

Cao ◽

Zhang ◽

Wang ◽

Li ◽

Guo ◽

...

Keyword(s):

Transcription Factor ◽

Leaf Senescence ◽

Transcriptional Activation ◽

Regulatory Elements ◽

Expression Level ◽

Transactivation Domain ◽

Specific Expression ◽

Nac Transcription Factors ◽

Oriental Melon ◽

Male Flowers

NAC transcription factors (TFs) play important roles in plants’ responses to abiotic stresses and developmental processes, including leaf senescence. Oriental melon (Cucumis melo var. makuwa Makino) is an important vegetable crop in China and eastern Asia countries. However, little is known about the functions of the melon NAC family members. In this study, a phylogenetic tree was constructed to show that CmNAC60 and the senescence regulator AtNAP were in the same cluster, which implied that CmNAC60 might be a NAC related to leaf senescence. The expression analysis of CmNAC60 in different melon organs showed that the expression of CmNAC60 was highest in the male flowers and lowest in the hypocotyl. In addition, the expression level of CmNAC60 in the senescing leaves was significantly higher than in the non-senescing leaves. Similarly, the expression level of CmNAC60 in the dark-treated leaves was significantly higher than in the untreated leaves. Furthermore, the subcellular localization and transcriptional activation assays indicated that CmNAC60 was a nucleus localized NAC transcription factor with a C-terminal transactivation domain. An analysis of the tissue specific expression showed that the promoter of CmNAC60 may contain cis-acting regulatory elements responsive to leaf senescence. CmNAC60 overexpressing lines of Arabidopsis showed a precocious senescence compared with the wild type (WT). Collectively, our results showed that CmNAC60 was associated with leaf senescence, and could be potentially utilized in molecular breeding to improve melon yield or to extend the postharvest shelf life by delaying leaf senescence.

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The DLK gene is a transcriptional target of PPARγ

Biochemical Journal ◽

10.1042/bj20101840 ◽

2011 ◽

Vol 438 (1) ◽

pp. 93-101 ◽

Cited By ~ 7

Author(s):

Jean-Philippe Couture ◽

Richard Blouin

Keyword(s):

Transcriptional Activation ◽

Binding Sites ◽

Leucine Zipper ◽

Transcriptional Start Site ◽

Peroxisome Proliferator ◽

Mobility Shift ◽

Peroxisome Proliferator Activated Receptor ◽

Bioinformatic Tools ◽

Pparγ Agonist ◽

Transcriptional Target

DLK (dual leucine zipper-bearing kinase) is a key regulator of development, cell differentiation and apoptosis. Interestingly, recent studies have shown that DLK expression is up-regulated in 3T3-L1 cells induced to differentiate into adipocytes and that DLK knockdown impairs the expression of PPARγ (peroxisome-proliferator-activated receptor γ), a master regulator of adipogenesis. Because the PPARγ agonist rosiglitazone was found to increase DLK expression in 3T3-L1 cells, we hypothesized that PPARγ is required for the transcriptional activation of the DLK gene. To test this hypothesis, we first examined the effects of pharmacological inhibition or shRNA (small-hairpin RNA)-mediated depletion of PPARγ on DLK accumulation in 3T3-L1 cells undergoing differentiation. In addition to blocking adipocyte conversion of 3T3-L1 cells, inhibition of PPARγ suppressed DLK expression at both the mRNA and protein levels. Moreover, supporting a role for PPARγ in DLK regulation, two potential PPARγ-binding sites identified by bioinformatic tools at positions −611 and −767 upstream of the DLK gene transcriptional start site were shown by electrophoretic mobility-shift assay and chromatin immunoprecipitation to bind PPARγ and its essential heterodimer partner retinoid X receptor as differentiation proceeds. Collectively, these results show that DLK is a novel transcriptional target of PPARγ with functional PPARγ-binding sites in its promoter.

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